MyoD regulates p57kip2 expression by interacting with a distant cis-element and modifying a higher order chromatin structure

Abstract

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 p57kip2 plays a critical role in coordinating differentiation and growth arrest during muscle development, as well as in other tissues. p57kip2 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 p57kip2 transcription in differentiating muscle cells. We show that the induction of p57kip2 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a chromatin loop involving p57kip2 promoter. We also show that differentiation-dependent regulation of p57kip2, 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 p57kip2 during development and, possibly, for its aberrant silencing in some pathologies.

Figure 1.

Expression of p57, Kcnq1 and Kcnq1ot1 during muscle differentiation. C2.7 myoblasts and MyoD-expressing fibroblasts were analyzed by qRT-PCR at different times (hours) after the shift to differentiation medium (h in DM). The expression levels are relative to the T0 value for each transcript. The results shown are representative of three independent experiments. Error bars indicate standard error of the mean for each sample analyzed in triplicate.

Figure 2.

MyoD interacts in vivo with KvDMR1. (a) Top: schematic diagram showing the reciprocal location of p57, Kcnq1 and Kcnq1ot1 genes and KvDMR1 region. Filled arrows indicate the direction of transcription. Dashed arrows point to an enlargement of the repressive KvDMR1 element previously described (31) extending from nt +1561 to +3420 of AF119385 sequence. The positions of the putative MyoD binding sites are also indicated. A further enlargement indicates the location of the three fragments (F1, F2 and F3) amplified in the ChIP assay shown in the bottom. Bottom: In vivo binding activity of MyoD during differentiation. Chromatin from C2.7 myoblasts and MyoD-expressing fibroblasts (responsive and unresponsive) kept either in growth (T0) or in differentiation medium for 24 h (T24) was immunoprecipitated using a specific antibody to MyoD or in the absence of antibody (NoAb). Input represents non-immunoprecipitated cross-linked chromatin. The indicated fragments were amplified by PCR as described in ‘Materials and Methods’ section. A p57 promoter fragment, which we have previously demonstrated not to be bound by MyoD (28), was used as a negative control. The results obtained with 30 amplification cycles are shown. (b) qRT-PCR analysis and quantitation of the ChIP assay for MyoD binding to fragment 3 (F3) and myogenin (myog) promoter. Values derived from three independent experiments were normalized for background signals (NoAb) and expressed as percentage of input chromatin (% input). Data are shown as mean ± standard deviation.

Figure 3.

MyoD relieves the enhancer-blocking activity of a KvDMR1 sub-region. (a) Schematic representation of the plasmid construct used for the enhancer-blocking assay, indicating the extent of the [11–22] test fragment (31) with respect to the KvDMR1 repressive element reported in Figure 2a. (b) Relative enhancer-blocking activity of the 11–22 fragment of KvDMR1 in K562 cells co-transfected with the MyoD expression vector (MyoD) or with the empty vector (Control). For each co-transfection, values, normalized relative to luciferase activity, were determined by dividing the number of G418-resistant colonies obtained with the indicated constructs by the colony number obtained with the Epneo construct. The results are the mean of three independent transfection experiments.

Figure 4.

KvDMR1 physically interacts with p57 promoter in undifferentiated and in unresponsive cells. (a) Schematic representation of the p57-KvDMR1 locus showing the locations of the NcoI sites and of the PCR primers (arrows) used for 3C analysis. (b) 3C analysis of the p57-KvDMR1 locus in C2.7 myoblasts and in responsive (C57BL) and in unresponsive (C3H10T1/2) mouse embryo fibroblasts expressing MyoD kept either in growth (T0) or in differentiation medium for 24 h (T24). Ctr consists of a plasmid construct containing a ligation product of p57 promoter and KvDMR1 sequences and represents a positive control for the pair of primers used. The results shown are representative of three independent experiments. (c) 3C analysis of the p57-KvDMR1 locus in responsive and unresponsive fibroblasts infected with either the MyoD retroviral vector (+MyoD) or with the empty vector (–MyoD) and analyzed 24 h after the shift to differentiation medium. The results shown are representative of three independent experiments. (d) Schematic model of MyoD effect on the putative chromatin looping between KvDMR1 and p57 promoter.

Figure 5.

MyoD binds biallelically to KvDMR1 but does not affect the imprinting status of the locus. (a) Allele-specific chromatin immunoprecipitation of MyoD. Hybrid mouse fibroblasts expressing MyoD, kept 24 h in differentiation medium, were treated for ChIP assay as described earlier. MyoD immunoprecipitates were then analyzed by radioactive PCR specific for F3, followed by SSCP analysis. M and P show the electrophoretic mobility of maternal- and paternal-specific bands, respectively. (b) Semiquantitative RT-PCR analysis of allele-specific expression of p57 and kcnq1. Hybrid mouse fibroblasts expressing MyoD were kept either in growth (T0) or in differentiation medium for 24 h (T24). Maternal and paternal alleles were distinguished by RFLP analysis as described in ‘Materials and Methods’ section. UD indicates the electrophoretic mobility of the undigested paternal-specific fragments (P). AvaI and PvuII fragments indicate that of digested maternal-specific fragments (M).