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Fig. 3 | Epigenetics & Chromatin

Fig. 3

From: Sporadic DUX4 expression in FSHD myocytes is associated with incomplete repression by the PRC2 complex and gain of H3K9 acetylation on the contracted D4Z4 allele

Fig. 3

The D4Z4 locus is bivalent in human stem cells and turns univalent in myoblast. a Sequential chromatin immunoprecipitation beginning with antibodies to H3K27me3 and followed by antibodies to H3K4me3 was utilized to investigate bivalency at the DUX4 locus. The reverse sequential ChIP with antibodies recognizing H3K4me3 first and antibodies recognizing H3K27me3s was also performed with similar results (data not shown). Isogenic human iPS cell clones isolated from individuals with mosaic distributions of D4Z4 lengths were used for this study. Chromatin purified from iPS clones with non-contracted (mosaic long clone) and contracted (mosaic short clone) iPS cell clones was utilized in ChIP and quantitative PCR amplification. The stem cell bivalent locus (POU4F3) [25] and the stem cell univalent locus (HOXA3) [24, 25] were utilized as controls for comparison to the LLP region of D4Z4 in the iPS cell clones. Similarly, sequential ChIP was performed on myoblasts from non-FSHD control and FSHD myoblasts again uniquely amplifying the 4qA161-L allele in these cells. The arrow indicates signal was not detected. The pull down for the POU4F3 locus in the mosaic long clone was statistically significant using the Student’s t test when compared to the HOXA3 locus in the mosaic long clone and the DUX4 locus in the normal primary myoblast (*p value ≤ 0.05). Similarly, the pull down for POU4F3 locus in mosaic short clone was significantly different when compared to the HOXA3 locus in the mosaic short clone (*p value ≤ 0.05). b ChIP-seq data [25] were reprocessed and aligned to the human genome for DUX4 analysis. Similar levels of H3K27me3 and H3K4me3 at the DUX4 locus supports a bivalent chromatin structure and is consistent with our ChIP results in (a). c Chromatin immunoprecipitation of D4Z4 DNA with antibodies recognizing H3K4me3, H3K27me3, an EZH2 and selective amplification of the permissive allele in non-FSHD control and FSHD1 myoblasts. Comparison by t test of the mean percent input normalized to H3 between non-FSHD control to FSHD for H3K4me3 and H3K27me3 revealed statistical significance (*p value ≤ 0.05). The error bars show standard deviations

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