Mapping the Chromatin State Dynamics in Myoblasts

Abstract

Background: Genome-wide mapping reveals chromatin landscapes unique to cell states. Histone marks of regulatory genes involved in cell specification and organ development provide a powerful tool to map regulatory sequences. H3K4me3 marks promoter regions; H3K27me3 marks repressed regions, and Pol II presence indicates active transcription. The presence of both H3K4me3 and H3K27me3 characterize poised sequences, a common characteristic of genes involved in pattern formation during organogenesis.

Results: We used genome-wide profiling for H3K27me3, H3K4me3, and Pol II to map chromatin states in mouse embryonic day 12 forelimbs in wild type (control) and Pitx2-null mutant mice. We compared these data with previous gene expression studies from forelimb Lbx1⁺ migratory myoblasts and correlated Pitx2-dependent expression profiles and chromatin states. During forelimb development, several lineages including myoblast, osteoblast, neurons, angioblasts etc., require synchronized growth to form a functional limb. We identified 125 genes in the developing forelimb that are Pitx2-dependent. Genes involved in muscle specification and cytoskeleton architecture were positively regulated, while genes involved in axonal path finding were poised.

Conclusion: Our results have established histone modification profiles as a useful tool for identifying gene regulatory states in muscle development, and identified the role of Pitx2 in extending the time of myoblast progression, promoting formation of sarcomeric structures, and suppressing attachment of neuronal axons.

Keywords: chromatin; homeobox; muscle development; myogenesis.

Figure 1. Chromatin state changes in Pitx2 Mutants

Venn diagrams illustrating the co-occurrence of enriched regions around identified Pitx2 target sequences in WT and Pitx2 MT forelimbs, respectively. The MT data are represented by darker shades. Green represents H3K4me3; red represents H3K27me3; and gray represents Pol II in each sample. Homer v4.7 was used to match each identified target sequence to its nearest peaks.

Figure 2. Constant tag density in Wild-Type and Pitx2 Mutants

(A) Normalized tag densities around each known Refseq TSS for all three marks in WT and MT. Homer v4.7 annotatePeaks.pl was used to count the number of aligned reads around Refseq TSSs by scanning in 5bp bins. The average number of reads per bin per sequence is plotted on the y axis against the distance of the bin from the TSS. (B) Normalized tag densities around only the 125 Pitx2 target sequences identified. (C) Normalized tag densities around only the subset of SSTFs from (B). (D) Tag densities around all sequences not in (C).

Figure 3. Pitx2-dependent chromatin state of mouse embryonic forelimbs

UCSC genome browser visualization of selected gene loci. Genes were selected based on strong H3K27me3 marked chromatin, and/or high relative fold changes determined from the GSE31945 array data. WT and MT tracks of the same mark are overlaid. H3K4me3 is represented by green, H3K27me3 red, and Pol II gray. MT tracks are illustrated by the darker shades. (A) Trivalent, poised genes H3K4me3/H3K27me3/Pol II. (B) Activated genes (H3K4me3/Pol II). (C) Bivalent genes (H3K4me3/H3K27me3). (D) Genes with open chromatin (H3K4me3). (E) Genes with close chromatin (H3K27me3). (F) Genes with no significant chromatin signature.