Bnab 014

Endocrine Reviews, 2022, Vol. 43, No.
1, 160–197
doi:10.1210/endrev/bnab014
Review
Review
How Protein Methylation Regulates Steroid

Receptor Function
Lucie Malbeteau,1,2,3,* Ha Thuy Pham,1,2,3,* Louisane Eve,1,2,3
Downloaded from https://academic.oup.com/edrv/article/43/1/160/6270645 by guest on 25 August 2022

Michael R. Stallcup,4 Coralie Poulard,1,2,3 and Muriel Le Romancer1,2,3
1
Université de Lyon, F-69000 Lyon, France; 2Inserm U1052, Centre de Recherche en Cancérologie de
Lyon, F-69000 Lyon, France; 3CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, F-69000
Lyon, France; and 4Department of Biochemistry and Molecular Medicine, Norris Comprehensive Center,
University of Southern California, Los Angeles, CA 90089, USA
*The authors contributed equally to the work.
ORCiD number: 0000-0002-8491-4015 (M. Le Romancer).
Abbreviations: AR, androgen receptor; BC, breast cancer; ER, estrogen receptor; GAR, glycine and arginine-rich motif;
GC, glucocorticoid; GR, glucocorticoid receptor; IGF, insulin-like growth factor; JHDM, JmjC domain-containing histone
demethylase; KMT, lysine methyltransferase; LBD, ligand binding domain; MS, mass spectrometry; NLS, nuclear localization
signal; NR, nuclear receptor; NTD, aminoterminal domain; PC, prostate cancer; PR, progesterone receptor; PRMT, protein
arginine methyltransferase; PTM, post-translational modification; SR, steroid receptor; TF, transcription factor.
Received: 16 February 2021; Editorial Decision: 15 April 2021; First Published Online: 5 May 2021; Corrected and Typeset:
23 July 2021.
Abstract
Steroid receptors (SRs) are members of the nuclear hormonal receptor family, many of
which are transcription factors regulated by ligand binding. SRs regulate various human
physiological functions essential for maintenance of vital biological pathways, including
development, reproduction, and metabolic homeostasis. In addition, aberrant expres-
sion of SRs or dysregulation of their signaling has been observed in a wide variety of
pathologies. SR activity is tightly and finely controlled by post-translational modifica-
tions (PTMs) targeting the receptors and/or their coregulators. Whereas major attention
has been focused on phosphorylation, growing evidence shows that methylation is also
an important regulator of SRs. Interestingly, the protein methyltransferases depositing
methyl marks are involved in many functions, from development to adult life. They have
also been associated with pathologies such as inflammation, as well as cardiovascular
and neuronal disorders, and cancer. This article provides an overview of SR methylation/
demethylation events, along with their functional effects and biological consequences.
An in-depth understanding of the landscape of these methylation events could provide
new information on SR regulation in physiology, as well as promising perspectives for
the development of new therapeutic strategies, illustrated by the specific inhibitors of
protein methyltransferases that are currently available.
ISSN Print: 0163-769X

ISSN Online: 1945-7189
Printed: in USA
© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial- https://academic.oup.com/edrv 160
NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction
and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and
that the work is properly cited. For commercial re-use, please contact [email protected]
Endocrine Reviews, 2022, Vol. 43, No. 1 161
Key Words: ERα, PR, AR, GR, protein arginine methyltransferases, lysine methyltransferases, protein demethylases,
coregulators, steroid receptors, methylation
Graphical Abstract

ESSENTIAL POINTS
• Steroid hormones and their receptors play many critical roles in human physiology and pathology through specific
gene regulation and modulation of cytoplasmic signaling pathways.
• In addition to other types of post-translational modifications, methylation of lysine and arginine is recently shown
to regulate steroid receptor activity.
• Methylation of histones and nonhistone proteins also participate indirectly in the activities of steroid receptors.
• Protein methyltransferases and demethylases play important roles in physiology and their dysregulation contributes
to human pathologies.
• Increased understanding of the biology of steroid receptor methylation, along with specific methylation enzyme
inhibitors, will result in new potential therapeutic options.
Introduction lifespan. The female hormone estrogen is well known for its
Steroid hormones play critical roles in various target tis- effects on mammary gland and reproductive tract develop-
sues regulating body homeostasis. Their ability to easily ment. Progesterone, the other female hormone, plays a vital
transit through cell membranes aroused interest of scien- role during pregnancy. In males, the androgen hormone is
tists for their therapeutic potential. Glucocorticoid (GC) was important for sexual development and reproductive func-
the first type of steroid to be used in the clinic. The phys- tion. The mediators of these hormones remained elusive
ician Philip Hench successfully treated rheumatoid arthritis until the early 1960s, when radiolabeled lipophilic ligands
symptoms with cortisone, a discovery for which the Nobel were developed by Jensen and Jacobson (2). Their innova-
Prize in Medicine was awarded in 1950 (1). Thereafter, a tive experiments led to the identification of “radioligand-
plethora of experiments documented the influence of steroid binding proteins,” now known as steroid receptors (SRs).
hormones on many biological processes throughout the Interestingly, these receptors were able to migrate from the
162 Endocrine Reviews, 2022, Vol. 43, No. 1
cytoplasm to the nucleus, implying that ligand-bound recep- and reversible process controlled by the activity of protein
tors could influence transcription. This hypothesis was con- demethylases, which are able to remove methyl marks.
firmed by Ashburner, who showed that the addition of the It is particularly relevant to note that KMTs and PRMTs
ecdysteroid hormone was responsible for the activation of are frequently overexpressed in cancer cells compared with
a specific subset of genes in drosophila salivary glands (3). normal cells, and that their upregulation is often associated
This “Ashburner hormonal model” was the foundation for with poor prognosis. In line with this, specific inhibitors
our current knowledge on the regulation of transcription by targeting the methyltransferase activities have recently been
these receptors and is still, nowadays, unexpectedly relevant. developed to assess the involvement of this PTM in tumori-
Since then, numerous clinical therapies have been devel- genesis, making these enzymes interesting targets to modu-
oped based on this hormone–receptor association. Among late SR properties (14, 15).
them, mifepristone targets the progesterone receptor (PR) to The aim of this review is to summarize current and
treat endometriosis (4); dexamethasone acts on the gluco- emerging knowledge about the influence of protein methy-

corticoid receptor (GR) to induce anti-inflammatory and lation in the biology of SRs. As these receptors and their
immunosuppressive effects (5), and is for instance currently signaling are involved in numerous pathologies, targeting
used in the treatment of SARS-Cov2 viral infection (6, 7); tam- protein methyltransferase activities could provide prom-
oxifen blocks the estrogen receptor (ERα), thus inhibiting es- ising new therapeutic tools.
trogen binding and consequently reducing the progression of
hormone-dependent breast cancers (BCs) (8); antiandrogens
targeting the androgen receptor (AR) are frequently associated
Steroid Receptors
with chemical or rarely used surgical castration for improving
the overall survival of prostate cancer (PC) patients (9). Structure
The nuclear receptor (NR) superfamily consists of evo- Phylogenetic studies in eukaryotic organisms have placed
lutionarily related DNA-binding transcription factors (TFs), the emergence of SRs a long time before the divergence be-
encoded by 48 genes in humans. Within this superfamily, a tween vertebrates and invertebrates (16). This universality
subgroup of 5 ligand-regulated receptors, the SRs, has been has been a powerful tool for scientists, allowing them to
extensively exploited for the development of selective modu- study and to decrypt the regulation of crucial hormone re-
lators and raised promising expectations for novel treatment ceptor–dependent physiological processes, such as develop-
strategies to address diverse medical conditions (10, 11). We ment, cell growth, reproduction, and homeostasis, in simple
will herein focus on SRs for which methylation events have organisms (compared with humans). Since the 1980s, many
been well documented, namely ERα, PR, AR, and GR. NR cDNAs have been cloned and characterized, including
Methylation has emerged as a major post-translational GR (16), ERα (17), PR (18), and AR (19).
modification (PTM) of proteins, with wide-ranging conse- The cloning of the first SR genes (ie, GR and ERα) was
quences on their activity, in modulating their properties, a major breakthrough in the field, highlighting a surprising
such as subcellular localization, stability, or interactions homology between them. This observation provided the
with partners. Most protein methyltransferases prefer- first chemical evidence that distinct hormones bind struc-
entially methylate arginine and lysine residues. Based turally related receptors. Indeed, SRs share a common or-
on the residue targeted, these enzymes are classified as ganization with 5 functional domains (A/B, C, D, E, F), with
lysine methyltransferases (KMTs) or protein arginine varying degrees of structural homology (20, 21) (Fig. 1A).
methyltransferases (PRMTs). Protein methylation was ini- The central DNA binding domain (DBD or C domain) is
tially identified on histone proteins, a process deeply in- the most conserved region and is crucial for the association
volved in local chromatin remodeling and then in gene between the receptor and specific hormone-response elem-
regulation. However, a growing number of studies have ents (HREs) (22, 23). The ligand binding domain (LBD) is
reported methylation events on nonhistone proteins, re- the second domain with considerable structural homology
vealing that methylation is involved in a large variety of among NRs (24) (Fig. 1A). It is essential for ligand binding,
biological effects. Currently, hundreds of methylated sub- but its role is much broader. The LBD structure allows the
strates have been identified, in various cellular processes LBD itself to undergo conformational changes leading to
including RNA metabolism, DNA repair, or gene tran- the recruitment of a SR partner for dimerization (25), as
scription. Indeed, SR-dependent transcription is strongly well as coregulators (26) through a ligand-dependent trans-
influenced by histone methylation (12, 13), as well as by activation domain (also called AF-2 for activation func-
the methylation of SRs themselves and their associated tion-2) (27). Between these 2 highly conserved regions, a
coregulators. Consistent with other PTMs (ie, phosphor- less-conserved interdomain linker is present in all members
ylation, acetylation, etc.), protein methylation is a dynamic of the superfamily (28) (Fig. 1A), called the hinge region

Figure 1. Common structural organization of steroid receptors. (A) Schematic representation of steroid receptors (SRs) structure. The amino-terminal
domain (NTD) is variable in size and composition and contains a ligand-independent transactivation domain (AF-1). The DNA binding domain (DBD)
is the most conserved region, in which 2 zinc fingers maintain the core of the domain and bind to DNA. A less-conserved hinge region is present be-
tween the DBD and the ligand binding domain (LBD) and contains a nuclear localization signal (NLS). The ligand associates with the receptor through
the LBD, which also contains a ligand-dependent transactivation domain (AF-2). The functions associated with the F domain are still not clearly
understood. (B) The members of the steroid receptor (ie, ERα, PR, GR, and AR) subgroup share a deeply conserved structure of functional domains
with some specificities. The main biological roles of these SRs, and the associated pathological disorders when SR signaling are dysregulated, are
pointed out on the right.
(or D domain). This domain supports the DBD in the tran- Biological and Pathological Roles
scriptional activity of SRs (29, 30) by ensuring their nuclear SRs are ligand-inducible TFs playing crucial roles in the
localization with a nuclear localization signal, and by co- regulation of diverse and essential aspects of mamma-
operating with the LBD to mediate SR dimerization (31) lian physiology, by controlling the expression of genetic
and its interactions with certain coregulators (32). At the programs to achieve synchronized and accurate func-
N-terminal end, each member exhibits an aminoterminal tional responses (36–38). Responding to endocrine hor-
domain (NTD or A/B domain), highly variable in size, mones, they trigger adaptive signals and serve as a potent
amino acid composition, and PTMs (33) (Fig. 1B). This regulatory interface between the cellular and organismal
A/B domain notably includes a ligand-independent trans- environment and the genome. However, they also drive
activation domain (or AF-1), which can act independently, pathological states when their signaling pathways become
although optimal receptor activity generally requires a dysregulated. This feature has led to the development of
synergistic cooperation between AF-1 and the ligand- numerous treatments to control endocrine-associated dis-
dependent transactivation domain AF-2 (34). At the other eases, including neurological disorders, chronic inflamma-
extremity, the F region is highly variable and not present tory diseases, or cancers (39).
in all members of the superfamily. Although little is known ERα acts in concert with PR to regulate the physio-
about it, mutations or complete deletions can strongly alter logical development of female reproductive tissues and
the transcriptional activity of the receptor, ligand binding, mammary glands. Variable concentrations of ovarian pro-
as well as interactions with coregulators (35). gesterone and estrogen hormones regulate decisive phases
of human female life (puberty, pregnancy, and menopause). growth, and glucose and lipid metabolism. It seems that
ERα knockout in pubescent mice totally impairs mammary the receptor acts as a negative regulator of adipocyte de-
gland development (40), whereas in mature women, ERα velopment in adult males, resulting in late-onset obesity
is a crucial modulator of breast cell proliferation and sur- when knocked out (53). Similarly to other SRs, the field
vival, strongly influencing their risk of developing BC. In of action of AR is vast, and a decisive role for androgen-
bone tissue, ERα modulators such as tamoxifen maintain activated receptors in neurodegenerative processes, such as
bone mineral density in postmenopausal women and pre- spinal bulbar muscular atrophy, has been depicted (54, 55).
vent osteoporosis. A set of studies has also reported that Interestingly, in vivo studies highlighted underestimated
ERα signaling is deeply involved in metabolic homeostasis and essential roles for AR and androgen signaling in female
and metabolic disorders, such as diabetes, dyslipidemia, reproduction, including ovarian and breast physiology,
or obesity, and influences numerous biological systems, such that dysregulation leads to dysfunctions and cancers
including the immune and cardiovascular systems, due to (51, 56–58). In contrast, a recent work showed that AR

the presence of ERα in all these sites (41). acts as a tumor suppressor in ERα-positive BC (59).
Progesterone is the “hormone of pregnancy” in
postpubescent women, as it is essential for the establish-
ment and maintenance of pregnancy (42). Hormone- Signaling Pathways
associated PRs target genes in multiple organs or systems The binding of steroid hormones to their receptors triggers
to prepare the body for pregnancy, including the endomet- a conformational change within the LBD and functions as
rial epithelium, the venous walls or in mammary glands a critical step, shifting the receptor function from an in-
(43). Another notable role for PR is the modulation of im- active to an active state (60, 61). Steroid-bound receptors
mune functions, with deep and distinct consequences in generally dimerize as homodimers and translocate to the
autoimmune diseases, including rheumatoid arthritis and nucleus (Fig. 2). They target specific HREs, generally pal-
systemic lupus erythematosus (44, 45). Recent studies have indromic, which fully or partially resemble 2 consensus
also highlighted the pleiotropic protective effects in brain half-sites (5′-AGGACA-3′ for GR, or 5′-AGGTCA-3′ for
cells exerted by progestin-liganded PR, making PR a key ERα), arranged as inverted repeats and typically separ-
mediator of neuroprotection after stroke, in both sexes ated by 3 nucleotides (20). The ligand-dependent conform-
(46, 47). ational change also primes the receptor for the recruitment
The effects of GCs, including the natural human GC of coregulators and chromatin-modifying complexes, 2
cortisol, are mediated by intracellular GR, which is ex- major components of NR signaling (27, 62, 63). Indeed,
pressed throughout the body, but with a substantial hetero- ligand binding ultimately turns the receptor into a po-
geneity in GC sensitivity and biological responses across tent transcriptional regulator, which then assembles huge
tissues. Once released into the bloodstream, GCs affect all multiprotein complexes, also called coregulator complexes,
of the major body systems, including cardiovascular, mus- to activate or repress the expression of target genes (Fig.
culoskeletal, immune, nervous, and reproductive systems 2). Alternatively, ligand-stimulated SRs can regulate gene
(48). GCs exert anti-inflammatory and immunosuppres- expression by an indirect binding on chromatin, whereby
sive effects, and have thus been widely used in the clinic they associate with other DNA-bound TFs, such as AP-1 or
for treating autoimmune diseases, inflammatory diseases, SP-1 (64–66) (Fig. 2).
and hematological cancers (49). Moreover, their ability to In addition to these agonist-induced transcriptional ef-
trigger apoptosis of immature B and T lymphocytes (50), fects, most SRs are able to act on chromatin in a ligand-
has been exploited by clinicians to treat cancers, including independent fashion. This “unliganded form” (ie, receptor
leukemia and lymphoma. without ligand), first demonstrated for the nonsteroid thy-
The most remarkable role of AR is the maintenance of roid hormone receptors and later for PR, GR, and ERα,
male reproductive tissues and spermatogenesis, as demon- maintains gene silencing prior to hormonal activation
strated by the complete ablation of the male reproductive (67–69) (Fig. 2). In spite of solid evidence demonstrating
tract (seminal vesicles, vas deferens, epididymis, and pros- that ligand-independent SRs resort to a passive repression,
tate; small inguinal testes with arrested spermatogenesis) either by competition for binding sites with coactivators
in male AR-knock out (KO) mice (51). Not surprisingly, or through the formation of inactive heterodimers with
androgen-associated AR is strongly involved in several liganded homodimers (70, 71), recent reports suggest that
aspects of PC, the second most common solid cancer in inhibition of gene transcription is brought about by a
men (52). Beyond the reproductive activity, AR signaling more active mechanism. Advances in high-resolution and
controls several homeostatic processes, such as bone comparative techniques, such as cistrome analysis, clearly

Figure 2. Steroid receptor signaling pathways. The steroid hormone enters into the cell by passive diffusion through the plasma membrane and
binds with high affinity to its specific receptor. (1) Classical steroid hormone nuclear signaling. The ligand–receptor complex undergoes conform-
ational changes triggering its dissociation from the chaperone heat-shock protein (HSP), receptor dimerization, and its translocation into the nuclear
compartment. Inside the nucleus, the ligand-associated SR binds to specific DNA sequences that serve as enhancer or silencer elements, recruits
coregulators and enzyme-modifying chromatin complexes to locally perturb the chromatin organization and regulate assembly or disassembly of
an active transcription complex. SR-dependent multiprotein complexes target selective hormone-response elements (HREs) on target promoters,
or indirectly interact with chromatin through transcription factors (TFs) on their response elements (REs). This could affect the level of growth
factor receptors (GFRs), calcium signaling actors, or cellular proliferation effectors, among many other cellular pathways regulated by SR target
genes. (2) Nongenomic signaling. The steroid ligand binds to SRs located at the plasma membrane or in the cytoplasm and triggers rapid post-
translational modifications, often dependent on activating kinase cascades (MAPK, PI3K, Src), that in turn result in the transcriptional activation of
the receptor. Conversely, SR genomic effects can regulate rapid nongenomic events, highlighting a potent crosstalk dependent on ligand-bound
SRs. (3) Nonclassical steroid hormone nuclear signaling. Apart from the binding of the specific steroid hormone, SRs can also be indirectly activated
by growth factors, leading the recruitment and the activity of cytoplasmic phosphorylation cascades, the same involved in the classical signaling,
namely MAPK and PI3K/Akt kinases. (4) Unliganded-receptor nuclear signaling. More recent data revealed that unliganded forms of SRs play critical
roles on chromatin and deeply take part in gene repression of a subset of target genes after recruitment of corepressors (CoR).
showed that unliganded receptors can bind their own tar- (73, 74). Nevertheless, it is not surprising that SRs can exert
gets, resulting in a proper and optimal expression of sin- strong and decisive effects independently of ligands, as they
gular genetic programs (growth, apoptosis, development, evolved from an ancestral SR unable to bind ligands (75).
etc.) (72). They act as active repressors and target the basal In addition to these nuclear effects, SRs exhibit
transcription machinery. How these unliganded forms are nongenomic regulatory properties (76) (Fig. 2). Generally,
addressed to the nucleus rather than to the membrane/ too rapid to be dependent on gene transcription and pro-
cytosol is still unknown, but some mechanistic studies have tein synthesis, and insensitive to mRNA or protein syn-
revealed that the presence of specific marks on chromatin thesis inhibitors, they involve SRs at the cell surface and/
(ie, histone methylation marks) most of the time associ- or in the cytoplasm. In human mammary cancer cell
ated with collaborating epigenetic silencers definitely could lines for instance, the addition of hormones (ie, estradiol
play a role in the targeting and the binding of native recep- and progesterone) leads to the formation of cytoplasmic
tors to chromatin (72). Interaction with the ligand reverses multiprotein complexes in which SRs coexist with pro-
the epigenetic landscape, consistent with the hypothesis tein kinases and adaptor proteins. The steroid ligand,
that liganded SRs undergo a conformational change that as well as some decisive growth factors acting through
enables the loading of coactivators needed for chromatin their receptors, like epidermal growth factor (77), acti-
remodeling and gene activation. Further investigations are vate a membrane-associated receptor and downstream-
required to fully understand this regulation, certainly de- associated pathways, such as the tyrosine kinase/p21ras/
pendent on kinase-inducing regulatory phosphorylation mitogen-activated protein kinase, Src, and PI3K/Akt
and growth factor signaling, similarly to ligand-induced SRs (phosphoinositide 3-kinase/protein kinase B) (78–80).
Importantly, the final and overall biological effects of the physiological responses that are dependent on a given TF
hormone in the target tissue correspond to the conver- (89). Since the first SR (ie, GR) was described, over 300
gence of both cytoplasmic and nuclear effects. Indeed, coregulators have been discovered (93). Different combin-
cytoplasmic kinase-dependent phosphorylation of SRs ations of these coregulators are required for hormonal regu-
converts them into a transcriptionally active form, sug- lation of different target genes of the same SR in a given cell
gesting a convergence between classic genomic and rapid, type, enabling the independent regulation of different subsets
nongenomic signaling pathways (81) (Fig. 2). While many of the SR target genes to control different physiological re-
studies have revealed a strong impact of the nongenomic sponse pathways (89).
effects on SR nuclear signaling, recent data illustrated
how, inversely, hormone-mediated gene activation could
affect nongenomic responses. The regulation of calcium Protein Methyltransferases/Demethylases
homeostasis, playing a crucial role in tumorigenic pro- Protein methylation is a common covalent PTM that

gression, is a highly relevant illustration. SRs broadly consists in the addition of methyl groups from a donor,
regulate the transcription of many genes encoding com- S-adenoslyl-L-methionine (AdoMet, or SAM) to specific
ponents of the calcium pathway (calcium channels, cal- substrates (94). These substrates are preferentially methy-
cium receptors, etc.), which once translated into proteins lated on either a lysine residue, and thus are modified by
and activated, propagate nongenomic signals into the cell, enzymes, KMTs, or an arginine residue, by PRMTs.
contributing to cancer progression and survival (82, 83). This PTM is a complex phenomenon, as lysine res-
idues can be mono-, di-, or tri-methylated and arginine
residues can be mono- or dimethylated, symmetrically
Mechanisms of Regulation or asymmetrically (95) (Fig. 3). The addition of methyl
Many different signals can influence the adaptive responses groups on both residues hardly affects the positive charge
mediated by SRs in cells, and, among them, PTMs trigger of the amino acids but deeply influences the bulkiness and
subtle but potent adjustments in SR signaling. Indeed, hydrophobicity of the targeted protein. As such, protein
PTMs introduce structural constraints into functional do- methylation mostly functions as a potent signal for the
mains that, in turn, alter their properties. recruitment of effector partners, called readers (because
For a long time, phosphorylation was the main modifica- they distinguish the modified from the unmodified form of
tion identified to regulate SR functions. More recently, several the target protein and translate that into a function) (96).
other PTMs, such as acetylation, methylation, palmitoylation, As for other PTMs, methylation is a reversible process,
ubiquitylation, and SUMOylation, that mostly target alkaline involving enzymes able to remove methyl marks on lysines
residues (ie, lysine and arginine) have been described (84, 85). (lysine demethylases or KDMs) or on arginines (arginine
They are involved in every step of the receptor signaling path- demethylases) (97, 98). Both protein methyltransferases
ways and contribute to the overall process of transcription. and protein demethylases are strong actors in transcrip-
For instance, receptor stability, hormone binding and sensi- tion, as they influence the histone methylation landscape
tivity, subcellular localization, and interactions with partners and consequently the opening or compaction of chromatin
or DNA are affected by PTMs (86). Most of the time, these (99). However, growing evidence highlights that methyla-
modifications are both dynamic and reversible, dependent tion of nonhistone proteins plays important roles in cells
on enzymes that deposit the marks and enzymes that remove and sharply affects crucial homeostatic cellular processes.
them. This tightly controlled dynamic is crucial for an optimal Inevitably, dysregulation of these processes can lead to di-
regulation of SR activity. Concerning transcriptional regula- verse pathologies, including cancer.
tion, receptor-modifying enzymes, like methyltransferases
or deacetylases, act as influential SR coregulators, allowing
a tight regulation of gene expression by directly modifying KMTs, or Lysine (K) Methyltransferases
receptors (87). Moreover, their interactions with SRs can be The first KMT was uncovered on a bacterial flagellar protein
direct or indirect, being recruited to enhancers or promoters in 1959 (100). Numerous other enzymes were discovered
by upstream coregulators (88, 89). They also remodel chro- since then, making this family of proteins 1 of the largest
matin structure by modifying histones, and they promote class of epigenetic enzymes (95). KMTs catalyze the transfer
(coactivator) or inhibit (corepressor) the recruitment and acti- of 1 or more methyl group(s) from the AdoMet donor to
vation of RNA polymerase II, depending on the specific genes the ε-nitrogen of a lysine residue, generating mono-, di-, or
and cellular environment (89-92). Depending on which genes trimethyllysine (Kme1, Kme2, and Kme3, respectively) (Fig.
need to be activated (or repressed), specific coregulators are 3A) (101). KMTs methylate a wide variety of substrates:
preferentially required for hormonal regulation of selected it has been predicted that human cells contain more than
(a)
( )
(b)

Figure 3. Process of protein methylation. Lysine residues are methylated by lysine methyltransferases (KMTs, green arrow) to generate, mono-
(Kme1), di- (Kme2), or tri-methyllysines (Kme3). (A) KMTs use the methyl donor S-Adenosylmethionine (AdoMet) to add methyl (-CH3) groups
on targets and produce S-adenosylhomocysteine (AdoHcy) in addition to methyllysines. This process is highly dynamic and can be reversed by
lysine demethylases (KDMs, red arrow). (B) Arginine methylation is catalyzed by a family of 9 PRMTs, divided into 3 subgroups (type I, II, or III,
green arrows). All use the methyl donor AdoMet to add methyl (-CH3) groups on targets and produce AdoHcy in addition to methylarginines.
PRMTs that promote monomethylation (MMA), symmetric dimethylation (sDMA), or asymmetric dimethylation (aDMA) lead to the production of
monomethylarginine, asymmetric dimethylarginine, or symmetric dimethylarginine respectively. JMJD6 is currently the only enzyme identified with
an arginine demethylase activity (red arrow). PRMTs on which we focus in this article are highlighted in bold.
1000 proteins with 1 form of methyllysine (102). To date, specificity (105). In their structure, SET KMTs also ex-
2 KMT families have been described: the SET KMT family, press the peptide binding and the AdoMet binding
containing the majority of KMTs (103), and the seven pockets (95). The SET KMT family is classified into sub-
β-strand methyltransferases (7βS) or class I family (104). families, based on the sequence of the pre- and post-SET
The SET KMT family is characterized by the presence domains.
of the evolutionarily conserved Su(var)3-9n Enhancer-of- The 7βS family differs from the SET KMTs family by the
zeste and trithorax (SET) domain. This is the catalytic catalytic domain. It is composed of 4 motifs, designated I,
core of the enzymes, flanked by the pre- and post-SET post-I, -II, and -III. Motifs I and post-I play an important
domains, each displaying a defined substrate and product role in the interaction with the methyl donor AdoMet.
However, many KMTs in this family do not possess all of its substrates, as only the LF-K motif has been identified
the motifs but may harbor different ones. Although this (178).
family of KMTs is smaller than the SET family, a proteomic SMYD2-deficient zebrafish show malformation of both
study predicted that the human genome encodes more than the atrium and ventricle, and a reduced heart rate and
100 7βS KMTs (106). cardiac function (179). Mechanistically, in the cytoplasm
KMT substrates are involved in various cellular path- of cardiomyocytes, SMYD2 monomethylates the heat
ways including transcription, cell proliferation, DNA shock protein 90 (HSP90) at K616 and interacts with the
damage repair, inflammation and immune response (Table sarcomeric I-band region at the titin N2A domain via its
1) (107). They were first described as histone KMTs, be- N-terminal and extreme C-terminal regions to influence
cause numerous KMTs methylate lysine residues in histone cardiac contraction (180). Since aberrant SMYD2 expres-
N-terminal tails (169). However, recent technical advances sion and its dysfunction are often closely related to car-
in mass spectrometry (MS)–based proteomics have high- diovascular diseases and cancer, SMYD2 is a promising

lighted that numerous nonhistone proteins are modified by candidate for the treatment of these pathologies.
lysine methylation, revealing that KMT substrates extend
far beyond histones (170). SET7/9
In this review, we will focus on the enzymes involved SET7/9 is a SET KMT that performs mono- and
in SR signaling, namely G9a, GLP (G9a-like protein), dimethylation (127). It was first identified to
SMYD2, SET7/9 (SETD7), and DOT1L (DOT1 like monomethylate H3K4, leading to the recruitment of
histone KMT). RNA polymerase II and thus maintains the transcription
of target genes and the structure of active or potentially
G9a/GLP active euchromatin (181). As other KMTs, SET7/9 also
G9a and GLP are SET KMTs belonging to the Suv39h has nonhistone substrates involved in DNA damage re-
family (171). They cooperatively play a predominant role sponse, cell cycle regulation, cell proliferation, chromatin
during early embryonic development of mice, as G9a or modulation and cell differentiation (Table 1). SET7/9
GLP knockout induced growth retardation and early le- preferentially methylates proteins containing the K/R-S/
thality (171, 172). G9a and GLP were first described as T/A sequence (105). Interestingly, SET7/9 knockout mice
histone methyltransferases, performing Kme1 and Kme2 are viable and develop normally (182). SET7/9 seems to
at lysine 9 of histone H3 (H3K9) (171). Once methy- have different effects on carcinogenesis depending on the
lated, H3K9me1/2 becomes a docking site for effectors, cancer type (183). SET7/9 also appears to play a role in
especially the heterochromatin proteins HP1α, HP1β, and diabetes and atherosclerosis by activating inflammatory
HP1γ that strongly influence gene silencing (173). Since genes (184).
then, nonhistone substrates have been identified including
trimethylation of substrates such as G9a itself (124) or DOT1L
ATF7IP (110). Most of their substrates are involved in DOT1L is a 7βS KMT that performs mono-, di-, and
DNA damage response, cell cycle regulation, cell prolif- trimethylation. For a long time, its unique substrate was
eration, and chromatin modulation, but also in skeletal H3K79, the methylation of which contributes to activating
muscle differentiation (Table 1). Although the role of G9a the transcription of genes involved in DNA damage re-
in the initiation and progression of cancer is well known sponse and cell cycle regulation (185), but also in immune
(174), it also appears to be implicated in other diseases, response (186, 187). In addition, DOT1L is involved in
such as Alzheimer’s disease (175). These enzymes prefer- neointimal hyperplasia development, as it is upregulated in
entially methylate a nonexclusive motif such as ARKS/T the rat injured artery wall (188). Contrary to the other KMTs
(124). presented before, DOT1L has no identified nonhistone sub-
strate, aside from AR which we will described later in this
SMYD2 review (189). Interestingly, the loss of DOT1L in mice in-
SMYD2 is a protein methyltransferase that is capable duces developmental problems (ie, growth retardation,
of performing Kme1 and Kme2 (Table 1) (125). Initially cardiac dilatation) and death in utero (190). Furthermore,
described to methylate histone H3 at lysine 36 (H3K36) DOT1L seems to be implicated in diseases such as obesity
(176), it seems that this KMT can also methylate diverse and cancer. Indeed, inhibition of DOT1L increases adi-
nonhistone proteins (Table 1) (177). For instance, SMYD2 posity, making it a potential therapeutic target for obesity
was reported to monomethylate p53 at K370 and pRb at (191), and DOT1L mislocalization in leukemia promotes
K860 (127, 128), to inhibit their activities. Large-scale ana- H3K79 methylation and activation of the leukemic tran-
lysis revealed a low level of specificity for SMYD2 towards scriptional program (185).
Table 1. Lysine methylated substrates
KMT Substrate Site Effect of lysine methylation References
GLP HIF-1α K674me1/2 Represses HIF-1α transcriptional activity (108)

p53 K373me2 Negatively regulates p53 activity (109)
ATF7IP K16me3 Stimulates formation of ATF7IP / MPP8 complex (110)
DNMT3A K47me2 Induces the formation of Dnmt3a–MPP8–GLP/G9a inactive complex (111)
LIG1 K126me2/3 Induces LIG1-mediated recruitment of UHRF1 to replication foci (112)
G9a CDYL1 K135 Negatively regulates its binding to chromatin (113)
C/EBPβ K39 Represses C/EBPβ transactivation (114)
HIF-1α K674me1/2 Represses HIF-1α transcriptional activity (108)
MEF2D K267 Inhibits its chromatin recruitment and transcriptional activity (115)
MTA1 K532 Positively regulates its corepressor activity in NuRD complex (116)
MyoD K104 Inhibits MyoD transcriptional activity (117)

Pontin K265, K267, K268, Enhances p300 recruitment and increases HIF1 transcriptional activity (118)
K274, K281, K285
Reptin K67me1 Negatively regulates transcription of hypoxia genes (119)
RUNX3 K129me1/2, Suppresses its transcriptional activity (120)
K171me1/2
PLK1 K209me1 Supports DNA damage repair (121)
ATF7IP K16me3 Stimulates formation of ATF7IP/MPP8 complex (110)
DNMT3A K47me2 Induces the formation of Dnmt3a–MPP8–GLP/G9a inactive complex (111)
FOXO1 K273me1/2 Decreases FOXO1 stability (122)
G9a K165me2/3 Induces G9a interaction with HP1γ (123)
K239me3 (124)
LIG1 K126me2/3 Induces recruitment of UHRF1 to replication foci (112)
SMYD2 EZH2 K307me1/2 Represses transcription (125)
GFI1 K8 Promotes GFI1-mediated transcriptional repression though LSD1 recruitment (126)
pRb K860me1 Regulates RB Binding to the Transcriptional Repressor L3MBTL1 (128)
K810me1 Promotes E2F transcriptional activity (129)
PARP1 K528me1 Enhances PARP1 activity in response to DNA damage (130)
β-catenin K133me1 Activates Wnt signaling (131)
MAPKAPK3 K355me1 Activates MAPKAPK3 (132)
PTEN K313me2 Activation of the phosphatidylinositol 3-kinase-AKT pathway (133)
HSP90AB1 K531, K574me1 Enhances its polymerization and the chaperone complex formation (134)
SET7/9 FoxO3 K271me1 Decreases FoxO3 protein stability and increasing transcriptional activity (135)
FXR K206 Supports the transactivation of FXR target genes (136)
HIV Tat K51me1 Activates HIV transcription (137)
K71me1 (138)
LIN28A K135me1 Modifies transcription of c-myc target genes (139)
PGC-1α K779 Allows transcription of PGC-1α target genes (140)
pRb K873 Supports Rb-dependent transcriptional repression (141)
RelA K314me1, K315me1 Negatively regulates NF-κB transcriptional activation (142)
K37me1 Stabilizes the DNA-RelA complexes and induces the transcription of a subset of (143)
NF-κB-regulated genes
RORα2 K87 Enhances its target gene transcription (144)
YY1 K173me1, K411me1 Positively regulates YY1 DNA-binding activity (145)
YY2 K247me1 Positively regulates YY2 DNA-binding activity (146)
p53 K372me1 Stabilizes p53 chromatin-bound fraction (147)
PARP1 K508 Stimulates ARTD1 mediated ADP-ribosylation (148)
SIRT1 K333, K235, K236, Enhances p53 acetylation in response to DNA damage (149)
K238
SUV39H1 K105me1, K123me1 Negatively regulates it activity in response to DNA damage (150)
UHRF1 K385me1 Enhances the formation of UHRF1–PARP1 complex at DNA damage site (151)
ATG16L1 K151me1 Inhibits autophagy by impairing the formation of the ATG12–ATG5- (152)
ATG16L1 complex
Table 1. Continued
KMT Substrate Site Effect of lysine methylation References
β-catenin K180me1 Decreases β-catenin stability (153)

DNMT1 K142me1 Facilitates DNMT1 ubiquitin-dependent degradation (154)
E2F1 K185me1 Promotes E2F1 ubiquitin-dependent degradation (155)
eL42 K53me1, K80me1, Enhances translation (156)
K100me1
HIF-1α K32 Enhances HIF-1α stability (157)
IFITM3 K88me1 Reduces IFITM3 antiviral activity (158)
MYPT1 K442me1 Increases MYPT1 stability (159)
PLK1 K191me2 Promotes dynamic kinetochore-microtubule attachments (160)
RIOK1 K411me1 Promotes ubiquitin-dependent degradation of RIOK1 (161)
Rpl29 K5me2 Facilitates Rpl29 nuclear localization (162)

Sam68 K208 Positively regulates Sam68 protein stability (163)
Smad7 K70me1 Induces Smad7 ubiquitination and proteasomal degradation (164)
Sox2 K119me1 Induces Sox2 ubiquitination and proteasomal degradation (165)
STAT3 K140me2 Promotes STAT3 binding to SOCS3 promoter (166)
TAF10 K189me1 Increases TAF10 interaction with RNA polymerase II (167)
Yap K494me1 Promotes Yap cytoplasmic sequestration by the Hippo pathway (168)
Abbreviations: K, lysine; Kme1, monomethyllysine; Kme2, dimethyllysine; Kme3, trimethyllysine.
KMTs targeting KDMs, or Lysine (K) DeMethylases

In view of these data, it has become evident that members The discovery of KMTs quickly raised the question of the
of the KMT family constitute attractive new therapeutic existence of lysine demethylating proteins. The first KDM
targets. BIX-01294, the first G9a inhibitor, was identi- was identified in the early 2000s, and numerous other
fied in 2009, and numerous inhibitors targeting G9a have KDMs have since been discovered (201). They are classi-
since been produced (192). Targeting this enzyme gave rise fied into 2 groups based on their structure and the type of
to promising results in bladder cancer, especially with the lysine demethylation they perform (Fig. 3A).
selective inhibitor CM-272 that leads to cancer regression The first group, called KDM1, includes KDM1A
in vivo (193). Similar results were obtained in non–small (LSD1) and KDM1B (LSD2). LSD1 contains a flavin ad-
cell lung cancer, where the selective inhibitor UNC0638 enine dinucleotide–dependent amine oxidase domain and
prevents tumor growth in vitro and in vivo (194). performs demethylation of Kme1 and Kme2 only (202,
The first SMYD2 inhibitor was identified in 2011, and 203) (Fig. 3A). The second group is larger and includes
the same year the SMYD2 crystal structure was reported JmjC domain-containing histone demethylases (JHDMs).
(195). Since then, increasingly potent and selective SMYD2 The demethylase activity of the JmjC domains requires
inhibitors have been produced (196). Inhibition of SMYD2 Fe2+, 2-oxoglutarate and oxygen. JHDMs are capable of
was shown to increase the sensitivity of ovarian cancer demethylating Kme1, Kme2, and Kme3 through hydrox-
cells to the PARP inhibitor olaparib (197) and to sensitize ylation (204) (Fig. 3A). JHDMs have been classified into
non–small cell lung cancer cells to the anticancerous agent subgroups (KDM2-7), according to their JmjC domain se-
cisplatin (198). quence and domain architecture. Indeed, KDMs contain
For SET7/9 inhibitors, cyproheptadine was demonstrated DNA and histone binding domains, such as zinc fingers,
to selectively inhibit SET7/9 activity (199), and many deriva- Tudor domains, and PH domains. For example, KDM4A,
tives of this inhibitor have since been studied (200). KDM4B, and KDM4C from the KDM4 subgroup have 2
Of the KMTs presented in this review, only the DOT1L PH domains and 2 Tudor domains in addition to their JmjC
inhibitor pinometostat is currently undergoing clinical trial and JmjN domains, but no zinc fingers; and KDM4D only
according to the clinical trials website (www.clinicltrials. has JmjC and JmjN domains (205).
gov). To date, pinometostat is registered in 2 phase Ib/II In this section, we will focus on LSD1 and KDM4 family
clinical trials for different hematological malignancies members, which are currently the main KDMs involved in
(NCT03724084 and NCT03701295). SR regulation.
LSD1 KDMs from the KDM4 subgroup are dysregulated in

Similarly to KMTs, the first KDM substrates described several diseases such as cancers, cardiovascular diseases,
were initially histones, and nonhistone substrates and mental retardation (217). Despite the therapeutic po-
were later identified. The first identified substrate for tential of the KDM4 subgroup, there are few potent and
LSD1 was H3K4. LSD1-mediated demethylation of selective inhibitors to date (218).
H3K4me1/2 induces a repression of target gene tran-
scription by interacting with TFs harboring a SNAG do-
main, a N-terminal highly conserved repressive domain PRMTs, or Protein Arginine (R)
(eg, SNAIL1/2 and GFI1/B) (206). In addition, LSD1 Methyltransferases
demethylates nonhistone proteins, among which p53, Arginine methylation was first described in the 1970s
DNMT1, and HSP90, as well as ERα, which we will (219–222), though the first PRMT was only identified
further explore later in this review (207). Nevertheless, in 1996 (223). Similarly to KMTs, PRMTs are struc-

LSD1 is also a coactivator of several SRs. The relief of turally defined as S-adenosylmethionine (AdoMet)-
repressive histone marks, such as the demethylation of dependent methyltransferases. PRMT enzymes belong
H3K9me1/2, triggers chromatin and DNA conform- to the class I methyltransferases, which is characterized
ational changes that are essential for promoting AR and by a 7-stranded β-sheet structure. They also harbor add-
ERα-dependent transcription (208, 209). itional conserved sequences, including the motifs I, post-I,
and the Thr-His-Trp (THW) loop that forms the AdoMet
KDM4 binding pocket (224). PRMTs transfer 1 or 2 methyl
Enzymes from the KDM4 subgroup can activate or re- group(s) from the AdoMet methyl donor to the guan-
press transcription depending on the targeted lysine idine nitrogen atoms of the targeted arginine, resulting in
residue. KDM4A was first identified as a H3K9me2/3 S-adenosylhomocysteine (AdoHcy) and methylarginine
and H3K36me2/3 demethylase (210). By demethylating production (Fig. 3B). Three main forms of methylated
H3K36me3, KDM4A antagonizes HP1γ and allows cell arginines exist in eukaryotes: monomethylarginine (MMA),
cycle progression (211). Like LSD1, nonhistone substrates ω-NG,NG-asymmetric dimethylarginine (aDMA) in which
of KDM4A have been identified. Indeed, KDM4A was re- 2 methyl groups are added to the same guanidine nitrogen,
cently identified in a complex with SCFFbxo22 and methy- and ω-NG,N’ G-symmetric dimethylarginine (sDMA) where
lated p53. Indeed, it was reported that KDM4A-mediated 1 methyl group is attached to each guanidine nitrogen (225)
p53 demethylation is necessary for the destabilization of (Fig. 3B). So far, 9 PRMTs have been characterized as active
methylated p53 induced by SCFFbxo22 (212). and structurally conventional arginine methyltransferases
in human cells (226). They are classified into 3 fundamental
KDMs targeting subgroups, based on the type of methylation they cata-
Some KDMs are very attractive therapeutic targets. For lyze. Type I PRMTs (PRMT1, PRMT2, PRMT3, CARM1
instance, LSD1 is dysregulated in many cancer types for Coactivator Associated Arginine Methyltransferase
including small cell lung cancer and acute myeloid leu- 1 [PRMT4], PRMT6, and PRMT8) produce MMA and
kemia (213). Since the discovery of LSD1, many potent aDMA, while type II enzymes (PRMT5 and PRMT9) de-
and selective inhibitors have been identified, including posit MMA and sDMA marks. Type III contains only
GSK2879552, which displays an antitumor activity in PRMT7 and is responsible for MMA only (227) (Fig. 3B).
small cell lung cancer xenograft mouse models (214). Here, we will focus on PRMT1, CARM1, PRMT5 and
Currently, the clinical trials website (www.clinicltrials. PRMT6 which are currently the 4 main PRMTs involved
gov), references 5 LSD1 inhibitors undergoing clinical in SR regulation.
trials mostly in cancer patients (215). Among them, the PRMTs are involved in many essential cellular pro-
GSK2879552 clinical trial in myelodysplastic syndrome cesses and KO of most of them causes embryonic lethality.
patients completed phase II in 2019, but the outcome does PRMT1 and PRMT5 KO are lethal (ie, induce embryonic
not advocate for completing such trials (NCT02929498). or post-natal death) (228). Moreover, CARM1-KO mice
In addition, the dual LSD1/MAO-B inhibitor ORY-2001 die at birth and display a reduced size (229). In contrast,
efficiently rescues memory and behavioral alterations in PRMT6-KO mice are viable (230). Tissue ablation of
mouse models of Alzheimer’s disease. Of note, the ob- PRMTs has contributed to determining their involvement
served effects were essentially attributed to the inhibition in metabolic, immune, muscular, and neurodegenerative
of LSD1 (216). Interestingly, ORY-2001 is currently under- disorders and cancers (228, 231). The different PRMTs
going a phase II clinical trial in patients with Alzheimer’s regulate a wide variety of important cellular processes (eg,
disease (NCT03867253). DNA repair, transcriptional regulation, immune system
response, RNA processing and signal transduction) (232) PRMT6

by methylating a growing number of substrates (Table Histone H3R2 was thought to be the major histone target
2). It is well known that proteins harboring glycine and site of PRMT6 in cells, and PRMT6 was widely considered
arginine-rich motifs (GARs) are often targets for PRMTs to be a transcriptional repressor (349, 350). However, in
(346). However, even if it is the case for PRMT1, 5, and 6, several cases, PRMT6 was reported to act as a transcrip-
CARM1 prefers to methylate its substrates within a PGM tional coactivator, by depositing the H3R17me2a mark,
(proline, glycine, methionine) motif (347). similarly to CARM1 (351). PRMT6 also methylates
nonhistone proteins regulating transcription, DNA repair
PRMT1 and cell signaling (Table 2).
PRMT1 was initially shown to catalyze the methylation
of H4R3, an epigenetic active mark (348). To date, many PRMTs targeting
nonhistones substrates have also been identified (Table 2). For The majority of PRMTs and their variants have been

instance, BReast CAncer 1 (BRCA1) methylation by PRMT1 shown to be overexpressed in cancer compared with
affects its tumor suppressive capacity in BC cells and samples normal tissues (352). PRMT1 overexpression has been
(233). More recently, PRMT1 was shown to dimethylate the reported in breast, prostate, lung, colon, and bladder
KMT EZH2 (236). This event inhibited its ubiquitylation and cancer, as well as in leukemia. Similarly, overexpression
consequently increased the stability of the protein, which fur- of CARM1 and PRMT5 has been observed in PC, colon
ther impaired expression of EZH2 target genes, contributing cancer, and BC, whereas PRMT6 has been shown to be
to a sustained and aggressive phenotype of BC cells (epithelial overexpressed in bladder, lung, and BC (15). Moreover,
mesenchymal transition, invasion, and metastasis). their expression is often associated with poor prognosis
(15). In view of these findings, it has become increasingly
CARM1 evident that members of the PRMT family constitute new
CARM1-dependent methylation of various substrates targets for treating pathologies, including cancer (353-
notably contributes to tumorigenesis (347). For instance, 355). The first PRMT inhibitor, AMI-1, was discovered in
methylation of the core subunit BAF155 of the chromatin 2004 by Bedford et al. (356). Many potent and selective
complex SWI/SNF promotes proliferation, migration and inhibitors have since been produced (357). Although
metastasis of BC cells in vivo. Moreover, BAF155 methy- none of the inhibitors selectively inhibit PRMT1, several
lation was associated with poor survival of BC patients inhibitors specific for other Type I PRMTs are available.
(282). CARM1 methylation of the lysine demethylase TP-064 (358) and GSK3359088 (359) targeting CARM1
LSD1 stabilizes the protein, activating vimentin transcrip- are effective in inhibiting tumor growth for multiple mye-
tion through histone demethylation, which triggers inva- loma. EPZ2020411 selectively inhibits PRMT6, but this
sion and metastasis of BC (286). inhibitor is under preclinical development (360). Some
inhibitors are currently undergoing clinical trials in pa-
PRMT5 tients with different types of cancers. On the clinical trials
PRMT5 was shown to methylate, among other sub- website (www.clinicltrials.gov), 6 clinical trials for agents
strates, the HOXA9 protein, a TF that plays a crucial role that target PRMTs are referenced (1 for type I enzymes,
in hematopoietic stem cell expansion and is commonly and 5 for PRMT5). Most of the inhibitors in clinical trials
dysregulated in acute leukemia (304). This modification is are in phase I, assessing their safety, pharmacokinetics,
involved in epithelial mesenchymal transition activation, pharmacodynamics, and clinical activity. GSK3368715 is
due to induced expression of proinflammatory endothelial– the only type I PRMT inhibitor in clinical trials (361).
leukocyte adhesion molecules, such as E-selectin. As such, It was shown to inhibit in vitro tumor cell growth in
PRMT5 seems to be a critical actor in the induction of the lymphoma, acute myeloid leukemia, and numerous solid
proinflammatory function of HOXA9, which is important tumor cell lines.
in the pathobiology of inflammation and cardiovascular JNJ64619178 is a PRMT5 inhibitor that provokes
inflammatory diseases. PRMT5 also controls carcinogen- tumor growth inhibition and regression in patient-derived
esis by methylating the TF E2F1. This PTM increases the xenografts. In addition, PF-06939999, PRT811, and
stability of the target, promoting cell cycle progression PRT543 are PRMT5 inhibitors with antiproliferative and
and cell growth (302). Despite its oncogenic role, new re- antineoplastic activities in cancer cell lines. GSK3326595
ports highlighted that PRMT5 is also involved outside of is the only inhibitor currently in a phase II clinical
the cancer field. For instance, PRMT5-induced methylation trial. Recently, this inhibitor was shown to circumvent
of SREBP1 (311) and SPT5 (310) participate in regulating palbociclib (CDK4/CDK6 inhibitor) resistance in mel-
lipid metabolism. anoma (362).
Table 2. Arginine methylated substrates
PRMT Substrate Site Effect of arginine methylation Reference
PRMT1 BRCA1 504–802 Facilitates its binding to promoters (233)

C/EBPα R35, R156, R165 Dissociates from SWI/SNF Mediator complex (234)
c-Myc R299, R346 Activates its transcriptional activity by promoting its binding to (235)
p300
EZH2 R342 Suppresses EZH2 target transcription (236)
FOXO1 R248, R250 Blocks FOXO1 phosphorylation by Akt (237)
FOXP3 R48, R51 Enhances its transcriptional activity (238)
GLI1 R597 Enhances its transcriptional activity (239)
MyoD R121 Activates its transcriptional activity by promoting its DNA-binding (240)
Nrf2 R437 Enhances its transcriptional activity (241)
RACO-1 R98, R109 Enhances its binding to c-jun, activates AP1 transcription (242)

RelA R30 Inhibits its binding to DNA (243)
RIP40 R240, R650, R948 Decreases its corepressor function (244)
RunX1 R206, R210 Abrogates Sin3a binding, promoting its transcriptional activity (245)
STAT1 R31 Dissociates from PIAS1 and enhances IFNα/β induced (246)
transcription
TAF15 R203 Enhances TAF15-depend transcription (247)
TLS R216, R218, R242, R394 Enhances transcription of surviving (248)
TOP3B R833, R835 Involved in interaction with TDRD3, promoting its topoisomerase (249)
activity
Twist 1 R34 Represses import into nucleus and E-cadherin expression (250)
53BP1 1319–1480 Localizes to DNA breaks (251)
APE1 R301 Protects mitochondrial DNA from oxidative damage (252)
DNA polβ R137me1 Inhibits its interaction with PCNA, enhances base excision repair (253)
E2F1 R109 Induces PARP cleavage in response to DNA damage (254)
hnRNPK R296, R299 Inactivates caspase 3 after DNA damage (255)
hnRNPUL1 R584, R618, R620, R645, R656 Stimulates its recruitment to DNA damage (256)
MRE11 GAR domain Enhances its exonuclease activity (251)
ASK1 R78, R80 Negatively regulates ASK1 signaling (257)
Axin R378 Increases Axin stability and inhibits Wnt signaling (258)
CaMKII R9, R275 Suppresses cardiac CaMKII hyperactivation (259)
EGFR R198, R200 Promotes EGFR activation (260)
p38 MAPK R49, R149 Enhances p38α activation (261)
Smad4 R272 Activates wnt signaling (262)
Smad6 R74, R81 Activates BMP signaling (263)
R74, R81 Inhibits NFkB signaling (264)
Smad7 R57, R67 Enhances TGF-β signaling (265)
TSC2 R1457, R1459 Regulates mTORC1 activity (266)
BAD R94, R96 Inhibits its association with 14-3-3 (267)
CDK4 R55, R73, R82, R163 Destabilizes CDK4-Cyclin-D3 complex and inhibits cell cycle (268)
progression
CNBP R25me1/2a, R27me1/2a Decreases its RNA binding (269)
cTnI R146me1/2a, R148me1/2a Inhibits cardiomyocytes hypertrophy (270)
EIF4G1 R689me1, R698me1 Contributes to its stability and facilitates translation initiation (271)
complex assembly
EZH2 R342me2a Enhances its stability (236)
G3BP1 R435 me1/2a, R447 me1/2a Prevents stress granule formation (272)
hnRNP A1 R214, R226, R223, R240 Enhances its RNA binding (273)
HSP70 R416, R447 Protects PDAC cells from apoptosis (274)
INCENP R887 Facilitates interaction with AURKB (maintains chromosomal (275)
alignment)
KCNQ R333, R345, R353, R435 Facilitates its ion channel activity by PIP2 interaction (276)
MYCN R65 Stabilizes MYCN protein (277)
RBM15 R578 Facilitates its degradation by CNOT4 (RNA splicing) (278)
rps3 R64, R65, R67 Targets rps3 into ribosomes (translation) (279)
Table 2. Continued
RunX1 R233, R237 Resists to apoptosis under stress condition (280)

TRAF6 R88, R125 Inhibits its ubiquitin ligase activity (281)
CARM1 BAF155 R1064 Regulates transcription related to c-Myc pathway (282)
C/EBPβ R3 Dissociates from SWI/SNF mediator complex (283)
CARM1 R551 Promotes its effect on transcription and mRNA splicing (284)
HSP70 R469me1 Activate RA-induced RARβ2 transcription (285)
LSD1 R838 Stabilizes LSD1, enhancing E-cadherin and decreasing vimentin (286)
transcription
Pax7 R161 Activates Myf5 transcription via MLL1/2 complex (287)
Pontin R333, R339 Activates Foxo3-induced autophagy gene expression (288)
PRMT5 R505 Enhances its enzymatic activity, decreasing γ-globin gene (289)

transcription
RNA pol II R1810 Activates the transcription of small nuclear RNAs (290)
RUNX1 R223 Induces the repressor complex formation (291)
SOX2 R113 Enhances Sox2-mediated transactivation by self-association (292)
p300 R754 Promotes BRCA1 recruitment to p21 promoter during DNA (293)
damage
GAPDH R234 Inhibits glycolysis by repressing its activity (294)
HuD R236, R248 Decreases p21 stability (295)
HuR R217 Stabilizes mRNAs (296)
MDH1 R248me1/2a Inhibits Gln metabolism (297)
PKM2 R445, R447 Enhances its pyruvate kinase activity (298)
RPIA R42 Enhances its enzymatic activity (pentose phosphate pathway) (299)
PRMT5 Actin R256me1 Either activates or represses transcription (300)
BCL6 R305 Facilitates its transcriptional repressive activity (301)
E2F1 R111, R113 Inhibits its transcriptional activity (302)
GATA4 R317 Inhibits its transcriptional activity (303)
HOXA9 R140 Promotes transcription of E-selectin (304)
RelA R30 Enhances NFKB transcriptional activity (305)
R30me1, R35 (306)
R174 (307)
RNA pol II R1810 Controls termination of transcription (308)
SHP R57 Facilitates its transcriptional repressive activity (309)
SPT5 ND Releases SPT5 from Bscl2 promoter (lipid metabolism) (310)
SREBP1a R321 Enhances SREBP1transcriptional activity (311)
53BP1 GAR motif (both ADMA and Enhances DNA repair process (312)
SDMA)
FEN1 R192 Facilitates DNA repair by binding to PCNA (313)
p53 R333me1, R335, R337 stimulates p53-dependent G1 arrest in response to DNA damage (314)
RAD9a R172, R174, R175 Regulates cell cycle checkpoints (315)
RUVBL1 R205 Removes 53BP1 from DNA breaks then enhances HR-mediated (316)
DSB repair
TDP1 R361, R586 Stimulates TDP1/XRCC1 recruitment to DNA breaks (317)
ASK1 R89 Inhibits H 2O2-induced ASK1 activation (318)
BRAF R671 Inhibits ERK activation (EGFR signaling) (319)
CRAF R563
DUSP14 R17me1/me2s R38, R45me1 Promotes its ubiquitination, inhibiting TCR signaling (320)
EGFR R1175me1 Inhibits EGF-induced ERK pathway (271)
YBX1 R205 Activates NF-κB signaling (321)
G3BP1 R460 Prevents stress granule assembly (272)
GLI1 R990, R1018 Stabilizes GLI1 protein (322)
GM 130 R18, R23 Regulates GA ribbons, maintaining Golgi architecture (323)
hnRNP A1 R218, R225 Enhances interaction with IREs RNA to promote translation (324)
Facilitates HIV-1 IRES-mediated translation (325)
HSP90A R345, R368 Suppresses the cell apoptosis (326)
Table 2. Continued
KLF-4 R374, R376, R377 Inhibits its ubiquitination, maintaining genome stability (327)
LSH R309 Decreases stem-like properties (328)
PDCD4 R110 Inhibits its tumor suppressive activity (329)
RPS10 R158, 160 Facilitates its assembly into ribosome (330)
ZNF326 R175 Regulates alternative splicing (331)
PRMT6 FOXO3 R188, R249 Activates transcriptional activity (332)
HIV-1 Tat R52, R53 Inhibits Tat transcriptional activation (333)
HIV-1 R10, R32 Inhibits reverse transcription (334)
nucleocapsid
RFX5 R466, R468 Down-regulates transcription (335)
TOP3B R833, R835 Promotes transcription (249)

DNA pol β R83, R152 Promotes Polβ activity in DNA strand break repair (336)
CRAF R100 Diminishes MEK/ERK signaling (337)
PTEN R159 Inhibits PI3K–AKT signaling (338)
BAG5 R15, R24 Represses cell autophagy (339)
GPS2 R323 Prevents GPS2 degradation (340)
HIV-1 Rev R38 Inhibits viral RNA export to the cytoplasm (341)
p21 R156me1/me2a Enhances cytoplasmic localization of p21 (132)
p16 R22, R131, R138 Weakens p16-mediated apoptosis (342)
PRMT6 R35 Stabilizes PRMT6 protein level (343)
RCC1 R214 Induces its association with chromatin and activation of RAN (344)
SIRT7 R388me1/me2a Inhibits its deacetylase activity (mitochondria biogenesis) (345)
When the type of methylation is not specified it is Rme2a for PRMT1, CARM1, and PRMT6, and Rme2s for PRMT5.
Arginine (R) Demethylases Interestingly, JMJD6 is upregulated in a large spectrum

JMJD6 of cancers and its enzymatic activities have been asso-
With regards to arginine methylation, only 1 demethylase ciated with tumorigenic roles, making JMJD6 a prom-
has so far been identified, and this role was initially a ising novel therapeutic target (370). However, so far
matter of controversy (363). Indeed, in 2007, JMJD6 only 1 inhibitor has been developed and its effect on
(Jumonji domain-containing protein 6) was described as JMJD6 demethylase activity has not been investigated,
a JmJC-containing iron-and 2 oxoglutarate-dependent although it displayed promising antiproliferative effects
dioxygenase, able to remove dimethyl groups from H3R2 on ovarian cancer cells (371).
and H4R3 (98) (Fig. 3B). However, at that time, Webby et al Of note, the fact that JMJD6 is unable to demethylate
did not confirm these results and reported that arginine- H3R8, H3R17, H3R26, or H2A sites (98) suggests that
rich (RS) domains of U2AF65 and LUC7-like2 synthesized other arginine demethylases may play a role in the dynamic
with dimethylated arginine residues could be Jmjd6 sub- regulation of arginine methylation. Indeed, other works dem-
strates for hydroxylation (364). JMJD6 also catalyzes the onstrated that some KDMs are also able to remove methyl
hydroxylation of lysine residues of histones H2A and H2B marks from arginine residues. KDM3A, KDM4A, KDM5,
(365). and KDM6B display arginine demethylase activity in vitro
Despite this multifaceted role for JMJD6, more re- on histones and on certain nonhistone peptides (372).
cent studies confirmed that this enzyme demethylates JMJD1B (or KDM3B) was recently reported to demethylate
arginines of some nonhistone substrates. Among them, H4R3me2s and its intermediate H4R3me1, during the de-
ERα (366), RNA helicase A (367), the TF PAX3 (368), velopment of hematopoietic stem cells (373). However,
HSP70 (285), the Ras-GTPase activating SH3-domain- its very narrow specificity strongly suggests that other un-
binding-protein 1 (G3BP1) (369) and the ubiquitin ligase known enzymes may display arginine demethylase proper-
TRAF6 (281). It is now broadly accepted that JMJD6 ties. In addition to true demethylation, arginine methylation
acts as a dual demethylase and lysyl hydroxylase, able levels are further modulated by the conversion of arginine
to modify proteins on both arginine and lysine residues. into citrulline by protein arginine deiminase (374).
Methylation/Demethylation of Steroid Recep We can thus speculate that the well-known association be-
tors: Biological Implications tween ERα and HSP90 in the cytoplasm before hormonal
stimulation could involve SMYD2 in order to maintain
Estrogen Receptor α, or ERα
ERα in an inactive state in the absence of estrogen.
Lysine methylation/demethylation K235. The latest described ERα methylation on lysine is
K302. In 2008, Vertino’s team identified the first lysine catalyzed by the KMT G9a, which dimethylates the re-
methylation of a SR (375). They found that SET7/9 cata- ceptor at K235, in its DBD (Fig. 4A and Table 3). This
lyzes ERα methylation on lysine 302 (K302), located in the modification functions as an activator for the expression of
hinge region, to promote ERα transactivation. Moreover, some estrogen target genes, activating the growth of ERα-
they linked K302 methylation with the stabilization of positive BC cells (377). K235 dimethylation is a recogni-
ERα protein levels (Fig. 4A and Table 3). Indeed, estrogen- tion site for the Tudor domain of PHF20, a member of the
induced ubiquitylation of ERα and its subsequent deg- MOF histone acetyltransferase complex, which catalyzes
radation by the proteasome is an important step in the

the acetylation of histone H4K16, as well as nonhistone
transcriptional activity of the receptor (390, 391). As such, proteins involved in transcription. The association of ERα
SET7/9 was recognized as a potent modulator of ERα- with PHF20 through its K235 methylation site then re-
dependent gene expression. Interestingly, K302 is located cruits the MOF complex to deposit acetylation to H4K16
in a PTM “hot-spot,” where acetylation (K299, K302 and of E2 target genes, supporting access of ERα to chromatin
K303), ubiquitylation (K302), and phosphorylation (S305) and improving its transcriptional activity. As previously
were previously reported (85). Similarly to what happens reported for other methylation events, in the vicinity of
on histone tails, the presence of other PTMs in the vicinity K235, S236 was reported to be phosphorylated by protein
of K302 could affect the methylation event. Consistently, kinase A. Unlike K235 methylation, S236 phosphorylation
previous research reported the existence of connections be- is an obstacle for ERα dimerization and its recruitment to
tween acetylation and phosphorylation, which markedly DNA. Therefore, these 2 adjacent modifications compete
regulates the phenotype of cells. ERα nonacetylated K303 to regulate the transactivation activity of ERα. Aside from
variant (K303R) was detected in primary ductal hyper- K235, K303 in the hinge domain was also revealed to be
plasia and aggressive BC (392, 393). K303R promotes methylated by G9a in vitro; however, this has not been con-
phosphorylation on the nearby serine 305 (S305) and pro- firmed in vivo.
motes high transcriptional activity, even with low estrogen Taken together, it seems that ERα activity is regulated
levels. Therefore, the crosstalk between K302 methylation by 3 different lysine methylations carried out by 3 dif-
and K303 acetylation can contribute to invasive breast tu- ferent enzymes, in which K302 and K235 methylation
mors, highlighting that PTMs deeply influence SR signaling strengthens ERα activity, whereas K266 methylation func-
in normal and malignant contexts. tions as a repressor. Importantly, these 3 ERα residues are
K266. Later, K266 was shown to be methylated by located in the same “hot-spot,” which undergoes intensive
SMYD2 (376). In the absence of estrogen, this modifica- posttranslational modifications, and it is thus fundamental
tion impairs ERα binding to chromatin to prevent gene to study the importance of context-dependent effects of
activation. Upon estrogen stimulation, K266 methylation methylation events to better understand their interplay
is diminished, enabling p300-induced acetylation on this with other modification marks within the context of ERα
lysine residue to activate ERα transcriptional activity (Fig. signaling.
4A and Table 3). Moreover, cells with ERα-K266R muta-
tion have a higher capacity to proliferate than wild-type Arginine methylation/demethylation
(WT) cells under estrogen-depleted growth conditions, R260. Our team was the first to identify an arginine methy-
likely indicating that SMYD2-mediated K266 methyla- lation event for a SR. Indeed, we showed that PRMT1
tion blocks the estradiol-induced (E2) cellular response. As dimethylates ERα on arginine 260 (R260), located at the
LSD1 has been shown to remove methyl marks on SMYD2 junction between the DBD and the hinge domain (Fig. 4A
substrates, including p53 and HSP90 (394, 395), the au- and Table 3). This modification is a crucial event for es-
thors investigated whether it could participate in the regu- trogen nongenomic signaling. Mechanistically, estrogen
lation of ERα K266 methylation. They showed that LSD1 triggers a rapid and transient ERα methylation, which is re-
is able to remove SMYD2-K266 methylation, allowing quired for its interaction with the kinases Src and PI3K. The
ERα acetylation on the same residue by p300, activating formation of this complex is a prerequisite for activating
its transcriptional activity (Fig. 4A). More recently, HSP90 the downstream Akt pathway (382). In addition, we dem-
and its cochaperone p23 have been shown to bind SMYD2, onstrated that insulin-like growth factor (IGF-1) also trig-
inducing an increase in its ability to methylate ERα (396). gers ERα methylation via PRMT1, an important event for
IGF-1 signaling in BC, highlighting that targeting PRMT1 is located in the AF-1 hormone-independent transactivation
activity could be a good strategy to concomitantly impact domain, previously narrowed down to a 91 amino acid
estrogen and IGF-1 pathways (384). sequence preceding the DBD in PRs (Fig. 4B) (401). Site-
Together, these results emphasize the importance directed mutagenesis on PR revealed that nonmethylable
of PRMT1 as a regulator of both estrogen and IGF-1 mutants display a higher ligand-independent activation, in
signaling, highlighting PRMT1 as a promising target for particular perceptible by an increase in PR phosphorylation
treating ERα-positive BC patients. at S400, a basally phosphorylated site (402). Importantly,
As ERα methylation is a transient event, we investigated K464 mutations have a significant effect on ligand-induced
whether a demethylase could be involved in the regulation activity of PR, implying that K464 methylation impedes the
of ERα methylation. We showed that, upon estrogen stimu- transcriptional activity of PR.
lation, JMJD6 is integrated into the hallmark nongenomic A more recent study identified K481me1 acting in co-
complex metER/Src/PI3K, where it demethylates ERα, operation with K464 in the ligand-induced transcriptional

causing dissociation of the complex and termination of activity of the receptor (379) (Fig. 4B). Although the KMT
downstream signaling (366). Moreover, JMJD6 expres- is not identified yet, this study is the first to argue in favor of
sion is associated with poor prognosis in BC (397), but the importance of methylation in regulating PR signaling.
its enzymatic activities have not yet been fully associated These studies suggest that methylation of lysine residues of
with BC. the AF-1 domain disrupts PR transcriptional activity, sup-
Most of the studies on SR methylation have been con- porting the notion that methylation is a modulator of PR
ducted in cancer cell models; however, metERα expression signaling in BC cells.
has been evaluated in human breast tissues concomitantly
with ERα/Src and ERα/PI3K complexes, hallmarks of ERα
nongenomic signaling. Their expression has been detected Arginine methylation
at low levels in human breast tissues and high levels in a R492. Within the same study conducted by Woo et al.,
subset of breast tumors. Interestingly, their high expression R492me1 was shown to synergize with the 2 preceding
is associated with BC aggressiveness (383). More recently, lysine methylation events (K464 and K481), in the tran-
we showed that ERα nongenomic signaling is increased in scriptional activity of PR (379) (Fig. 4B and Table 3).
BC resistant to tamoxifen treatment (398). Later, using a When residues were substituted to neutral polar glu-
mouse model harboring ERα mutated R264A (equivalent tamine (K464Q/K481Q/R492Q), the defective triple-
to R260 in human), the role of metERα in physiology was methylation mutant exhibited a strong increase in
investigated. It was shown that although this arginine is transcriptional activity in response to progestin, com-
not required for the physiological regulation of the skeleton pared with WT PR, suggesting that positive charge due
(399) nor for fertility (385), this residue is involved in ERα to methylation could act as a brake for transcription ef-
activity, such as the rapid dilatation of mesenteric arteries ficiency. Moreover, data showed that these key residues
and the endothelial repair of carotid (385). provide not only the interaction interface with major
Altogether, these findings highlight the importance of ar- PR coregulators, like SRC-1, but also with the AF-2-
ginine methylation in ERα nongenomic pathways. Probably containing LBD, described as acting with AF-1 to bring
because R260 is not conserved among NR, this modifica- PR towards a full transcriptional activation.
tion has never been involved in nongenomic signaling trig- R637. Our team recently demonstrated a functional
gered by other members of the family. crosstalk between arginine methylation, PR transcrip-
tional activity, and progestin-induced proliferation in
BC cells (386). We showed that PRMT1 is an important
modulator of the transcriptional activity of PR, at least
Progesterone Receptor, or PR in part through methylation in the hinge region (Fig.
Lysine methylation 4B and Table 3). Under progestin treatment, PRMT1
K464, K481. PR is largely post-translationally modified, es- dimethylates PR in the nucleus, at R637, within a RGG
pecially by phosphorylation on serine and threonine residues methylation consensus motif (346, 403). This methyla-
(400). Methylation on lysine residues has also been reported, tion event modulates PR oncogenic properties, as cells
in the NTD close to the DBD. Chung et al. (378) reported expressing a nonmethylable mutant (R637K) displayed a
that K464 methylation is essential in PR ligand–independent retarded cell growth and a reduced expression of a subset
and –dependent transcriptional activities (Fig. 4B and Table of genes that promote proliferation and survival of BC
3). Using MS, they showed that both PR is endogenously cells. R637 methylation facilitates PR degradation, which
monomethylated in T47D BC cells (378). Interestingly, K464 in turn constitutes a critical stimulatory switch that
Endocrine Reviews, 2022, Vol. 43, No. 1
Neurodegenerative
178
Table 3. Lysine and arginine methylation of steroid receptors
Steroid receptor methylation by lysine methyltransferases
Receptor Enzyme Residue Biological effect References
ERα SET7/9 K302me1 Promotes transcriptional activity by protein stabilization (375)

SMYD2 K266me1 Represses transcriptional activity (376)
G9a K235me2 Promotes transcriptional activity (377)
PR ND K464me1 Decreases ligand sensitivity (378)
K481me1 Represses AF1 activity (379)
AR SET7/9 K632me1 Promotes its transcriptional activity (380)
K630me1 (381)
AR DOT1L K349 Activates its transcriptional activity (189)

Steroid receptor methylation by protein arginine methyltransferases
ERα PRMT1 R260me2a Participates in E2 non genomic signaling (382, 383)
Participates in IGF-1 signaling (384)
Participates in vascular protective effects (385)
PR ND R492me1 Decreases transcriptional efficiency (379)
PRMT1 R637me2a Regulates stability and transcriptional activity (386)
AR PRMT5 R761me1/2s Represses genes involved in differentiation (387)
PRMT6 R210me2a, R212me2a, Activates its transcriptional activity in SBMA, by (388)
R787me2a, R789me2a inhibiting phosphorylation by Akt
GR PRMT5 Rme2s ND (389)
K, lysine; R, arginine; Kme1, monomethyllysine; Kme2, dimethyllysine; Rme1, monomethyarginine; Rme2a, asymmetric dimethyarginine; Rme2s, symmetric
dimethylarginine; ND, nondetermined; IGF-1, insulin-like growth factor; SBMA, spinal and bulbar muscular atrophy. ND, not determined
accelerates the recycling of PR from pre-initiation com- Androgen Receptor, or AR

plexes, which is required for active hormone-dependent Lysine methylation
transcription (390, 404, 405). Moreover, BC patients K630, 632. In 2011, 2 different teams reported that AR
with PR-positive tumors expressing high level of PRMT1 activity can be regulated by SET7/9. The first 1 reported
show a worse survival than patients with low PRMT1, that AR interacts with the methyltransferase, which in turn
suggesting that targeting PRMT1 could constitute a new monomethylates the K632 residue within a KLKK motif,
therapeutic option. similarly to the sequence methylated in the hinge domain
Interestingly, SUMOylation of PR on K388 has also been of ERα (407) (Fig. 4C and Table 3). This methylation in-
shown to regulate PR stability by competing with ubiqui- duced upon ligand binding is necessary for enhancing
tination. Indeed, the E3 ubiquitin ligase CUEDC2 binding AR transcriptional activity. As SET7/9 functions as a
attenuates sumoylation SUMOylation of K388, while pro- proproliferative and antiapoptotic factor, highly expressed
moting ubiquitination (406). As crosstalk between different in PC, it could constitute a potential target to treat these
PTMs have largely been described (85), we can hypothesize tumor (407). In parallel, a study from another research
that the integration of these different signals tightly regu- group demonstrated that K630, and not K632, is methy-
lates PR stability and transcriptional activity. lated by SET7/9, and globally linked with the same func-
Taken together, methylation is an indisputable mech- tions (381). The discrepancy between these 2 works has so
anism involved in the regulation of the transcriptional far not been resolved. K632 modification better matches
activity of PR, sometimes acting as a repressor or as an the consensus site of SET7/9, but the identification was
activator, once again highlighting the importance of performed with a small peptide. MS analyses on the full
context-dependent effects of this PTM in cells. protein or specific antibodies are needed to clarify this
Figure 4: continued
Figure 4. Biological consequences of SR methylation. All the methylation events targeting the steroid receptors on arginine (R) and lysine (K) res-
idues and reported at this time are represented for (A) ERα, (B) PR, (C) AR, and (D) GR. When identified, the protein methyltransferases involved
are noted in black and the demethylases in brown. The methylation events leading to repressive functions are represented in red and the activating
functions are in green. For ERα, we enlarged the hinge domain as it is the main region modified by methylation. When decrypted and reported, the
biological consequences of the methylation event on the physiology/pathology have been indicated (in green for activating functions, red for repres-
sive functions and blue when no effect). NTD, N-terminal domain; DBD, DNA-binding domain; h, hinge; LBD, ligand binding domain; NLS, nuclear
localization signal; NES, nuclear export signal; BC, breast cancer; PC, prostate cancer.
point. Nonetheless, these 2 methylation sites belong to a AR (binding to an androgen response element present in
rich acetylation target domain. Indeed, K630, 632, and 633 PRMT6 promoter) is necessary for AR-controlled sperm-
residues are acetylated by p300, p/CAF (408), and TIP60 atogenesis (415).
(409), suggesting, similarly to ERα, a crosstalk between R761. PRMT5 has been shown to methylate AR on R761
acetylation and methylation. in the LBD (Fig. 4C and Table 3). This modification was
K349. Lysine methylation is also involved in the regulation described in PC cases expressing the TMPRSS2:ERG fu-
of AR transcriptional activity, through the binding of 2 long sion gene. Mechanistically, the TF ERG recruits PRMT5 to
noncoding RNAs, namely PRNCR1 and PCGEM1, in PC AR target genes involved in prostate differentiation, where
cells. The KMT DOT1L was required for AR binding to the enzyme methylates the receptor (387). This event at-
PCGEM1. Indeed, DOT1L methylates AR on K349, located tenuates AR recruitment to these sites and subsequent tran-
in the N-terminal region, a critical step for the recruitment scription from the associated gene promoter. Consistently,
of PCGEM1 to AR (189) (Fig. 4C and Table 3). Moreover, R761 methylation supports proliferative functions of the

DOT1L is overexpressed in PC and is associated with poor oncoprotein ERG. Thus, R761 methylation could serve
clinical outcome, and this KMT selectively regulates the as a potent biomarker for AR-dependent proliferation in
tumorigenicity of AR-positive PC cells in vitro and in vivo, TMPRSS2:ERG-positive PC, and inhibiting PRMT5 ac-
making a promising therapeutic target (410). However, the tivity could be beneficial for treating this tumor subtype.
results about AR methylation on K349 are matter of debate Altogether, it appears that AR and its variants are
since they were refuted by Chinnaiyan’s team (411). highly methylated on both lysine and arginine residues,
participating actively in its full transcriptional activity,
Arginine methylation even though the interplay between these modifications is
R210, R212, R787, R789. The first link between PRMT6 still unknown.
and AR was unveiled by Sun et al. (412), showing a direct
PRMT6/AR interaction in Cos7 cells overexpressing AR.
Furthermore, PRMT6 was able to methylate AR, although Glucocorticoid Receptor, or GR
specific methylation sites were not explored. More recently, GR was not known to undergo methylation until recently.
this interaction was confirmed with the AR mutant con- Indeed, using pan methyl antibodies recognizing only
taining polyglutamine stretch in its NTD and implicated dimethylated arginines, our team found that GR is methy-
in the X-linked transmitted spinal and bulbar muscular at- lated by PRMT5 within the nucleus of the ERα-positive BC
rophy (388). For this interaction, the required regions were cell line MCF-7 (Fig. 4D and Table 3) (389). Although the
the AF-2 domain of AR, the catalytic domain of PRMT6 as methylated arginine residue(s) and the role of this modifica-
wells as a LXXLL motif present in PRMT6. PRMT6 proved tion in GR activity is still under investigation, our finding is
to methylate AR and, to a higher extent, polyglutamine- highly promising as it suggests a new molecular regulation
expanded AR. The AR arginine methylation sites were of GR activity involving PRMT5.
identified in both the NTD and the LBD (R210/212 and
R787/789) (Fig. 4C and Table 3), within RXRXXS motifs
involved in Akt-mediated AR phosphorylation. Overall, Indirect Role of Methylation/Demethylation
AR transactivation by PRMT6-mediated methylation was in Steroid Receptor Transcriptional Activity
regulated by phosphorylation through a mutually exclusive Additional indirect methylation events modulate the
interaction, making PRMT6 a modifier of polyglutamine- transcriptional activity of SRs. On the one hand, SR
expanded AR neurotoxicity. A model is suggested in which coregulators are also methylated. These methylation events
arginine methylation of polyglutamine-expanded AR by regulate, among other processes, molecular interactions,
PRMT6 at the Akt consensus site motif enhances func- stability, and subcellular localization of SRs, in order to
tion and toxicity leading to neurodegeneration, whereas tightly modulate their transcriptional activity. Specific
phosphorylation by Akt prevents binding to testosterone, coregulators are recruited on the promoter/enhancer re-
thereby protecting neurons from degeneration (388). gions of their target genes to locally remodel the chromatin
Exemplified in this disease model, the PRMT6/AR inter- structure and to orchestrate the assembly or disassembly
action is likely to play relevant roles in other diseases, such of an active transcription complex. On the other hand,
as PC, where PRMT6 is overexpressed in comparison with lysine or arginine methyltransferases, or demethylases,
normal prostate tissue (413, 414) or even physiological modify residues in histone tails and thereby participate in
conditions. Thus, overexpression of PRMT6 has been SR-dependent target gene expression. In this section, we
observed in the testes of AR-KO mice (51, 415). Results selected key examples of these indirect methylation events
from this study suggest that downregulation of PRMT6 by to illustrate their impact on SR transcriptional activity.
Lysine Methylation/Demethylation of both AR and its main splice variant, AR-V7, which
G9a/GLP methylation and GR regulation has been implicated in resistance to androgen deprivation
G9a and its paralogue GLP are interesting illustrations therapy in castration-resistant PC (427). Overall, through
of KMTs involved in SR transcriptional activity through enhanced transactivation of AR (and possibly AR variants),
more indirect mechanisms than those previously described. LSD1 may favor proliferation and invasiveness of PC cells
First identified as KMTs methylating the repressive mark and impair apoptosis under androgen deprivation therapy
H3K9, these enzymes also act as GR coactivators under (425, 428).
certain circumstances (90). The self-methylation of G9a/ Interestingly, phosphorylation of H3T6 by pro-
GLP on K185 and K205, respectively, provide a binding tein kinase C beta I prevents LSD1 from demethylating
site for HP1γ, which facilitates the recruitment of RNA H3K4me1/2 and induces a switch in substrate specificity
polymerase II, activating the transcription of a subset of from H3K4me1/2 to H3K9me1/2, orienting LSD1 towards
GR target genes in specific cell contexts (Fig. 5A). In con- a transcriptional coactivator role (208, 429). Again, this set

trast, phosphorylation of the adjacent threonine (T186 for of experiments clearly underlines that protein methylation
G9a and T206 for GLP) by Aurora kinase B (AURKB) pre- can interfere with other PTMs and induce a switch in the
vents binding to HP1γ and reduces coactivator functions of functions of SR coregulators, resulting in a fine-tuning of
G9a and GLP (416), resulting in distinct biological effects gene expression.
in a tissue-specific manner. For instance, the coactivating
activity of G9a/GLP regulates migration of the lung cancer
Arginine Methylation/Demethylation
cell line A549 (416), and GC-induced cell death in B-acute
lymphoblastic leukemia (417) (Fig. 5A). Moreover, a panel Similarly to lysine residues, methylation on arginine res-
of KDM inhibitors identified the JmjC KDM family as idues of histone tails and coregulators has been reported to
demethylases for G9a and GLP. JmjC inhibitors increased indirectly regulate SR transcriptional activities.
G9a methylation, expression of G9a-HP1γ-dependent GR
target genes and GC-induced cell death in B-acute lympho- Methylation of histone marks by PRMT1 and CARM1
blastic leukemia cell lines (418). In vitro demethylation as- Twenty years ago, numerous studies demonstrated the
says unveiled the KDM4 family as demethylases for G9a strong impact of PRMT1 and CARM1 as coregulators that
and GLP (Fig. 5A). This study was a proof of concept modify histone tails in order to modulate SR-dependent
that a methylation/phosphorylation event could influence transcription. Historically, CARM1 was characterized
a coregulator, functioning as a coactivator or corepressor, as a methyltransferase owing to its capacity to methylate
tightly regulating the transcriptional activity of a SR. the N-terminal tail of H3 in vitro (430). This finding was
then confirmed in vivo on hormone-regulated promoters.
LSD1 regulation of histone marks Indeed, the transcriptional activities of ERα, GR, and AR
Among the well-known coregulators that influence SR are known to be regulated by CARM1-induced methyla-
signaling by altering protein methylation status, LSD1 was tion of both R17 and R26 of histone H3 (431-433) (Fig.
the first demethylase described to regulate histone methyla- 5A). As CARM1, PRMT1 also acts as a coactivator of SR
tion in a hormone-dependent context. Indeed, the recruit- through histone methylation (12, 13, 434). H4R3 is methy-
ment of LSD1 and subsequent H3K4me1/2 demethylation lated by PRMT1 and plays a critical role in transcriptional
induces repression of androgen-dependent AR target gene activation, making PRMT1 a potent coactivator of AR
expression (including AR itself) (419). In addition to its transcriptional activity (13). Interestingly, as it was de-
well-established corepressor functions (203, 420), LSD1 scribed for lysine methylation on histone tails, cross-talks
acts as a coactivator for several TFs, including AR and between arginine methylation and other PTMs, namely
ERα (208, 421-424). This effect was not only observed lysine acetylation, were reported to be involved in the
in PC cells but also in kidney cancer cells, which are usu- regulation of SR-dependent transcription. For instance,
ally not considered to be androgen-sensitive. In this study, stimulation by estrogens results in the acetylation of H3K18
using pargyline, an inhibitor of LSD1, investigators were by the acetyltransferase CREB binding protein (CBP).
able to block demethylation of H3K9 and subsequently This event stimulates CARM1 recruitment to the H3 tail,
AR-dependent transcriptional activation (425). In another which methylates H3R17, leading to the implementation
recent study, LSD1 demethylated the pioneer TF FOXA1, of estrogen-regulated gene expression (435). More recently,
thereby facilitating its DNA-binding, notably in androgen PRMT5 associated with pICln was described as an epigen-
response element and estrogen response element, where it etic activator of AR transcription in castration-resistant PC
is a well-known active partner in AR- and ER-mediated cells, suggesting that targeting its enzymatic activity could
transactivation (426). Lastly, LSD1 acts as a coactivator represent a novel therapeutic approach (436).
Methylation of coregulators by CARM1 CARM1 methylome), such as acetyltransferases (P300,

Aside from direct histone methylation, some transcrip- P400), KMTs (KMT2C and KMT2D) and components of
tional coregulators are also methylated on arginine res- the SWI/SNF, NuRD, and mediator complexes (232). Of
idues, making the arginine methyltransferases potent note, JMJD6 regulates also the activation of ERα enhan-
transcriptional modulators, such as CARM1 for ERα- and cers by participating in the interaction between MED12
AR-dependent transcription (437). For example, CARM1 and CARM1 (446) (Fig. 5B).
dimethylates CBP on R742 in vivo, playing a role in estrogen- Aside from CARM1, PRMT1 also dimethylates
induced gene activation (438) (Fig. 5B). P300 is methylated coregulators to modulate SR signaling. For example,
by CARM1 on R2142, localized in its C-terminal GRIP1 PRMT1 dimethylates arginines on the peroxisome
binding domain, inhibiting p300 binding to the coregulator proliferator-activated receptor γ coactivator 1α (PGC-1α),
glucocorticoid receptor interacting protein 1 (GRIP1/SRC2), a coactivator of many SRs, including ERα (447). PRMT1
a key coregulator of SRs in vitro and in vivo (439). It was and its catalytic activity enhances PGC-1α coactivator

later shown that CARM1-dependent CBP methylation on activity on ERα reporter genes (Fig. 5B). Endogenously,
R742, 768, and 2151 stimulates its histone acetyltransferase PRMT1 regulates the expression of PGC-1α target genes
activity by increasing its autoacetylation (440). Interestingly, that are important for mitochondrial biogenesis (447).
using antibodies specific for the individual methylation sites, Altogether, these results highlight that methylation of
they showed that methylation of CBP is required for its re- coregulators and histones is heavily involved in the fine
cruitment by ERα to specific target genes (Fig. 5B). Using regulation of the transcriptional activity of SRs, making
genome-wide analyses, different patterns of binding to ERα these enzymes promising targets to modulate SR functions.
target genes were observed for the various methylated CBP
species (440). These data suggest that CARM1-dependent
CBP methylation induces the expression of specific target Outlook
genes, diversifying the ERα transcriptional program. There is increasing evidence that SRs are tightly regulated by
CARM1 was also shown to methylate SR coactivator pro- protein interactions and PTMs targeting the receptors them-
teins of the SRC/p160 family, including SRC-3 (441, 442). SRC selves, but also histone tails and coregulatory proteins. Among
proteins serve as primary coregulators to recruit secondary these PTMs, methylation has assumed an increasingly im-
coactivators, such as p300/CBP or CARM1, to the promoters portant role, and given the crosstalk between PTMs, we can
of specific estrogen-dependent target genes (443). SRC-3 extrapolate that the effects extend beyond current knowledge.
methylation on R1171 takes place in its C-terminal domain Even if ERα appears to be the most post-translationally
containing its binding sites with p300/CBP and CARM1 (441, modified SR (probably because it is the most studied), it is
442). Consequently, SRC-3 methylation induced by estrogens likely that other receptors are also methylated on residues that
triggers its dissociation from CBP and CARM1, a decrease in remain to be identified. This review focused on 4 SRs, but we
its stability and a subsequent decrease in ERα-mediated tran- can easily imagine that other NRs are modified by methylation.
scription (Fig. 5B). The authors suggested that this methyla- It has already been shown for the orphan receptor HNF4, the
tion event serves as a molecular switch for disassembly of the transcriptional activity of which is regulated by PRMT1 in 2
SRC-3 transcriptional coactivator complex (441). ways (448): it methylates HNF4-DBD, enhancing its binding
More recently, 2 new substrates for CARM1 to chromatin, and methylates H4R3. In addition, RARα is also
were identified: BAF155 in the SWI/SNF chromatin methylated on lysine residues, impacting its transcriptional ac-
remodeling complex and MED12, a component of the tivity (449). Although the thyroid hormone receptor has so
mediator complex that facilitates RNA polymerase II re- far not been shown to be methylated, PRMT1 can regulate its
cruitment (282). CARM1-methylation of MED12 regu- transcriptional activity through H4R3 methylation, contrib-
lates its binding to the chromatin in order to regulate uting to T3-induced metamorphosis of Xenopus laevis (450).
p21 gene expression, increasing BC cell sensitivity to LSD1 is also suspected to be a functionally important cofactor
chemotherapy in vitro and in vivo (282, 444) (Fig. 5B). for the mineralocorticoid (MR) and MR-related disease, such
An additional study confirmed that MED12 is methy- as high blood pressure (451).
lated by CARM1 in a cluster of arginine residues located In this review, we focused on some methyltransferases
in its C-terminal domain, regulating MED12 recruitment though other enzymes may also regulate SR function via
to ERα enhancers (445) (Fig. 5B). These methylated res- methylation. For instance, PRMT2 regulates the transcrip-
idues are recognized by the coactivator protein TDRD3 tional activities of ERα (452) and AR (453), likely through
to activate ERα target genes. Interestingly, a high- its enzymatic activity. In addition, PRMT6 enhances ERα
resolution MS analysis of CARM1 substrates identified a ligand–dependent and –independent activity (412), even
list of coregulators involved in ERα transcription (called if the mechanism involved has not been clearly identified.
Figure 5. Indirect methylation events regulating SR signaling. Here, we highlight 2 examples, in (A) GR and in (B) ERα, of indirect methylation events
(ie, not directly on SRs), regulating the transcriptional activity of these 2 receptors. This concerns the methylation of histone tails on chromatin and/
or the methylation of coregulators. When identified, the targeted lysines (K) or arginines (R) and the methyltransferases are noted in black and the
demethylases in brown. The methylation events leading to repressive functions are represented with red lines and the activating functions with green
arrows. Me, methylation; GRE, GR response elements; ERE, estrogen response elements; H3, histone H3; H4, histone H4; CoA, coactivators; Dex,
dexamethasone; E2, estrogens; BC, breast cancer.
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We thank V. Vlaemink-Guillem for her advice and B. Manship
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for proofreading the manuscript. The illustrations were created
arginine 3 facilitating transcriptional activation by nuclear hor-
by using Servier Medical Art.
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Financial Support: M.L.R, L.M., H.T.P, L.E., and C.P.’s laboratory is
14. Husmann D, Gozani O. Histone lysine methyltransferases in
funded with grants from “La Ligue contre le Cancer,” the “Fondation
ARC Cancer,” and the “Fondation de France.” L.M. was supported biology and disease. Nat Struct Mol Biol. 2019;26(10):880-889.
by a fellowship from “Fondation ARC Cancer.” L.E. was supported 15. Poulard C, Corbo L, Le Romancer M. Protein arginine

by a fellowship from “La Ligue contre le Cancer.” H.T.P. was sup- methylation/demethylation and cancer. Oncotarget.
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Endocrine Reviews, 2022, Vol. 43, No.

How Protein Methylation Regulates Steroid

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ORCiD number: 0000-0002-8491-4015 (M. Le Romancer).

ISSN Print: 0163-769X

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Table 1. Lysine methylated substrates

KMT Substrate Site Effect of lysine methylation References

GLP HIF-1α K674me1/2 Represses HIF-1α transcriptional activity (108)

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Table 1. Continued

KMT Substrate Site Effect of lysine methylation References

β-catenin K180me1 Decreases β-catenin stability (153)

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Abbreviations: K, lysine; Kme1, monomethyllysine; Kme2, dimethyllysine; Kme3, trimethyllysine.

KMTs targeting KDMs, or Lysine (K) DeMethylases

LSD1 KDMs from the KDM4 subgroup are dysregulated in

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response, RNA processing and signal transduction) (232) PRMT6

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Table 2. Arginine methylated substrates

PRMT Substrate Site Effect of arginine methylation Reference

PRMT1 BRCA1 504–802 Facilitates its binding to promoters (233)

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Table 2. Continued

PRMT Substrate Site Effect of arginine methylation Reference

RunX1 R233, R237 Resists to apoptosis under stress condition (280)

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Table 2. Continued

PRMT Substrate Site Effect of arginine methylation Reference

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Arginine (R) Demethylases Interestingly, JMJD6 is upregulated in a large spectrum

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Table 3. Lysine and arginine methylation of steroid receptors

Steroid receptor methylation by lysine methyltransferases

Receptor Enzyme Residue Biological effect References

ERα SET7/9 K302me1 Promotes transcriptional activity by protein stabilization (375)

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accelerates the recycling of PR from pre-initiation com- Androgen Receptor, or AR

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Methylation of coregulators by CARM1 CARM1 methylome), such as acetyltransferases (P300,

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23. Schwabe JW, Chapman L, Finch JT, Rhodes D. The crystal

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133. Nakakido M, Deng Z, Suzuki T, Dohmae N, Nakamura Y,

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