Epigenetics and Regeneration

Nobuyasu Maki and Hironobu Kimura

Abstract During newt lens regeneration a unique transdifferentiation event

occurs. In this process, dorsal iris pigmented epithelial cells transdifferentiate into
lens cells. This system should provide a new insight into cellular plasticity in basic
and applied research. Recently, a series of approaches to study epigenetic repro-
gramming during transdifferentiation have been performed. In this review, we
introduce the regulation of dynamic regulation of core-histone modifications and
the emergence of an oocyte-type linker histone during transdifferentiation. Finally,
we show supporting evidence that there are common strategies of reprogramming
between newt somatic cell in transdifferentiation and oocytes after somatic cell
nuclear transfer.

Contents

1 Introduction........................................................................................................................ 238
2 Newt Lens Transdifferentiation ........................................................................................ 238
2.1 A Key Biological Event ........................................................................................... 238
2.2 Structural Changes in the Nucleus........................................................................... 240
2.3 Gene Expression ....................................................................................................... 240
3 Epigenetics in Newt Lens Transdifferentiation................................................................ 241
3.1 Core Histone Modifications ..................................................................................... 241

N. Maki (&)
H. Kimura

Institute of Protein Research,
Osaka University, 3-2 Yamadaoka, Suita-Shi, Osaka 565-0871, Japan
e-mail: [email protected]
N. Maki
PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi,
Saitama 332-0012, Japan

Current Topics in Microbiology and Immunology (2013) 367: 237252 237

DOI: 10.1007/82_2012_293
Springer-Verlag Berlin Heidelberg 2012
Published Online: 30 November 2012
238 N. Maki and H. Kimura

3.2 Oocyte-Type Linker Histone B4.............................................................................. 244

4 Discussion .......................................................................................................................... 246
References................................................................................................................................ 248

1 Introduction

The developmental program is controlled by genetic and epigenetic regulation.

Epigenetic regulation provides the diversity of cell differentiation in development.
After fertilization, the zygote differentiates into diverse cells depending on inter-
actions between their cell lineage and differentiation signals. Although differen-
tiated cells have identical DNA sequences, they exhibit different profiles of gene
expression. Epigenetics involves heritable alterations of gene expression without
changes in DNA sequence, and contributes to the diversity of gene expression and
memory of cell lineage. One major mechanism of epigenetics is the chemical
modifications to the nucleosome, including DNA methylation and histone
modification.
It is clear that epigenetics plays a major role in development, the field of
epigenetics research during regeneration has just started. There are pioneering
studies in DNA methylation during Xenopus limb regeneration (Yakushiji et al.
2007) and during zebrafish pancreatic b-cell and liver regeneration (Anderson
et al. 2009; Sadler et al. 2007). In this review, we focus on two mechanisms of
epigenetic changes, core- and linker-histone regulation, during newt lens
transdifferentiation.

2 Newt Lens Transdifferentiation

Urodele amphibians have a strong regenerative ability. In particular, the newt can
regenerate almost all tissues in its body including lens, retina, limbs, jaw, tail,
small intestine, heart, and brain. Understanding the mechanism of amphibian
regeneration will provide crucial information for both basic and applied biology.
Especially, the understanding of unique events in regenerative animals will be
important. In this chapter, we introduce a unique phenomenon of transdifferenti-
ation identified in newt lens regeneration.

2.1 A Key Biological Event

In newt lens regeneration, dorsal iris pigmented epithelial cells (PECs) transdif-
ferentiate into lens cells (Fig 1a). Newt lens regeneration can be divided into three
major steps. The initial step after lentectomy (from day 0 to day 3) involves
Epigenetics and Regeneration 239

Fig. 1 Newt lens regeneration. a Dorsal PECs transdifferentiate into lens cells. About 4 days
after lentectomy, PECs begin to re-enter the cell cycle and shed their pigments. PECs change to
transparent cells by around day 8. After day 14, lens differentiation occurs from dorsal iris.
Although the ventral PECs show depigmentation and cell cycle re-entry, they never regenerate
lens. b Structural change in the nucleus during lens transdifferentiation. Original PECs have a
small and shrunken nucleus. During dedifferentiation, the PEC nucleus swells and its nucleoli
become huge. N, nucleus; arrow head, nucleolus. Illustration of nucleus is reproduced from
(Eguchi 1980) with permission

molecular and cellular events that precede PECs re-entering the cell cycle. The
next step (days 412) is the point at which PECs start cell cycle re-entry 45 days
after lentectomy. At this time, PECs start shedding their pigment granules. PECs
continue depigmentation and proliferation and finally change to transparent cells
by day 8. This process, where PECs lose their original tissue characteristics, is
called dedifferentiation. On days 1012 depigmented PECs form a vesicle but do
not express lens-specific markers.
In the last step, lens differentiation begins. After day 14, posterior cells of the
dorsal vesicle elongate and start to express lens markers. The vesicle grows and
differentiates into lens, which is of a considerable size and normal morphology by
day 20. Embryologically, lens cells and PEC are derived from surface and neural
ectoderm origin, respectively (Coulombre 1965), suggesting that lens transdiffer-
entiation is accomplished by a different mechanism from that seen in embryogenesis.
The transdifferentiation of PECs has been demonstrated in clonal culture experi-
ments. A single PEC dissociated from dorsal iris transdifferentiates into a lentoid
body, which expresses lens-specific markers in culture (Abe and Eguchi 1977).
240 N. Maki and H. Kimura

It is note worthy that the transdifferentiation of PEC is one of the best and most
suitable systems to study epigenetics in regeneration because its cell lineage is so
simple and so obvious. The ventral PECs show depigmentation and cell cycle
re-entry. The number of BrdU-positive cells in the ventral iris is comparable with
that in the dorsal iris by day 6 (Maki et al. 2007). In contrast to dorsal PECs,
however, the ventral PECs never differentiate to lens in vivo (Grogg et al. 2005;
Hayashi et al. 2006). Therefore, there is a dorso-ventral selectivity in lens
regeneration.

2.2 Structural Changes in the Nucleus

The PEC nucleus dynamically changes its structure during lens transdifferentiation
(Maki et al. 2007, 2010b) (Fig. 1b). The nucleus of the original PEC is small
(about 10 lm in a diameter) and shrunken in shape and has highly developed
heterochromatin, which is the transcriptionally inactive region of Chromatin (Maki
et al. 2010b). During the dedifferentiation of the PEC, nuclear swelling occurs, and
finally the nucleus changes its shape to become round with its diameter reaching
more than 20 lm by around day 10. In parallel with the nuclear swelling,
euchromatic regions, transcriptionally active regions, increase dramatically (Maki
et al. 2010b). The structure of the nucleoli also changes during the dedifferenti-
ation (Fig. 1b). Although the nucleoli in the original PEC are small, they become
huge during the dedifferentiation. After the onset of lens differentiation, the nuclei
of the cells become smaller and elongated, and have small nucleoli. Therefore
during transdifferentiation, the nucleus dynamically changes its structure in cor-
relation with the cellular state. It is suggested that the nuclear swelling with the
enlargement of euchromatin during the dedifferentiation is due to reprogramming
of the cellular state from differentiated to a stem cell-like state.

2.3 Gene Expression

2.3.1 Nucleostemin

Nucleostemin (NS), a member of the nucleolar GTPase family, is highly expressed

in stem cells, progenitor cells, and most cancer cells (Baddoo et al. 2003; Nikpour
et al. 2009; Ohmura et al. 2008; Tsai and McKay 2002). Both knocking down and
over-expression of NS reduces cell proliferation in cultured cells. The major
function of NS is as a regulator of proliferation in both p53-dependent (Dai et al.
2008; Tsai and McKay 2002) and p53-independent pathways (Beekman et al.
2006; Romanova et al. 2009).
To understand the cellular state during newt dedifferentiation, expression of NS
during the early process of lens regeneration has been analyzed. After lens
removal, the expression of NS is activated and NS accumulates in nucleoli of
Epigenetics and Regeneration 241

dedifferentiated cells (Maki et al. 2007). Importantly, the increase of NS accu-

mulating cells occurs prior to S-phase re-entry, suggesting that the increase of NS
accumulating cells is not due to proliferation of pre-existing stem cells but due to
changing of the cellular state of PECs during dedifferentiation.

2.3.2 Stem Cell Pluripotency Factors

Embryonic stem (ES) cells are in a pluripotent state which allows them to dif-
ferentiate into all types of cells in three germ layers (Evans and Kaufman 1981;
Martin 1981). Another significant property of the ES cell is an ability to reprogram
somatic cells (Cowan et al. 2005; Tada et al. 2001). By electrofusion with ES cells,
somatic cell nuclei can be reprogrammed and express ES cell markers such as
Oct4, and the hybrid cells contribute to all three primary layers of chimeric
embryos, suggesting the existence of reprogramming factors in ES cells. On the
basis of this fact, the reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, have
been screened and pluripotent stem cells have been induced from fibroblasts by
introducing these four factors (Okita et al. 2007; Takahashi et al. 2007; Takahashi
and Yamanaka 2006; Wernig et al. 2007; Yu et al. 2007).
Retinal PECs from chicken embryo dedifferentiate and transdifferentiate into
lens cells in culture. It has been shown that dedifferentiated cells from the retinal
PECs express c-Myc (Agata et al. 1993). During newt lens regeneration, expres-
sion of Sox2 at the lens differentiating stage has been reported (Hayashi et al.
2004). To further investigate newt dedifferentiation, expression of the stem cell
factors during dedifferentiation of PECs have been examined (Maki et al. 2009).
Although Oct4 and Nanog are not expressed, Sox2, Klf4, and c-Myc are expressed
in a stage-dependent manner during dedifferentiation of PEC. Sox2 and Klf4 are
upregulated at a very early step (day 2). The expression of c-Myc reaches a peak at
a later stage (day 8). In addition to NS, the expression of those stem cell factors
suggests that dedifferentiated cells have a stem cell-like state.

3 Epigenetics in Newt Lens Transdifferentiation

3.1 Core Histone Modifications

In the nuclei of eukaryotic cells, genomic DNA is highly organized as chromatin.

The nucleosome is a basic unit of chromatin, which consists of a histone octamer, a
linker histone, and approximately 147 base pairs of DNA wrapped around the
histone octamer. The histone octamer consists of an H3H4 tetramer and two sets of
H2AH2B dimers (Kornberg 1974). Linker histones are bound to the linker DNA
which is found between nucleosomes and are responsible for forming higher-order
chromatin structure (Fig. 2a). Core-histones, histone H2A, H2B, H3, and H4, are
242 N. Maki and H. Kimura

Fig. 2 Chromatin structure and histone modifications. a Approximately 147 base pairs of DNA
wrap around a histone octamer consisting of an H3-H4 tetramer and two H2A-H2B dimers to
form a single nucleosome. The nucleosome is packed with histone H1 to form higher order
chromatin structure. b Histone tail modifications. Acetylation (Ac) and methylation (Me) of
lysine residues at N-terminus of histone H3 and H4 are shown. c There are four types of linker-
histones identified

small basic proteins consisting of a flexible N-terminus called a histone tail and a
fold domain that interacts with DNA. The histone tail is subject to several post-
translational modifications (Kouzarides 2007; Ruthenburg et al. 2007) (Fig. 2b).
Epigenetics and Regeneration 243

Generally, histone acetylation is a positive mark for gene expression and

associated with euchromatin (Shahbazian and Grunstein 2007). Histone acetyl-
transferases (HATs) transfer an acetyl group to a lysine residue and thus neu-
tralizes the positive charge on lysine, thereby reducing the interaction between
DNA and core-histones. As a result, histone acetylation promotes transcriptional
activation (Grant et al. 1999; Kuo et al. 1996; Schiltz et al. 1999; Spencer et al.
1997). Contrary to HATs, histone deacetylases (HDACs) remove an acetyl group
from lysine and induce transcriptional silencing (Rundlett et al. 1996). The acet-
ylation status of a promoter region, which is accomplished by a balance between
HATs and HDACs, regulates gene expression.
Histone methylation is involved in various biological aspects. The compre-
hensive analysis of methylated histones in the genome reveals that methylated
histones are associated with both transcriptionally active and inactive regions
(Barski et al. 2007; Bernstein et al. 2006; Guenther et al. 2007; Mendenhall and
Bernstein 2008; Mikkelsen et al. 2007; Wang et al. 2008). The lysine residue can be
mono-, di-, or tri-methylated and each methylation state is associated with a dif-
ferent effect on gene expression. TriMeH3K4 (tri-methylated histone H3 lysine 4)
and TriMeH3K36 are associated with actively transcribed genes (Krogan et al.
2003; Li et al. 2002; Nishioka et al. 2002; Schaft et al. 2003; Wang et al. 2001).
In contrast to those active marks, MeH3K9 and MeH3K27 are marks associated
with a repressive state (Fischle et al. 2003; Lachner et al. 2003). Mono- and di-
methylation on H3K9 are related to facultative heterochromatin, whose formation
is developmentally regulated depending on cellular differentiation (Rice et al.
2003). TriMeH3K9 is associated with constitutive heterochromatin such as cen-
tromeric heterochromatin (Peters et al. 2003; Rice et al. 2003; Schotta et al. 2004).
TriMeH3K27 is associated with facultative heterochromatin. By genome-wide
mapping, it has been shown that TriMeH3K27 is associated with more than 1000
silenced genes, including HOX genes, which are repressed for proper embryonic
development and cell fate decisions (Bracken et al. 2006).
To understand whether histone modifications are involved in dedifferentiation
of PECs and dorsal selectivity of lens differentiation, changes in global histone
modification have been analyzed (Maki et al. 2010b). DiMeH3K9 and TriM-
eH3K9, which are marks for gene repression, are almost constant in both irises
during dedifferentiation. However, TriMeH3K27, which is also a mark for gene
repression, shows a significant difference between the dorsal and ventral iris during
dedifferentiation (Fig. 3). Although not much changes in the dorsal iris, the level
of TriMeH3K27 increases in the ventral iris. Because this modification is enriched
in the genes which should be repressed for proper development (Bracken et al.
2006), the up-regulation of TriMeH3K27 in the ventral iris suggests its partici-
pation in inhibition of lens formation from the ventral iris. TriMeH3K4, AcH3K9,
and AcH4 are histone modifications for gene activation. TriMeH3K4 and AcH4
(K5, 8, 12, 16) in the dedifferentiating cell are increased in both irises toward to
day 8. In contrast to these modifications, AcH3K9 is decreased during the dedif-
ferentiation in both irises (Fig. 3). Those facts suggest that each histone modifi-
cation for gene activation is independently regulated during dedifferentiation of
244 N. Maki and H. Kimura

Fig. 3 Summary of changes in global histone modifications of PEC during dedifferentiation in

lens regeneration

PEC. The increasing of TriMeK4 and AcH4 could be related to gene activation for
cell cycle reentry and reprogramming of cellular fate during dedifferentiation. The
decrease of AcH3K9 is an interesting point. It should be noted that Di- and
TriMeH3K9 do not change at the same time suggesting that a modification state of
H3K9 is not repressive. The combination of such histone modifications, increasing
levels of TriMeK4 and AcH4 and decreasing levels of AcH3K9, could be a
hallmark of the chromatin state during newt dedifferentiation.
Bivalent histone modification with TriMeH3K27 and TriMeH3K4 is a
remarkable feature of the ES cell. The comprehensive analysis of histone mod-
ifications shows that a vast majority of genes modified with TriMeH3K27, a
repressive mark, are co-modified with TriMeH3K4, an active mark, in ES cells and
that the co-modified fraction is enriched in genes that function during development
(Azuara et al. 2006; Bernstein et al. 2006; Mikkelsen et al. 2007; Pan et al. 2007;
Zhao et al. 2007). The bivalent histone modification is thought to poise genes for
later activation, while keep them inactivated (Bernstein et al. 2006). It has been
reported that in intact zebrafish developmental regulatory genes are silenced by
the bivalent modifications and the silenced genes are activated by loss of
TriMeH3K27 modification in the fin regeneration (Stewart et al. 2009). However,
during newt lens regeneration, the bivalent modification is not observed. This
might be due to a difference in the mode of regeneration between dedifferentiation
versus stem cell differentiation. Recently, it has been demonstrated that the
zebrafish heart is regenerated by dedifferentiation of cardiomyocytes using a Cre/
lox system (Jopling et al. 2010). Thus, histone modifications during dediffer-
entiation in different regenerative animals could be investigated in the future.

3.2 Oocyte-Type Linker Histone B4

Linker histones are classified into four types, i.e., somatic-, oocyte-, testis-, and
erythrocyte-type linker histones, according to their cellular specificity and
sequence homology (Fig. 2c). Oocyte-type linker histones have been identified in
human (referred as H1oo or H1foo), mouse (H1oo, H1foo), cow (H1foo), newt
Epigenetics and Regeneration 245

Fig. 4 Oocyte-type linker histone B4 is required for newt lens transdifferentiation. a Detection
of B4 protein by Western blot analysis using B4 antibody or neutralized antibody with the
antigen. Lane 1, ovary; lane 2, dorsal iris 10 days after lentectomy. b Immunostaining of ovary
using B4 and H1 antibody. Bar, 200 lm. Note that germinal vesicle (GV) was stained by B4
antibody and the nucleus of follicle cells was stained by H1 antibody c immunostaining of iris
during lens regeneration. Bar, 20 lm. Note that the staining intensities of each panel are not
comparative because images were processed to show nuclear distribution of each protein.
d Changes in the ratio of B4 to histone H1 during lens regeneration. After immunostaining, the
intensities of B4 and H1 signals in each nucleus were measured, and the ratio of B4 to histone H1
was calculated. e Knocking down of B4 altered gene expression of key genes of lens
differentiation. Using a vivo-morpholino technique, the amount of B4 in dorsal iris during lens
regeneration decreased by nearly 50 %. In this condition, expression levels of structural and
regulatory genes in lens differentiation were analyzed by qPCR. The expression of each gene was
normalized with that of ribosomal protein L27. Asterisks indicate a significant difference at
p \ 0.0342, Students t test
246 N. Maki and H. Kimura

(B4), frog (B4, H1X), zebrafish (H1M), and sea urchin (cs-H1) (Cho and Wolffe
1994; Maki et al. 2010a; Mandl et al. 1997; McGraw et al. 2006; Ohsumi and
Katagiri 1991; Tanaka et al. 2001, 2003; Wibrand and Olsen 2002). The oocyte-
type linker histones are predominant linker histones during oogenesis and early
embryogenesis. After the onset of zygotic gene expression, oocyte-type linker
histone disappears in parallel with an initiation of somatic-type linker histone H1
expression.
Epigenetic reprogramming occurs after somatic cell nuclear transfer (SCNT)
into oocyte. During reprogramming, the somatic nucleus regains pluripotency to
differentiate into all the cell types in the animal (Gurdon et al. 1958; Wilmut et al.
1997; Wakayama et al. 1998). Following nuclear transfer, somatic-type linker
histone H1 is rapidly replaced by oocyte-type linker histone (Becker et al. 2005;
Gao et al. 2004; Teranishi et al. 2004). Incorporation of oocyte-type linker histone
into the nucleus is required for the reactivation of pluripotency genes such as Oct4
and Sox2 in reprogramming after SCNT (Jullien et al. 2010). Furthermore, in
assembled chromatin in vitro, B4 allows the chromatin to be remodeled by ATP-
dependent chromatin remodeling factor, whereas somatic-type histone H1 prevents
the remodeling (Saeki et al. 2005). Thus, oocyte-type linker histone has a func-
tional significance in chromatin remodeling and is required for the reprogramming
after SCNT.
Unlike other animals analyzed, only the newt expresses B4 in somatic cells
during lens regeneration (Fig. 4) (Maki et al. 2010a). After lens removal, B4 is
reactivated and incorporated into the nucleus of dedifferentiating PECs. The ratio
of B4H1 in dorsal iris PEC starts to increase 8 days after lentectomy. The ratio
reaches a peak at day 12, when the cells are still undifferentiated. On day 15, when
lens differentiation occurs, the ratio starts to decreases and reaches a basal level by
day 18. However, such a peak is not observed in the ventral iris. If B4 is knocked
down, the regenerated lens is considerably small because of inhibited proliferation
and induced apoptosis. Moreover, B4 knockdown represses gene expression of
pax6 and MafB, transcriptional factors in lens differentiation, and almost abolishes
expression of c-crystalline, a lens differentiation marker (Fig. 4). Thus, expression
of B4 in somatic cells is required in newt lens transdifferentiation and it is sug-
gested that reprogramming in the newt somatic cell during transdifferentiation and
in the oocyte after SCNT share common strategies.

4 Discussion

In this review, we have shown a dynamic change of core-histone modifications,

emergence of oocyte-type linker histone B4, and expression of stem cell factors
during newt lens transdifferentiation. Using those markers, the cellular state during
lens transdifferentiation can be dissected in detail. In fact, such changes have
modified the previous concepts of dedifferentiation during the process of lens
transdifferentiation. In the past, it has been thought that the reprogramming of PEC
Epigenetics and Regeneration 247

Fig. 5 Reprogramming in newt lens transdifferentiation. a Expression prolife of B4 and stem

cell factors in dorsal iris PECs during lens transdifferentiation. Note that these genes are activated
sequentially through lens transdifferentiation. b New model for reprogramming in newt lens
transdifferentiation. Thus far, it has been thought that the reprogramming in which differentiated
PEC change to stem cell-like cell, occurs only at an early step of lens regeneration (earl step
model). However, this model is not based on the gene expression profile in lens transdifferen-
tiation. Based on the expression profile of those genes, we propose a new model in which the
somatic nucleus is reprogrammed in a stepwise fashion through the lens transdifferentiation
process (whole step model)

is completed by about 8 days after lentectomy and that the reprogrammed cells
already have a restored ability for lens differentiation (Fig. 5a), since the cells have
lost the morphological characteristics of PEC and have re-entered the cell cycle. In
fact, however, those gene markers related to nuclear reprogramming are expressed
sequentially throughout lens transdifferentiation and not just limited to the period
prior to cell cycle re-entry. Especially, oocyte-type linker histone, which is
required for the reprogramming after SCNT, shows a peak of expression on day
12. Based on the expression profile of reprogramming-related genes, we propose a
248 N. Maki and H. Kimura

whole step reprogramming model during newt lens transdifferentiation

(Fig. 5b). In this model, the nucleus of PEC is reprogrammed in a stepwise fashion
through the transdifferentiation process.
Even though other animals cannot express oocyte-type linker histone in somatic
cells, newts can do this. It is of great interest to know how newts gained the ability
of to re-express B4 in somatic cells. Analysis of the newt B4 promoter might
answer this question. The B4 promoter in Xenopus laevis, which cannot express
B4 in somatic cells, has been analyzed (Cho and Wolffe 1994). Consistent with
oocyte expression of B4, two Y-box elements exist in the Xenopus B4 promoter.
The Y-box element interacts with trans-acting factors such as FRGY2, abundant
oocyte-specific trans-acting factor. One possible reason for newt B4 expression in
somatic cells is that FRGY2 or other factor(s), which can interact with Y-box, is
expressed during newt transdifferentiation. The other possibility is that some
element(s) for somatic expression is inserted in the newt B4 promoter. Under-
standing the mechanism of somatic B4 expression will not only shed light on
evolutional differences between regenerative and non-regenerative animals, but
could also be adapted to future regenerative medicine.

Acknowledgments We would like to thank Kiyoe Ura for critical reading and suggestions, and
Rinako Maki for making illustrations.

References

Abe S, Eguchi G (1977) An analysis of differentiative capacity of pigmented epithelial cells of

adult newt iris in clonal cell culture. Dev Growth Differ 19:309317
Agata K, Kobayashi H, Itoh Y, Mochii M, Sawada K, Eguchi G (1993) Genetic characterization
of the multipotent dedifferentiated state of pigmented epithelial cells in vitro. Development
118:10251030
Anderson RM, Bosch JA, Goll MG, Hesselson D, Dong PD, Shin D, Chi NC, Shin CH, Schlegel A,
Halpern M, Stainier DY (2009) Loss of Dnmt1 catalytic activity reveals multiple roles for
DNA methylation during pancreas development and regeneration. Dev Biol 334:213223
Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, Gouti M, Casanova M,
Warnes G, Merkenschlager M, Fisher AG (2006) Chromatin signatures of pluripotent cell
lines. Nat Cell Biol 8:532538
Baddoo M, Hill K, Wilkinson R, Gaupp D, Hughes C, Kopen GC, Phinney DG (2003)
Characterization of mesenchymal stem cells isolated from murine bone marrow by negative
selection. J Cell Biochem 89:12351249
Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K (2007)
High-resolution profiling of histone methylations in the human genome. Cell 129:823837
Becker M, Becker A, Miyara F, Han Z, Kihara M, Brown DT, Hager GL, Latham K, Adashi EY,
Misteli T (2005) Differential in vivo binding dynamics of somatic and oocyte-specific linker
histones in oocytes and during ES cell nuclear transfer. Mol Biol Cell 16:38873895
Beekman C, Nichane M, De Clercq S, Maetens M, Floss T, Wurst W, Bellefroid E, Marine JC
(2006) Evolutionarily conserved role of nucleostemin: controlling proliferation of stem/
progenitor cells during early vertebrate development. Mol Cell Biol 26:92919301
Epigenetics and Regeneration 249

Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M,
Plath K, Jaenisch R, Wagschal A, Feil R, Schreiber SL, Lander ES (2006) A bivalent chromatin
structure marks key developmental genes in embryonic stem cells. Cell 125:315326
Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K (2006) Genome-wide mapping of
Polycomb target genes unravels their roles in cell fate transitions. Genes Dev 20:11231136
Cho H, Wolffe AP (1994) Xenopus laevis B4, an intron-containing oocyte-specific linker histone-
encoding gene. Gene 143:233238
Coulombre AJ (1965) The eye. In: DeHaan RL, Ursprung H (ed) Organogenesis. New Yolk:
Holt, Rinehart and Winston, pp 219251
Cowan CA, Atienza J, Melton DA, Eggan K (2005) Nuclear reprogramming of somatic cells after
fusion with human embryonic stem cells. Science 309:13691373
Dai MS, Sun XX, Lu H (2008) Aberrant expression of nucleostemin activates p53 and induces
cell cycle arrest via inhibition of MDM2. Mol Cell Biol 28:43654376
Eguchi G (1980) Lens regeneration: transdifferentiation of tissue cells. Iwanami shoten, Tokyo
Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse
embryos. Nature 292:154156
Fischle W, Wang Y, Allis CD (2003) Histone and chromatin cross-talk. Curr Opin Cell Biol
15:172183
Gao S, Chung YG, Parseghian MH, King GJ, Adashi EY, Latham KE (2004) Rapid H1 linker
histone transitions following fertilization or somatic cell nuclear transfer: evidence for a
uniform developmental program in mice. Dev Biol 266:6275
Grant PA, Eberharter A, John S, Cook RG, Turner BM, Workman JL (1999) Expanded lysine
acetylation specificity of Gcn5 in native complexes. J Biol Chem 274:58955900
Grogg MW, Call MK, Okamoto M, Vergara MN, Del Rio-Tsonis K, Tsonis PA (2005) BMP
inhibition-driven regulation of six-3 underlies induction of newt lens regeneration. Nature
438:858862
Guenther MG, Levine SS, Boyer LA, Jaenisch R, Young RA (2007) A chromatin landmark and
transcription initiation at most promoters in human cells. Cell 130:7788
Gurdon JB, Elsdale TR, Fischberg M (1958) Sexually mature individuals of Xenopus laevis from
the transplantation of single somatic nuclei. Nature 182:6465
Hayashi T, Mizuno N, Takada R, Takada S, Kondoh H (2006) Determinative role of Wnt signals
in dorsal iris-derived lens regeneration in newt eye. Mech Dev 123:793800
Hayashi T, Mizuno N, Ueda Y, Okamoto M, Kondoh H (2004) FGF2 triggers iris-derived lens
regeneration in newt eye. Mech Dev 121:519526
Jopling C, Sleep E, Raya M, Marti M, Raya A, Izpisua Belmonte JC (2010) Zebrafish heart
regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 464:606609
Jullien J, Astrand C, Halley-Stott RP, Garrett N, Gurdon JB (2010) Characterization of somatic
cell nuclear reprogramming by oocytes in which a linker histone is required for pluripotency
gene reactivation. Proc Natl Acad Sci U S A 107:54835488
Kornberg RD (1974) Chromatin structure: a repeating unit of histones and DNA. Science
184:868871
Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693705
Krogan NJ, Kim M, Tong A, Golshani A, Cagney G, Canadien V, Richards DP, Beattie BK,
Emili A, Boone C, Shilatifard A, Buratowski S, Greenblatt J (2003) Methylation of histone
H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA
polymerase II. Mol Cell Biol 23:42074218
Kuo MH, Brownell JE, Sobel RE, Ranalli TA, Cook RG, Edmondson DG, Roth SY, Allis CD
(1996) Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines.
Nature 383:269272
Lachner M, OSullivan RJ, Jenuwein T (2003) An epigenetic road map for histone lysine
methylation. J Cell Sci 116:21172124
Li J, Moazed D, Gygi SP (2002) Association of the histone methyltransferase Set2 with RNA
polymerase II plays a role in transcription elongation. J Biol Chem 277:4938349388
250 N. Maki and H. Kimura

Maki N, Suetsugu-Maki R, Sano S, Nakamura K, Nishimura O, Tarui H, Del Rio-Tsonis K,

Ohsumi K, Agata K, Tsonis PA (2010a) Oocyte-type linker histone B4 is required for
transdifferentiation of somatic cells in vivo. FASEB J 24:34623467
Maki N, Suetsugu-Maki R, Tarui H, Agata K, Del Rio-Tsonis K, Tsonis PA (2009) Expression of
stem cell pluripotency factors during regeneration in newts. Dev Dyn 238:16131616
Maki N, Takechi K, Sano S, Tarui H, Sasai Y, Agata K (2007) Rapid accumulation of
nucleostemin in nucleolus during newt regeneration. Dev Dyn 236:941950
Maki N, Tsonis PA, Agata K (2010b) Changes in global histone modifications during
dedifferentiation in newt lens regeneration. Mol Vis 16:18931897
Mandl B, Brandt WF, Superti-Furga G, Graninger PG, Birnstiel ML, Busslinger M (1997) The
five cleavage-stage (CS) histones of the sea urchin are encoded by a maternally expressed
family of replacement histone genes: functional equivalence of the CS H1 and frog H1 M
(B4) proteins. Mol Cell Biol 17:11891200
Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in
medium conditioned by teratocarcinoma stem cells. In: Proceedings of the national academy
of sciences of the United States of America, vol 78. pp 76347638
McGraw S, Vigneault C, Tremblay K, Sirard MA (2006) Characterization of linker histone
H1FOO during bovine in vitro embryo development. Mol Reprod Dev 73:692699
Mendenhall EM, Bernstein BE (2008) Chromatin state maps: new technologies, new insights.
Curr Opin Genet Dev 18:109115
Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W,
Kim TK, Koche RP, Lee W, Mendenhall E, ODonovan A, Presser A, Russ C, Xie X,
Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander ES, Bernstein BE (2007) Genome-
wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448:553560
Nikpour P, Mowla SJ, Jafarnejad SM, Fischer U, Schulz WA (2009) Differential effects of
Nucleostemin suppression on cell cycle arrest and apoptosis in the bladder cancer cell lines
5637 and SW1710. Cell Prolif 42:762769
Nishioka K, Chuikov S, Sarma K, Erdjument-Bromage H, Allis CD, Tempst P, Reinberg D
(2002) Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding
histone tail modifications required for heterochromatin formation. Genes Dev 16:479489
Ohmura M, Naka K, Hoshii T, Muraguchi T, Shugo H, Tamase A, Uema N, Ooshio T, Arai F,
Takubo K, Nagamatsu G, Hamaguchi I, Takagi M, Ishihara M, Sakurada K, Miyaji H, Suda T,
Hirao A (2008) Identification of stem cells during prepubertal spermatogenesis via monitoring
of nucleostemin promoter activity. Stem cells 26:32373246
Ohsumi K, Katagiri C (1991) Occurrence of H1 subtypes specific to pronuclei and cleavage-stage
cell nuclei of anuran amphibians. Dev Biol 147:110120
Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent
stem cells. Nature 448:313317
Pan G, Tian S, Nie J, Yang C, Ruotti V, Wei H, Jonsdottir GA, Stewart R, Thomson JA (2007)
Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic
stem cells. Cell Stem Cell 1:299312
Peters AH, Kubicek S, Mechtler K, OSullivan RJ, Derijck AA, Perez-Burgos L, Kohlmaier A,
Opravil S, Tachibana M, Shinkai Y, Martens JH, Jenuwein T (2003) Partitioning and
plasticity of repressive histone methylation states in mammalian chromatin. Mol Cell
12:15771589
Rice JC, Briggs SD, Ueberheide B, Barber CM, Shabanowitz J, Hunt DF, Shinkai Y, Allis CD
(2003) Histone methyltransferases direct different degrees of methylation to define distinct
chromatin domains. Mol Cell 12:15911598
Romanova L, Grand A, Zhang L, Rayner S, Katoku-Kikyo N, Kellner S, Kikyo N (2009) Critical
role of nucleostemin in pre-rRNA processing. J Biol Chem 284:49684977
Rundlett SE, Carmen AA, Kobayashi R, Bavykin S, Turner BM, Grunstein M (1996) HDA1 and
RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and
transcription. Proc Natl Acad Sci USA 93:1450314508
Epigenetics and Regeneration 251

Ruthenburg AJ, Li H, Patel DJ, Allis CD (2007) Multivalent engagement of chromatin

modifications by linked binding modules. Nat Rev Mol Cell Biol 8:983994
Sadler KC, Krahn KN, Gaur NA, Ukomadu C (2007) Liver growth in the embryo and during liver
regeneration in zebrafish requires the cell cycle regulator, uhrf1. In: Proceedings of the
national academy of sciences of the United States of America, vol 104. pp 15701575
Saeki H, Ohsumi K, Aihara H, Ito T, Hirose S, Ura K, Kaneda Y (2005) Linker histone variants
control chromatin dynamics during early embryogenesis. Proc Natl Acad Sci USA 102:
56975702
Schaft D, Roguev A, Kotovic KM, Shevchenko A, Sarov M, Neugebauer KM, Stewart AF (2003)
The histone 3 lysine 36 methyltransferase, SET2, is involved in transcriptional elongation.
Nucleic Acids Res 31:24752482
Schiltz RL, Mizzen CA, Vassilev A, Cook RG, Allis CD, Nakatani Y (1999) Overlapping but
distinct patterns of histone acetylation by the human coactivators p300 and PCAF within
nucleosomal substrates. J Biol Chem 274:11891192
Schotta G, Lachner M, Sarma K, Ebert A, Sengupta R, Reuter G, Reinberg D, Jenuwein T (2004)
A silencing pathway to induce H3K9 and H4K20 trimethylation at constitutive
heterochromatin. Genes Dev 18:12511262
Shahbazian MD, Grunstein M (2007) Functions of site-specific histone acetylation and
deacetylation. Annu Rev Biochem 76:75100
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai
SY, Tsai MJ, OMalley BW (1997) Steroid receptor coactivator-1 is a histone
acetyltransferase. Nature 389:194198
Stewart S, Tsun ZY, Belmote JC (2009) A histone demethylase is necessary for regeneration in
zebrafish. Proc Natl Acad Sci U S A 106:1988919894
Tada M, Takahama Y, Abe K, Nakatsuji N, Tada T (2001) Nuclear reprogramming of somatic
cells by in vitro hybridization with ES cells. Curr Biol: CB 11:15531558
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007)
Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell
131:861872
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and
adult fibroblast cultures by defined factors. Cell 126:663676
Tanaka M, Hennebold JD, Macfarlane J, Adashi EY (2001) A mammalian oocyte-specific linker
histone gene H1oo: homology with the genes for the oocyte-specific cleavage stage histone
(cs-H1) of sea urchin and the B4/H1 M histone of the frog. Development 128:655664
Tanaka Y, Kato S, Tanaka M, Kuji N, Yoshimura Y (2003) Structure and expression of the
human oocyte-specific histone H1 gene elucidated by direct RT-nested PCR of a single
oocyte. Biochem Biophys Res Commun 304:351357
Teranishi T, Tanaka M, Kimoto S, Ono Y, Miyakoshi K, Kono T, Yoshimura Y (2004) Rapid
replacement of somatic linker histones with the oocyte-specific linker histone H1foo in
nuclear transfer. Dev Biol 266:7686
Tsai RY, McKay RD (2002) A nucleolar mechanism controlling cell proliferation in stem cells
and cancer cells. Genes Dev 16:29913003
Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R (1998) Full-term development
of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394:369374
Wang H, Cao R, Xia L, Erdjument-Bromage H, Borchers C, Tempst P, Zhang Y (2001)
Purification and functional characterization of a histone H3-lysine 4-specific methyltransfer-
ase. Mol Cell 8:12071217
Wang Z, Zang C, Rosenfeld JA, Schones DE, Barski A, Cuddapah S, Cui K, Roh TY, Peng W,
Zhang MQ, Zhao K (2008) Combinatorial patterns of histone acetylations and methylations in
the human genome. Nat Genet 40:897903
Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE,
Jaenisch R (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.
Nature 448:318324
252 N. Maki and H. Kimura

Wibrand K, Olsen LC (2002) Linker histone H1 M transcripts mark the developing germ line in
zebrafish. Mech Dev 117:249252
Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH (1997) Viable offspring derived from
fetal and adult mammalian cells. Nature 385:810813
Yakushiji N, Suzuki M, Satoh A, Sagai T, Shiroishi T, Kobayashi H, Sasaki H, Ide H, Tamura K
(2007) Correlation between Shh expression and DNA methylation status of the limb-specific
Shh enhancer region during limb regeneration in amphibians. Dev Biol 312:171182
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA,
Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived
from human somatic cells. Science 318:19171920
Zhao XD, Han X, Chew JL, Liu J, Chiu KP, Choo A, Orlov YL, Sung WK, Shahab A, Kuznetsov VA,
Bourque G, Oh S, Ruan Y, Ng HH, Wei CL (2007) Whole-genome mapping of histone H3 Lys4
and 27 trimethylations reveals distinct genomic compartments in human embryonic stem cells.
Cell Stem Cell 1:286298