Figure 4

MyoD and myogenin are enriched at the MCK -SIE in skeletal myocytes. (A) Diagram of the 6.5-kb MCK regulatory region with the three known active regulatory regions: the 5'-enhancer, PP, MR1 (white boxes), the MCK-SIE (light gray box) exons 1 and 2 (black boxes) and other regions (gray), including the 33.7-kb Mark4 gene (located approximately 40 kb 3' of the MCK-SIE and transcribed in the opposite direction). E-box CAnnTG core motifs (arrowheads) occur throughout the 6.5-kb sequence. Among the thirty-five total E-boxes are two functional E-boxes within the 5'-enhancer, one functional E-box within the proximal promoter and two E-box motifs within the MCK-SIE (longer arrows). The less frequent MEF2 motifs (full diamonds) are found only in the 5'-enhancer and MCK-SIE and as a possible nonconsensus MEF2 site (open diamond) in the proximal promoter. The chromatin immunoprecipitation (ChIP) primer pairs (black lines) that span the 5'-enhancer sequence were used as positive controls for MyoD and myogenin binding to functional E-boxes. Negative controls consist of genomic regions containing either no core E-box motifs (region within the Mark4 intron 1 (M4, dagger)) or core E-box motifs with no proven transcriptional function (MCK gene exon 1/intron 1 boundary (two E-boxes) and exon 2 (one E-box); see Results, section-5). (B) MyoD and myogenin bind MCK gene E-box motifs. ChIP analyses using antibodies for MyoD, myogenin, MEF2 and control immunoglobulin G (IgG) were performed using chromatin from differentiated MM14 cell myocytes. The graph shows data from one of three ChIP experiments that is representative of the enrichment detected at each position by antibodies to myogenin (black bars), MyoD (gray bars) or MEF2 (white bars) over nonspecific rabbit IgG as determined by quantitative polymerase chain reaction (qPCR) assay. Error bars represent ±1 standard deviation of triplicate samples. (C) Electrophoretic mobility shift assay (EMSA) of MEF2 binding to the MCK-SIE MEF2 control element. Nuclear extracts from differentiated MM14 cultures were incubated with a ³²P-labeled probe containing the MCK-SIE-MEF2 sequence with no competitor (lane 1), wild-type MEF2 competitor (lane 2), two different mutant MEF2 competitors (lanes 3 and 4), pan-MEF2 antibodies (lane 5), transcriptional enhancer factor 1 (TEF-1)-specific antibodies (lane 6) or nonspecific rabbit IgG (lane 7). Arrows indicate the MEF2-containing complex and free probe. (D) MEF2 ChIP-Seq occupancy at the 6.5-kb MCK regulatory region in differentiated C₂ C₁₂ cells shows that MEF2 is present at all three control regions. The 6.5-kb region is shown in schematic at the top (5'-enhancer, proximal promoter and MR1 are shown in white; MCK-SIE is shown in gray). Sequences that match the MEF2 canonical motif (CTAWWWWTAG) at the 80%, 85% and 100% thresholds are mapped throughout the 6.5-kb region. The sequenced and mapped ChIP signals (reads per million (rpm)) for the two pan-MEF2 antibodies 1 and 2 and the control (input DNA) are indicated as black histograms (scale shown at the right). Two different ChIP-Seq region finders (Model-based Analysis of ChIP-Seq data and Enhanced Read Analysis of Gene Expression) define the sequence range in which MEF2 is predicted to bind (see Materials and methods), and these are shown below each signal track as black bars. Conservation across the regions is shown from the University of California Santa Cruz (UCSC) Genome Browser plot of phastCons scores for the 20 default placental mammals.