Rossella Maione

The discovery of the skeletal muscle-specific transcription factor MyoD represents a milestone in the field of transcriptional regulation during differentiation and cell-fate reprogramming. MyoD was the first tissue-specific factor found capable of converting non-muscle somatic cells into skeletal muscle cells. A unique feature of MyoD, with respect to other lineage-specific factors able to drive trans-differentiation processes, is its ability to dramatically change the cell fate even when expressed alone. The present review will outline the molecular strategies by which MyoD reprograms the transcriptional regulation of the cell of origin during the myogenic conversion, focusing on the activation and coordination of a complex network of co-factors and epigenetic mechanisms. Some molecular roadblocks, found to restrain MyoD-dependent trans-differentiation, and the possible ways for overcoming these barriers, will also be discussed. Indeed, they are of critical importance not only to expand our knowledge of basic muscle biology but also to improve the generation skeletal muscle cells for translational research.

The growing knowledge on several classes of non-coding RNAs (ncRNAs) and their different functional roles has aroused great interest in the scientific community. Beyond the Central Dogma of Biology, it is clearly known that not all RNAs code for protein products, and they exert a broader repertoire of biological functions. As described in this review, ncRNAs participate in gene expression regulation both at transcriptional and post-transcriptional levels and represent critical elements driving and controlling pathophysiological processes in multicellular organisms. For this reason, in recent years, a great boost was given to ncRNA-based strategies with potential therapeutic abilities, and nowadays, the use of RNA molecules is experimentally validated and actually exploited in clinics to counteract several diseases. In this review, we summarize the principal classes of therapeutic ncRNA molecules that are potentially implied in disease onset and progression, which are already used in clinics or under clinical trials, highlighting the advantages and the need for a targeted therapeutic strategy design. Furthermore, we discuss the benefits and the limits of RNA therapeutics and the ongoing development of delivery strategies to limit the off-target effects and to increase the translational application.

The myogenic factor MyoD regulates skeletal muscle differentiation by interacting with a variety of chromatin-modifying complexes. Although MyoD can induce and maintain chromatin accessibility at its target genes, its binding and trans-activation ability can be limited by some types of not fully characterized epigenetic constraints. In this work we analysed the role of PARP1 in regulating MyoD-dependent gene expression. PARP1 is a chromatin-associated enzyme, playing a well recognized role in DNA repair and that is implicated in transcriptional regulation. PARP1 affects gene expression through multiple mechanisms, often involving the Poly(ADP-ribosyl)ation of chromatin proteins. In line with PARP1 down-regulation during differentiation, we observed that PARP1 depletion boosts the up-regulation of MyoD targets, such as p57, myogenin, Mef2C and p21, while its re-expression reverts this effect. We also found that PARP1 interacts with some MyoD-binding regions and that its presence, independently of the enzymatic activity, interferes with MyoD recruitment and gene induction. We finally suggest a relationship between the binding of PARP1 and the loss of the activating histone modification H3K4me3 at MyoD-binding regions. This work highlights not only a novel player in the epigenetic control of myogenesis, but also a repressive and catalytic-independent mechanisms by which PARP1 regulates transcription.

Revealing the interactions of long noncoding RNAs (LncRNAs) with specific genomic regions is of basic importance to explore the mechanisms by which they regulate gene expression. Chromatin oligo-affinity precipitation (ChOP) technique was the first method developed to analyze the association of LncRNAs with genomic regions in the chromatin context. The first step of the procedure is cell cross-linking, aimed at stabilizing the RNA–protein–DNA complexes. Next, after chromatin fragmentation, the RNA complexes are pulled down through hybridization with antisense oligonucleotides tagged with biotin and purification with anti-biotin antibody. After extensive wash, the RNA-interacting chromatin is eluted by RNase treatment. Subsequent protein elimination and DNA purification allow to retrieve DNA fragments for following analyses such as qPCR or sequencing. In the present chapter, we describe the ChOP protocol, as used in our laboratory for investigating the interaction of the LncRNA Kcnq1ot1 with chromatin at specific regulatory regions of the Cdkn1c locus.

The cell-cycle inhibitor p57kip2 plays a critical role in mammalian development by coordinating cell proliferation and differentiation in many cell types. p57kip2 expression is finely regulated by several epigenetic mechanisms, including paternal imprinting. Kcnq1ot1, a long non-coding RNA (LncRNA), whose gene maps to the p57Kip2 imprinting domain, is expressed exclusively from the paternal allele and participates in the cis-silencing of the neighboring imprinted genes through chromatin-level regulation. In light of our previous evidence of a functional interaction between myogenic factors and imprinting control elements in the regulation of the maternal p57Kip2 allele during muscle differentiation, we examined the possibility that also Kcnq1ot1 could play an imprinting-independent role in the control of p57Kip2 expression in muscle cells. We found that Kcnq1ot1 depletion by siRNA causes the upregulation of the maternal and functional p57Kip2 allele during differentiation, suggesting a previously undisclosed role for this LncRNA. Consistently, Chromatin Oligo-affinity Precipitation assays showed that Kcnq1ot1 physically interacts not only with the paternal imprinting control region of the locus, as already known, but also with both maternal and paternal alleles of a novel p57Kip2 regulatory region, located intragenically and containing two binding sites for the muscle-specific factor MyoD. Moreover, chromatin immunoprecipitation assays after Kcnq1ot1 depletion demonstrated that the LncRNA is required for the accumulation of H3K27me3, a chromatin modification catalyzed by the histone-methyl-transferase EZH2, at the maternal p57kip2 intragenic region. Finally, upon differentiation, the binding of MyoD to this region and its physical interaction with Kcnq1ot1, analyzed by ChIP and RNA immunoprecipitation assays, correlate with the loss of EZH2 and H3K27me3 from chromatin and with p57Kip2 de-repression. These findings highlight the existence of an imprinting-independent role of Kcnq1ot1, adding new insights into the biology of a still mysterious LncRNA. Moreover, they expand our knowledge about the molecular mechanisms underlying the tight and fine regulation of p57Kip2 during differentiation and, possibly, its aberrant silencing observed in several pathologic conditions.

P57kip2 is the most complex member of the CIP/KIP family of cyclin-dependent kinase inhibitors and plays a fundamental role in regulating cell cycle and differentiation during mammalian development. Consistently with a key role for p57kip2 in the spatial and temporal control of cell proliferation, its expression is fine-tuned by multiple regulatory mechanisms, resulting in a tissue-, developmental phase- and cell type-specific pattern. Moreover, p57kip2 is an imprinted gene, further supporting the importance of its proper expression dosage. Importantly, misregulation of p57kip2 expression has been associated, more frequently than mutations in its coding region, to human growth disorders, such as Beckwith-Wiedemann and Silver-Russell syndromes, as well as to the onset of several types of cancers. This review will summarize the molecular mechanisms regulating p57kip2 transcription during differentiation and development, their relationship with the imprinting control and their alterations in growth-related diseases and cancer. Particular attention will be given to the role of epigenetic mechanisms, involving DNA methylation, histone modifications, long-range chromatin interactions and non-coding RNAs in modulating and integrating the functions of cis-regulatory elements and trans-acting factors.

The cdk inhibitor p57^kip2, encoded by the Cdkn1c gene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1c silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1c expression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1c induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1c promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1c is silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1c and, possibly, of its aberrant silencing in some pathological conditions.

Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation.

Higher-order chromatin structures appear to be dynamically arranged during development and differentiation. However, the molecular mechanism underlying their maintenance or disruption and their functional relevance in gene regulation are poorly understood. We recently described a dynamic long-range chromatin interaction between the gene-promoter of the cdk inhibitor p57kip2 and the imprinting control region KvDMR1 in muscle cells. Here we show that CTCF, the best characterized organizer of long-range chromatin interactions, binds to both p57kip2 promoter and KvDMR1 and is necessary for the maintenance of their physical contact. Moreover, we show that CTCF-mediated looping is required for preventing p57kip2 expression before differentiation. Finally, we provide evidence that the induction of p57kip2 during myogenesis involves the physical interaction of the muscle regulatory-factor MyoD with CTCF at KvDMR1, the displacement of the cohesin complex subunit Rad21 and the destabilization of the chromatin loop. The finding that MyoD affects chromatin looping at CTCF-binding sites represents the first evidence that a differentiation factor regulates chromatin loop dynamics and provides a useful paradigm for gaining insights into the developmental regulation of long-range chromatin contacts.

The bHLH transcription factor MyoD, the prototypical master regulator of differentiation, directs a complex program of gene expression during skeletal myogenesis. The up-regulation of the cdk inhibitor p57 ^kip2 plays a critical role in coordinating differentiation and growth arrest during muscle development, as well as in other tissues. p57 ^kip2 displays a highly specific expression pattern and is subject to a complex epigenetic control driving the imprinting of the paternal allele. However, the regulatory mechanisms governing its expression during development are still poorly understood. We have identified an unexpected mechanism by which MyoD regulates p57 ^kip2 transcription in differentiating muscle cells. We show that the induction of p57 ^kip2 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a chromatin loop involving p57 ^kip2 promoter. We also show that differentiation-dependent regulation of p57 ^kip2 , while involving a region implicated in the imprinting process, is distinct and hierarchically subordinated to the imprinting control. These findings highlight a novel mechanism, involving the modification of higher order chromatin structures, by which MyoD regulates gene expression. Our results also suggest that chromatin folding mediated by KvDMR1 could account for the highly restricted expression of p57 ^kip2 during development and, possibly, for its aberrant silencing in some pathologies.