Effects of histone modifications on chromatin regulation
Student thesis: Doctoral Thesis
Post-translational modifications (PTMs) to histones have long been associated with the regulation of gene transcription, a crucial mechanism for cell differentiation. Significant progress has been made towards our understanding of how these modifications affect transcription, through genome-wide mapping (ChIP-seq) and advances in proteomics methodologies but there remains limited evidence pertaining to certain modifications (e.g. H3K4me1 and the combinatorial effects of two or more modifications within the same histone. Here, ChIP was used to determine the spatio-temporal distributions of H3K4 methylations and H3K27ac across the developmentally regulated ßglobin locus in primitive (5DCE) and definitive chicken erythrocytes (15DCE). Surprisingly high enrichments of H3K27ac were found together with H3K4me1 at the inactive ßH and ßA genes in 5DCE. Since these genes will never be expressed in 5DCE, these PTMs may be related to their specific repression. However, in 15DCE the inactive genes carried lower H3K27ac than the active genes, so this modification is not simply linked to inactivity. In 5DCE, the enhancer was enriched in both H3K4me3 and K27ac but in 15DCE, increased enrichment of H3K4me3 and loss of K27ac was observed. These observations are consistent with the suggestion that H3K4me1 is a pioneering mark for establishment of the enhancer and an increase in modification status (to me3) is required for enhancer activity. H3K4me1 may also represent an additional mechanism to ensure gene repression. As observed in previous studies, H3K4me3 marked the active genes in both 5 and 15DCE. To identify effector proteins that bind H3K4me1 and/or K27ac to regulate transcription, histones carrying defined modifications were produced by reductive alkylation and genetic encoding and assembled into mono-nucleosomes for use in stable isotope labelling by amino acids in cell culture (SILAC) based nucleosome affinity pull downs (SNAP) coupled with tryptic MS analysis. These data show that the facilitates active chromatin transcription (FACT) complex binding was reduced by H3K4me1-containing nucleosomes, implicating H3K4 as a key residue for modulating FACT interaction with chromatin revealing at least one mechanism for transcriptional repression at inactive genes in this system.
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