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1.  Tackling Skeletal Muscle Cells Epigenome in the Next-Generation Sequencing Era 
Recent advances in high-throughput technologies have transformed methodologies employed to study cell-specific epigenomes and the approaches to investigate complex cellular phenotypes. Application of next-generation sequencing technology in the skeletal muscle differentiation field is rapidly extending our knowledge on how chromatin modifications, transcription factors and chromatin regulators orchestrate gene expression pathways guiding myogenesis. Here, we review recent biological insights gained by the application of next-generation sequencing techniques to decode the epigenetic profile and gene regulatory networks underlying skeletal muscle differentiation.
doi:10.1155/2012/979168
PMCID: PMC3371680  PMID: 22701348
2.  TNF/p38 alpha/Polycomb signalling to Pax7 locus in satellite cells links inflammation to the epigenetic control of muscle regeneration 
Cell stem cell  2010;7(4):455-469.
How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified a novel inflammation-activated signalling in muscle stem (satellite) cells, by which the Polycomb Repressive Complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38alpha kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EzH2, the enzymatic sub-unit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. Anti-TNF antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signalling – p38alpha and EzH2 - invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signalling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signalling to Pax7 and satellite cell decision to proliferate or differentiate.
doi:10.1016/j.stem.2010.08.013
PMCID: PMC2951277  PMID: 20887952
Pax7; p38; muscle stem (satellite) cells; regeneration; chromatin; Polycomb complex
3.  Phosphoryl-EZH-ion 
Cell stem cell  2011;8(3):262-265.
Polycomb group (PcG) proteins regulate gene expression in embryonic and adult stem cells, but the mechanisms responsible for PcG gene targeting and regulation remain largely unknown. Recent evidence shows that EZH2, the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), is a nuclear phosphoprotein linking cell-cycle-intrinsic or extracellular signals to specific epigenetic signatures.
doi:10.1016/j.stem.2011.02.012
PMCID: PMC3166223  PMID: 21362566
4.  Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity 
Molecular Biology of the Cell  2008;19(12):5203-5213.
NF-Y binds to CCAAT motifs in the promoter region of a variety of genes involved in cell cycle progression. The NF-Y complex comprises three subunits, NF-YA, -YB, and -YC, all required for DNA binding. Expression of NF-YA fluctuates during the cell cycle and is down-regulated in postmitotic cells, indicating its role as the regulatory subunit of the complex. Control of NF-YA accumulation is posttranscriptional, NF-YA mRNA being relatively constant. Here we show that the levels of NF-YA protein are regulated posttranslationally by ubiquitylation and acetylation. A NF-YA protein carrying four mutated lysines in the C-terminal domain is more stable than the wild-type form, indicating that these lysines are ubiquitylated Two of the lysines are acetylated in vitro by p300, suggesting a competition between ubiquitylation and acetylation of overlapping residues. Interestingly, overexpression of a degradation-resistant NF-YA protein leads to sustained expression of mitotic cyclin complexes and increased cell proliferation, indicating that a tight regulation of NF-YA levels contributes to regulate NF-Y activity.
doi:10.1091/mbc.E08-03-0295
PMCID: PMC2592644  PMID: 18815279
5.  Mechanisms underlying the transcriptional regulation of skeletal myogenesis 
During skeletal myogenesis, chromatin-modifying enzymes are engaged at discrete genomic regions by transcription factors that recognize sequence-specific DNA motifs located at muscle gene regulatory regions. The composition of the chromatin-bound protein complexes and their temporally and spatially regulated recruitment influence gene expression. Recent findings are consistent with the concept that chromatin modifiers play an important role in regulating skeletal muscle gene expression and cellular differentiation.
doi:10.1016/j.gde.2005.04.015
PMCID: PMC1283108  PMID: 16055324
6.  Fgfr4 is required for effective muscle regeneration in vivo: Delineation of a MyoD-Tead2-Fgfr4 transcriptional pathway 
The Journal of biological chemistry  2005;281(1):429-438.
Fgfr4 has been shown to be important for appropriate muscle development in chick limb buds, however, Fgfr4 null mice show no phenotype. Here, we show that staged induction of muscle regeneration in Fgfr4 null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed. By 7 days of regeneration, differentiation of myotubes became poorly coordinated and delayed by both histology and embryonic myosin heavy chain staining. By 14 days, much of the muscle was replaced by fat and calcifications. To begin to dissect the molecular pathways involving Fgfr4, we queried the promoter sequences for transcriptional factor binding sites, and tested candidate regulators in a 27 time point regeneration series. The Fgfr4 promoter region contained a Tead protein binding site (M-CAT 5′-CATTCCT-3′), and Tead2 showed induction during regeneration commensurate with Fgfr4 regulation. Co-transfection of Tead2 and Fgfr4 promoter reporter constructs into C2C12 myotubes showed Tead2 to activate Fgfr4, and mutation of the M-CAT motif in the Fgfr4 promoter abolished these effects. Immunostaining for Tead2 showed timed expression in myotube nuclei consistent with the mRNA data. Query of the expression timing and genomic sequences of Tead2 suggested direct regulation by MyoD, and, consistent with this, MyoD directly bound to two strong E-boxes in the first intron of Tead2 by chromatin immunoprecipitation assay. Moreover, co-transfection of MyoD and Tead2 intron reporter constructs into 10T1/2 cells activated reporter activity in a dose dependent manner. This activation was greatly reduced when the two E-boxes were mutated. Our data suggest a novel MyoD-Tead2-Fgfr4 pathway important for effective muscle regeneration.
doi:10.1074/jbc.M507440200
PMCID: PMC1892582  PMID: 16267055
Muscle regeneration; Tead; TEF; Fgfr; MyoD; Microarray
7.  Transcriptional Activation of the Cyclin A Gene by the Architectural Transcription Factor HMGA2 
Molecular and Cellular Biology  2003;23(24):9104-9116.
The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120E4F, interfering with p120E4F binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation.
doi:10.1128/MCB.23.24.9104-9116.2003
PMCID: PMC309667  PMID: 14645522
8.  Cloning and characterization of the histone-fold proteins YBL1 and YCL1 
Nucleic Acids Research  2000;28(19):3830-3838.
Histones are among the most conserved proteins in evolution, sharing a histone fold motif. A number of additional histonic proteins exist and are involved in the process of transcriptional regulation. We describe here the identification, cloning and characterization of two small members of the H2A–H2B sub-family (YBL1 and YCL1) related to the NF-YB and NF-YC subunits of the CCAAT-binding activator NF-Y and to the TATA-binding protein (TBP) binding repressor NC2. Unlike the latters, YBL1 and YCL1 have no intrinsic CCAAT or TATA-binding capacity. In nucleosome reconstitution assays, they can form complexes with histones in solution and on DNA and they are part of relatively large complexes, as determined by glycerol gradient experiments. Our data support the idea that YBL1 and YCL1 are divergent with respect to NF-YB and NF-YC for specific functions, but have coevolved the capacity to interact with nucleosomal structures.
PMCID: PMC110757  PMID: 11000277
9.  A Functionally Essential Domain of RFX5 Mediates Activation of Major Histocompatibility Complex Class II Promoters by Promoting Cooperative Binding between RFX and NF-Y 
Molecular and Cellular Biology  2000;20(10):3364-3376.
Major histocompatibility complex class II (MHC-II) molecules occupy a pivotal position in the adaptive immune system, and correct regulation of their expression is therefore of critical importance for the control of the immune response. Several regulatory factors essential for the transcription of MHC-II genes have been identified by elucidation of the molecular defects responsible for MHC-II deficiency, a hereditary immunodeficiency disease characterized by regulatory defects abrogating MHC-II expression. Three of these factors, RFX5, RFXAP, and RFXANK, combine to form the RFX complex, a regulatory protein that binds to the X box DNA sequence present in all MHC-II promoters. In this study we have undertaken a dissection of the structure and function of RFX5, the largest subunit of the RFX complex. The results define two distinct domains serving two different essential functions. A highly conserved N-terminal region of RFX5 is required for its association with RFXANK and RFXAP, for assembly of the RFX complex in vivo and in vitro, and for binding of this complex to its X box target site in the MHC-II promoter. This N-terminal region is, however, not sufficient for activation of MHC-II expression. This requires an additional domain within the C-terminal region of RFX5. This C-terminal domain mediates cooperative binding between the RFX complex and NF-Y, a transcription factor binding to the Y box sequence of MHC-II promoters. This provides direct evidence that RFX5-mediated cooperative binding between RFX and NF-Y plays an essential role in the transcriptional activation of MHC-II genes.
PMCID: PMC85629  PMID: 10779326
10.  NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms 
Molecular and Cellular Biology  1999;19(12):8591-8603.
NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB–NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB–NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB–NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB–NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.
PMCID: PMC84987  PMID: 10567583

Results 1-10 (10)