Background: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting... more Background: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. Results: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina.
Background The high-mobility group Hmga family of proteins are non-histone chromatin-interacting ... more Background The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. Results We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place ...
The development of therapeutic approaches based on direct cardiac reprogramming of fibroblasts in... more The development of therapeutic approaches based on direct cardiac reprogramming of fibroblasts into induced-cardiomyocytes (iCM) has emerged as an attractive strategy to repair the injured myocardium. The identification of the mechanisms driving lineage conversion represents a crucial step toward the development of new and more efficient regenerative strategies. To this aim, here we show that pre-treatment with the Bmi1 inhibitor PTC-209 is sufficient to increase the efficiency of Chemical-induced Direct Cardiac Reprogramming both in mouse embryonic fibroblasts and adult cardiac fibroblasts. PTC-209 induces an overall increase of spontaneously beating iCM at end-stage of reprogramming, expressing high levels of late cardiac markers Troponin T and myosin muscle light chain-2v. The inhibition of Bmi1 expression occurring upon PTC-209 pre-treatment was maintained throughout the reprogramming protocol, contributing to a significant gene expression de-regulation. RNA profiling revealed t...
Naïve pluripotent embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent distinct... more Naïve pluripotent embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent distinctive developmental stages, mimicking the pre- and the post-implantation events during the embryo development, respectively. The complex molecular mechanisms governing the transition from ESCs into EpiSCs are orchestrated by fluctuating levels of pluripotency transcription factors (Nanog, Oct4, etc.) and wide-ranging remodeling of the epigenetic landscape. Recent studies highlighted the pivotal role of microRNAs (miRNAs) in balancing the switch from self-renewal to differentiation of ESCs. Of note, evidence deriving from miRNA-based reprogramming strategies underscores the role of the non-coding RNAs in the induction and maintenance of the stemness properties. In this review, we revised recent studies concerning the functions mediated by miRNAs in ESCs, with the aim of giving a comprehensive view of the highly dynamic miRNA-mediated tuning, essential to guarantee cell cycle progression, pl...
HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity pe... more HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity per se, but are able to modify chromatin structure by interacting with the transcriptional machinery and thus negatively or positively regulate the transcription of several genes. They have been extensively studied in cancer where they are often found to be overexpressed but their functions under physiologic conditions have still not been completely addressed. Hmga1 and Hmga2 are expressed during the early stages of mouse development, whereas they are not detectable in most adult tissues. Hmga overexpression or knockout studies in mouse have pointed to a key function in the development of the embryo and of various tissues. HMGA proteins are expressed in embryonic stem cells and in some adult stem cells and numerous experimental data have indicated that they play a fundamental role in the maintenance of stemness and in the regulation of differentiation. In this review, we discuss available ...
Cardiovascular diseases represent the first cause of morbidity in Western countries, and chronic ... more Cardiovascular diseases represent the first cause of morbidity in Western countries, and chronic heart failure features a significant health care burden in developed countries. Efforts in the attempt of finding new possible strategies for the treatment of CHF yielded several approaches based on the use of stem cells. The discovery of direct cardiac reprogramming has unveiled a new approach to heart regeneration, allowing, at least in principle, the conversion of one differentiated cell type into another without proceeding through a pluripotent intermediate. First developed for cancer treatment, nanotechnology-based approaches have opened new perspectives in many fields of medical research, including cardiovascular research. Nanotechnology could allow the delivery of molecules with specific biological activity at a sustained and controlled rate in heart tissue, in a cell-specific manner. Potentially, all the mediators and structural molecules involved in the fibrotic process could be...
To describe a second hereditary sensory autonomic neuropathy type VI (HSAN-VI) family harboring 2... more To describe a second hereditary sensory autonomic neuropathy type VI (HSAN-VI) family harboring 2 novel heterozygous mutations in the dystonin (DST) gene and to evaluate their effect on neurons derived from induced pluripotent stem cells (iPSC). The family consisted of 3 affected siblings from nonconsanguineous healthy parents. All members underwent clinical and electrophysiologic evaluation and genetic analysis. Two patients underwent quantitative sensory testing (QST), cardiovascular reflexes, dynamic sweat test, and skin biopsy to evaluate somatic and autonomic cutaneous innervation and to get fibroblast cultures for developing iPSC-derived neurons. Onset occurred in the first decade, with painless and progressive mutilating distal ulcerations leading to amputation and joint deformity. Sensation to pain, touch, and vibration was reduced. Autonomic disturbances included hypohidrosis, pupillary abnormalities, and gastrointestinal and sexual dysfunction. Nerve conduction studies sho...
We report on very soft X-ray sources (VSSs) in M31. In a survey which was most sensitive to soft ... more We report on very soft X-ray sources (VSSs) in M31. In a survey which was most sensitive to soft sources in four 8 ′ × 8 ′ regions covered by Chandra's ACIS-S S3 CCD, we find 33 VSSs that appear to belong to M31. Fifteen VSSs have spectral characteristics mirroring the supersoft X-ray sources studied in the Magellanic Cloud and Milky Way (k T e f f ≤ 100 eV); we therefore call these "classical" supersoft sources, or simply supersoft sources (SSSs). Eighteen VSSs may either have small (< 10%) hard components, or slightly higher effective temperatures (but still < 350 eV). We refer to these VSSs as quasisoft sources (QSSs). While hot white dwarf models may apply to SSSs, the effective temperatures of QSSs are too high, unless, e.g., the radiation emanates from only a small portion of surface. Two of the SSSs were first detected and identified as such through ROSAT observations. One SSS and one QSS may be identified with symbiotics, and 2 SSSs with supernova remnants. Both SSSs and QSSs in the disk are found near star-forming regions, possibly indicating that they are young. VSSs in the outer disk and halo are likely to be old systems; in these regions, there are more QSSs than SSSs, which is opposite to what is found in fields closer to the galaxy center. The largest density of bright VSSs is in the bulge; some of the bulge sources are close enough to the nucleus to be remnants of the tidal disruption of a giant by the massive central black hole. By using Chandra data in combination with ROSAT and XMM observations, we find most VSSs to be highly variable, fading from or brightening toward detectability on time scales of months. There is evidence for VSSs with low luminosities (∼ 10 36 erg s −1).
The kinase suppressor of Ras 1 (KSR1) has a fundamental role in mitogenic signaling by scaffoldin... more The kinase suppressor of Ras 1 (KSR1) has a fundamental role in mitogenic signaling by scaffolding components of the Ras/MAP kinase pathway. In response to Ras activation, KSR1 assembles a tripartite kinase complex that optimally transfers signals generated at the cell membrane to activate ERK. We describe a novel mechanism of ERK attenuation based on ubiquitin-dependent proteolysis of KSR1. Stimulation of membrane receptors by hormones or growth factors induced KSR1 polyubiquitination, which paralleled a decline of ERK1/2 signaling. We identified praja2 as the E3 ligase that ubiquitylates KSR1. We showed that praja2-dependent regulation of KSR1 is involved in the growth of cancer cells and in the maintenance of undifferentiated pluripotent state in mouse embryonic stem cells. The dynamic interplay between the ubiquitin system and the kinase scaffold of the Ras pathway shapes the activation profile of the mitogenic cascade. By controlling KSR1 levels, praja2 directly affects compart...
Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryoni... more Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryonic development and tumorigenesis. It regulates gene expression through two different post-transcriptional mechanisms. The first one is based on the regulation of miRNA biogenesis, in particular that of the let-7 family, whose expression is suppressed by Lin28. Thus, loss of Lin28 leads to the upregulation of mRNAs that are targets of let-7 species. The second mechanism is based on the direct interaction of Lin28 with a large number of mRNAs, which results in the regulation of their translation. This second mechanism remains poorly understood. To address this issue, we purified high molecular weight complexes containing Lin28a in mouse embryonic stem cells (ESCs). Numerous proteins, co-purified with Lin28a, were identified by proteomic procedures and tested for their possible role in Lin28a-dependent regulation of the mRNA encoding DNA methyltransferase 3a (Dnmt3a). The results show that L...
Cell reprogramming can either refer to a direct conversion of a specialized cell into another or ... more Cell reprogramming can either refer to a direct conversion of a specialized cell into another or to a reversal of a somatic cell into an induced pluripotent stem cell (iPSC). It implies a peculiar modification of the epigenetic asset and gene regulatory networks needed for a new cell, to better fit the new phenotype of the incoming cell type. Cellular reprogramming also implies a metabolic rearrangement, similar to that observed upon tumorigenesis, with a transition from oxidative phosphorylation to aerobic glycolysis. The induction of a reprogramming process requires a nexus of signaling pathways, mixing a range of local and systemic information, and accumulating evidence points to the crucial role exerted by the Hippo pathway components Yes-Associated Protein (YAP) and Transcriptional Co-activator with PDZ-binding Motif (TAZ). In this review, we will first provide a synopsis of the Hippo pathway and its function during reprogramming and tissue regeneration, then we introduce the l...
The multigenic family of mammalian Fe65s encodes three highly similar proteins with the same modu... more The multigenic family of mammalian Fe65s encodes three highly similar proteins with the same modular organisation: a WW domain and two phosphotyrosine-binding domains. The PTB2 domain of these proteins binds to the cytosolic domains of the Alzheimer's β-amyloid precursor protein APP and related proteins APLP1 and APLP2, generating a highly redundant system that is hard to dissect by reverse genetics. By searching potential Fe65-like genes in the nematode Caenorhabditis elegans, we identified a single gene, feh-1 (Fe65 homolog-1), encoding a protein with a high sequence similarity to mammalian Fe65s. FEH-1 is also functionally related to mammalian orthologues;in fact its PTB2 domain binds to APL-1, the product of the C. elegansorthologue of APP. Staining with specific antibodies show that the neuromuscular structures of the pharynx are the sites in which FEH-1 is present at highest levels. Expression studies with reporters indicate that the feh-1 gene is also expressed by a subse...
The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its... more The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its related protein TAZ, mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and TAZ regulate a large number of target genes, acting as co-activators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis we identified two molecules which could have a role in the altered genome-wide methylation profile: the long non-coding RNA Ephemeron, whose rapid upregulation is crucial for ESCs transition into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Mar 5, 2016
Lin28 RNA-binding proteins play important roles in pluripotent stem cells, but the regulation of ... more Lin28 RNA-binding proteins play important roles in pluripotent stem cells, but the regulation of their expression and the mechanisms underlying their functions are still not definitively understood. Here we address the above-mentioned issues in the first steps of mouse embryonic stem cell (ESC) differentiation. We observed that the expression of Lin28 genes is transiently induced soon after the exit of ESCs from the naive ground state and that this induction is due to the Hmga2-dependent engagement of Otx2 with enhancers present at both Lin28 gene loci. These mechanisms are crucial for Lin28 regulation, as demonstrated by the abolishment of the Lin28 accumulation in Otx2 or Hmga2 knockout cells compared to the control cells. We have also found that Lin28 controls Hmga2 expression levels during ESC differentiation through a let-7-independent mechanism. Indeed, we found that Lin28 proteins bind a highly conserved element in the 3' UTR of Hmga2 mRNA, and this provokes a down-regula...
Background: A crucial event in the differentiation of mouse embryonic stem cells (ESCs) is the ex... more Background: A crucial event in the differentiation of mouse embryonic stem cells (ESCs) is the exit from the pluripotent ground state that leads to the acquisition of the 'primed' pluripotent phenotype, characteristic of the epiblast-like stem cells (EpiLCs). The transcription factors Oct4 and Otx2 play a key role in this phenomenon. In particular, Otx2 pioneers and activates new enhancers, which are silent in ESCs and which control the transcription of genes responsible for the acquisition of the EpiLC phenotype. An important point that remains to be addressed is the mechanism through which Otx2 engages the new enhancers and stably associates with them. Hmga2 is a member of the high-mobility group family of proteins, non-histone components of chromatin whose expression is high during embryogenesis and becomes low or undetectable in adults. Its high expression during embryogenesis suggests that Hmga2 fulfills important roles in development. Results: Here, we demonstrate that Hmga2 accumulates soon after the induction of ESC differentiation. Its suppression hampers the exit of ESCs from the pluripotent ground state and their differentiation into EpiLCs. Mechanistically, Hmga2 controls the differentiation process by cooperating with Otx2 in the pioneering of new enhancers. In Hmga2 null induced pluripotent stem cells we observe that Otx2 fails to regulate its target genes upon the induction of differentiation. Hmga2 associates to Otx2-bound loci in EpiLCs, and in Hmga2 KO cells Otx2 is unable to engage and activate the new enhancers, thus indicating that Hmga2 is required for the binding of Otx2 to its cis-elements. We find that this mechanism also operates on the Hmga2 gene, which is one of the targets of Otx2, thus indicating the existence of a positive feedback loop. Conclusions: Our findings reveal a novel mechanism necessary for the exit of ESCs from the pluripotent ground state. Upon the induction of ESC differentiation, Otx2 alone or in combination with Oct4 engages new enhancers, which are silent in undifferentiated ESCs. The Hmga2 gene is activated by Otx2 and Hmga2 protein binds to the enhancers targeted by Otx2, thus facilitating the engagement and/or the stable association of Otx2. Therefore, our results demonstrate that Hmga2 is a key element of the regulatory network that governs the exit of ESCs from the pluripotent ground state.
We previously demonstrated that Fe65 protein is one of the ligands of the cytoplasmic domain of β... more We previously demonstrated that Fe65 protein is one of the ligands of the cytoplasmic domain of β-amyloid precursor protein (APP). Another ligand of this molecule was recently identified; it is similar to Fe65, so it was named Fe65-like (Fe65L1). Herein we describe the cloning of another Fe65-like cDNA (Fe65L2), similar to Fe65 and to Fe65L1, which encodes a protein of approx. 50 kDa. Its cognate mRNA is expressed in various rat tissues, particularly in brain and testis. The three members of the Fe65 protein family share several structural and functional characteristics. The primary structures of the three proteins can be aligned in three regions corresponding to the protein-protein interaction domains of Fe65 [the protein-protein interaction domain containing two conserved tryptophan residues and the two phosphotyrosine interaction domain/phosphotyrosine binding (PID/PTB) domains], whereas the remaining sequences are poorly related. Like Fe65, Fe65L1 and Fe65L2 genes encode two dif...
Fe65 is an adaptor protein that interacts with the Alzheimer β-amyloid precursor protein and is e... more Fe65 is an adaptor protein that interacts with the Alzheimer β-amyloid precursor protein and is expressed mainly in the neurons of several regions of the nervous system. The FE65 gene has a TATA-less promoter that drives an efficient transcription in cells showing a neuronal phenotype, whereas its efficiency is poor in non-neuronal cells. A short sequence encompassing the transcription start site contains sufficient information to drive the transcription in neuronal cells but not in non-neural cells. Electrophoretic mobility-shift assays performed with rat brain nuclear extracts showed that three major DNA-protein complexes, named BI, BII and BIII, are formed by the FE65 minimal promoter. The proteins present in complexes BI and BII were purified from bovine brain; internal microsequencing of the purified proteins demonstrated that they corresponded to the previously isolated single-stranded-DNA-binding protein Pur α, abundantly expressed in the brain. In Chinese hamster ovary (CHO)...
Background: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting... more Background: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. Results: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina.
Background The high-mobility group Hmga family of proteins are non-histone chromatin-interacting ... more Background The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. Results We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place ...
The development of therapeutic approaches based on direct cardiac reprogramming of fibroblasts in... more The development of therapeutic approaches based on direct cardiac reprogramming of fibroblasts into induced-cardiomyocytes (iCM) has emerged as an attractive strategy to repair the injured myocardium. The identification of the mechanisms driving lineage conversion represents a crucial step toward the development of new and more efficient regenerative strategies. To this aim, here we show that pre-treatment with the Bmi1 inhibitor PTC-209 is sufficient to increase the efficiency of Chemical-induced Direct Cardiac Reprogramming both in mouse embryonic fibroblasts and adult cardiac fibroblasts. PTC-209 induces an overall increase of spontaneously beating iCM at end-stage of reprogramming, expressing high levels of late cardiac markers Troponin T and myosin muscle light chain-2v. The inhibition of Bmi1 expression occurring upon PTC-209 pre-treatment was maintained throughout the reprogramming protocol, contributing to a significant gene expression de-regulation. RNA profiling revealed t...
Naïve pluripotent embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent distinct... more Naïve pluripotent embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent distinctive developmental stages, mimicking the pre- and the post-implantation events during the embryo development, respectively. The complex molecular mechanisms governing the transition from ESCs into EpiSCs are orchestrated by fluctuating levels of pluripotency transcription factors (Nanog, Oct4, etc.) and wide-ranging remodeling of the epigenetic landscape. Recent studies highlighted the pivotal role of microRNAs (miRNAs) in balancing the switch from self-renewal to differentiation of ESCs. Of note, evidence deriving from miRNA-based reprogramming strategies underscores the role of the non-coding RNAs in the induction and maintenance of the stemness properties. In this review, we revised recent studies concerning the functions mediated by miRNAs in ESCs, with the aim of giving a comprehensive view of the highly dynamic miRNA-mediated tuning, essential to guarantee cell cycle progression, pl...
HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity pe... more HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity per se, but are able to modify chromatin structure by interacting with the transcriptional machinery and thus negatively or positively regulate the transcription of several genes. They have been extensively studied in cancer where they are often found to be overexpressed but their functions under physiologic conditions have still not been completely addressed. Hmga1 and Hmga2 are expressed during the early stages of mouse development, whereas they are not detectable in most adult tissues. Hmga overexpression or knockout studies in mouse have pointed to a key function in the development of the embryo and of various tissues. HMGA proteins are expressed in embryonic stem cells and in some adult stem cells and numerous experimental data have indicated that they play a fundamental role in the maintenance of stemness and in the regulation of differentiation. In this review, we discuss available ...
Cardiovascular diseases represent the first cause of morbidity in Western countries, and chronic ... more Cardiovascular diseases represent the first cause of morbidity in Western countries, and chronic heart failure features a significant health care burden in developed countries. Efforts in the attempt of finding new possible strategies for the treatment of CHF yielded several approaches based on the use of stem cells. The discovery of direct cardiac reprogramming has unveiled a new approach to heart regeneration, allowing, at least in principle, the conversion of one differentiated cell type into another without proceeding through a pluripotent intermediate. First developed for cancer treatment, nanotechnology-based approaches have opened new perspectives in many fields of medical research, including cardiovascular research. Nanotechnology could allow the delivery of molecules with specific biological activity at a sustained and controlled rate in heart tissue, in a cell-specific manner. Potentially, all the mediators and structural molecules involved in the fibrotic process could be...
To describe a second hereditary sensory autonomic neuropathy type VI (HSAN-VI) family harboring 2... more To describe a second hereditary sensory autonomic neuropathy type VI (HSAN-VI) family harboring 2 novel heterozygous mutations in the dystonin (DST) gene and to evaluate their effect on neurons derived from induced pluripotent stem cells (iPSC). The family consisted of 3 affected siblings from nonconsanguineous healthy parents. All members underwent clinical and electrophysiologic evaluation and genetic analysis. Two patients underwent quantitative sensory testing (QST), cardiovascular reflexes, dynamic sweat test, and skin biopsy to evaluate somatic and autonomic cutaneous innervation and to get fibroblast cultures for developing iPSC-derived neurons. Onset occurred in the first decade, with painless and progressive mutilating distal ulcerations leading to amputation and joint deformity. Sensation to pain, touch, and vibration was reduced. Autonomic disturbances included hypohidrosis, pupillary abnormalities, and gastrointestinal and sexual dysfunction. Nerve conduction studies sho...
We report on very soft X-ray sources (VSSs) in M31. In a survey which was most sensitive to soft ... more We report on very soft X-ray sources (VSSs) in M31. In a survey which was most sensitive to soft sources in four 8 ′ × 8 ′ regions covered by Chandra's ACIS-S S3 CCD, we find 33 VSSs that appear to belong to M31. Fifteen VSSs have spectral characteristics mirroring the supersoft X-ray sources studied in the Magellanic Cloud and Milky Way (k T e f f ≤ 100 eV); we therefore call these "classical" supersoft sources, or simply supersoft sources (SSSs). Eighteen VSSs may either have small (< 10%) hard components, or slightly higher effective temperatures (but still < 350 eV). We refer to these VSSs as quasisoft sources (QSSs). While hot white dwarf models may apply to SSSs, the effective temperatures of QSSs are too high, unless, e.g., the radiation emanates from only a small portion of surface. Two of the SSSs were first detected and identified as such through ROSAT observations. One SSS and one QSS may be identified with symbiotics, and 2 SSSs with supernova remnants. Both SSSs and QSSs in the disk are found near star-forming regions, possibly indicating that they are young. VSSs in the outer disk and halo are likely to be old systems; in these regions, there are more QSSs than SSSs, which is opposite to what is found in fields closer to the galaxy center. The largest density of bright VSSs is in the bulge; some of the bulge sources are close enough to the nucleus to be remnants of the tidal disruption of a giant by the massive central black hole. By using Chandra data in combination with ROSAT and XMM observations, we find most VSSs to be highly variable, fading from or brightening toward detectability on time scales of months. There is evidence for VSSs with low luminosities (∼ 10 36 erg s −1).
The kinase suppressor of Ras 1 (KSR1) has a fundamental role in mitogenic signaling by scaffoldin... more The kinase suppressor of Ras 1 (KSR1) has a fundamental role in mitogenic signaling by scaffolding components of the Ras/MAP kinase pathway. In response to Ras activation, KSR1 assembles a tripartite kinase complex that optimally transfers signals generated at the cell membrane to activate ERK. We describe a novel mechanism of ERK attenuation based on ubiquitin-dependent proteolysis of KSR1. Stimulation of membrane receptors by hormones or growth factors induced KSR1 polyubiquitination, which paralleled a decline of ERK1/2 signaling. We identified praja2 as the E3 ligase that ubiquitylates KSR1. We showed that praja2-dependent regulation of KSR1 is involved in the growth of cancer cells and in the maintenance of undifferentiated pluripotent state in mouse embryonic stem cells. The dynamic interplay between the ubiquitin system and the kinase scaffold of the Ras pathway shapes the activation profile of the mitogenic cascade. By controlling KSR1 levels, praja2 directly affects compart...
Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryoni... more Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryonic development and tumorigenesis. It regulates gene expression through two different post-transcriptional mechanisms. The first one is based on the regulation of miRNA biogenesis, in particular that of the let-7 family, whose expression is suppressed by Lin28. Thus, loss of Lin28 leads to the upregulation of mRNAs that are targets of let-7 species. The second mechanism is based on the direct interaction of Lin28 with a large number of mRNAs, which results in the regulation of their translation. This second mechanism remains poorly understood. To address this issue, we purified high molecular weight complexes containing Lin28a in mouse embryonic stem cells (ESCs). Numerous proteins, co-purified with Lin28a, were identified by proteomic procedures and tested for their possible role in Lin28a-dependent regulation of the mRNA encoding DNA methyltransferase 3a (Dnmt3a). The results show that L...
Cell reprogramming can either refer to a direct conversion of a specialized cell into another or ... more Cell reprogramming can either refer to a direct conversion of a specialized cell into another or to a reversal of a somatic cell into an induced pluripotent stem cell (iPSC). It implies a peculiar modification of the epigenetic asset and gene regulatory networks needed for a new cell, to better fit the new phenotype of the incoming cell type. Cellular reprogramming also implies a metabolic rearrangement, similar to that observed upon tumorigenesis, with a transition from oxidative phosphorylation to aerobic glycolysis. The induction of a reprogramming process requires a nexus of signaling pathways, mixing a range of local and systemic information, and accumulating evidence points to the crucial role exerted by the Hippo pathway components Yes-Associated Protein (YAP) and Transcriptional Co-activator with PDZ-binding Motif (TAZ). In this review, we will first provide a synopsis of the Hippo pathway and its function during reprogramming and tissue regeneration, then we introduce the l...
The multigenic family of mammalian Fe65s encodes three highly similar proteins with the same modu... more The multigenic family of mammalian Fe65s encodes three highly similar proteins with the same modular organisation: a WW domain and two phosphotyrosine-binding domains. The PTB2 domain of these proteins binds to the cytosolic domains of the Alzheimer's β-amyloid precursor protein APP and related proteins APLP1 and APLP2, generating a highly redundant system that is hard to dissect by reverse genetics. By searching potential Fe65-like genes in the nematode Caenorhabditis elegans, we identified a single gene, feh-1 (Fe65 homolog-1), encoding a protein with a high sequence similarity to mammalian Fe65s. FEH-1 is also functionally related to mammalian orthologues;in fact its PTB2 domain binds to APL-1, the product of the C. elegansorthologue of APP. Staining with specific antibodies show that the neuromuscular structures of the pharynx are the sites in which FEH-1 is present at highest levels. Expression studies with reporters indicate that the feh-1 gene is also expressed by a subse...
The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its... more The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its related protein TAZ, mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and TAZ regulate a large number of target genes, acting as co-activators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis we identified two molecules which could have a role in the altered genome-wide methylation profile: the long non-coding RNA Ephemeron, whose rapid upregulation is crucial for ESCs transition into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Mar 5, 2016
Lin28 RNA-binding proteins play important roles in pluripotent stem cells, but the regulation of ... more Lin28 RNA-binding proteins play important roles in pluripotent stem cells, but the regulation of their expression and the mechanisms underlying their functions are still not definitively understood. Here we address the above-mentioned issues in the first steps of mouse embryonic stem cell (ESC) differentiation. We observed that the expression of Lin28 genes is transiently induced soon after the exit of ESCs from the naive ground state and that this induction is due to the Hmga2-dependent engagement of Otx2 with enhancers present at both Lin28 gene loci. These mechanisms are crucial for Lin28 regulation, as demonstrated by the abolishment of the Lin28 accumulation in Otx2 or Hmga2 knockout cells compared to the control cells. We have also found that Lin28 controls Hmga2 expression levels during ESC differentiation through a let-7-independent mechanism. Indeed, we found that Lin28 proteins bind a highly conserved element in the 3' UTR of Hmga2 mRNA, and this provokes a down-regula...
Background: A crucial event in the differentiation of mouse embryonic stem cells (ESCs) is the ex... more Background: A crucial event in the differentiation of mouse embryonic stem cells (ESCs) is the exit from the pluripotent ground state that leads to the acquisition of the 'primed' pluripotent phenotype, characteristic of the epiblast-like stem cells (EpiLCs). The transcription factors Oct4 and Otx2 play a key role in this phenomenon. In particular, Otx2 pioneers and activates new enhancers, which are silent in ESCs and which control the transcription of genes responsible for the acquisition of the EpiLC phenotype. An important point that remains to be addressed is the mechanism through which Otx2 engages the new enhancers and stably associates with them. Hmga2 is a member of the high-mobility group family of proteins, non-histone components of chromatin whose expression is high during embryogenesis and becomes low or undetectable in adults. Its high expression during embryogenesis suggests that Hmga2 fulfills important roles in development. Results: Here, we demonstrate that Hmga2 accumulates soon after the induction of ESC differentiation. Its suppression hampers the exit of ESCs from the pluripotent ground state and their differentiation into EpiLCs. Mechanistically, Hmga2 controls the differentiation process by cooperating with Otx2 in the pioneering of new enhancers. In Hmga2 null induced pluripotent stem cells we observe that Otx2 fails to regulate its target genes upon the induction of differentiation. Hmga2 associates to Otx2-bound loci in EpiLCs, and in Hmga2 KO cells Otx2 is unable to engage and activate the new enhancers, thus indicating that Hmga2 is required for the binding of Otx2 to its cis-elements. We find that this mechanism also operates on the Hmga2 gene, which is one of the targets of Otx2, thus indicating the existence of a positive feedback loop. Conclusions: Our findings reveal a novel mechanism necessary for the exit of ESCs from the pluripotent ground state. Upon the induction of ESC differentiation, Otx2 alone or in combination with Oct4 engages new enhancers, which are silent in undifferentiated ESCs. The Hmga2 gene is activated by Otx2 and Hmga2 protein binds to the enhancers targeted by Otx2, thus facilitating the engagement and/or the stable association of Otx2. Therefore, our results demonstrate that Hmga2 is a key element of the regulatory network that governs the exit of ESCs from the pluripotent ground state.
We previously demonstrated that Fe65 protein is one of the ligands of the cytoplasmic domain of β... more We previously demonstrated that Fe65 protein is one of the ligands of the cytoplasmic domain of β-amyloid precursor protein (APP). Another ligand of this molecule was recently identified; it is similar to Fe65, so it was named Fe65-like (Fe65L1). Herein we describe the cloning of another Fe65-like cDNA (Fe65L2), similar to Fe65 and to Fe65L1, which encodes a protein of approx. 50 kDa. Its cognate mRNA is expressed in various rat tissues, particularly in brain and testis. The three members of the Fe65 protein family share several structural and functional characteristics. The primary structures of the three proteins can be aligned in three regions corresponding to the protein-protein interaction domains of Fe65 [the protein-protein interaction domain containing two conserved tryptophan residues and the two phosphotyrosine interaction domain/phosphotyrosine binding (PID/PTB) domains], whereas the remaining sequences are poorly related. Like Fe65, Fe65L1 and Fe65L2 genes encode two dif...
Fe65 is an adaptor protein that interacts with the Alzheimer β-amyloid precursor protein and is e... more Fe65 is an adaptor protein that interacts with the Alzheimer β-amyloid precursor protein and is expressed mainly in the neurons of several regions of the nervous system. The FE65 gene has a TATA-less promoter that drives an efficient transcription in cells showing a neuronal phenotype, whereas its efficiency is poor in non-neuronal cells. A short sequence encompassing the transcription start site contains sufficient information to drive the transcription in neuronal cells but not in non-neural cells. Electrophoretic mobility-shift assays performed with rat brain nuclear extracts showed that three major DNA-protein complexes, named BI, BII and BIII, are formed by the FE65 minimal promoter. The proteins present in complexes BI and BII were purified from bovine brain; internal microsequencing of the purified proteins demonstrated that they corresponded to the previously isolated single-stranded-DNA-binding protein Pur α, abundantly expressed in the brain. In Chinese hamster ovary (CHO)...
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Papers by Tommaso Russo