Genetic and epigenetic variation in histone H1

grantor: University of TorontoHistone proteins play a fundamental role in DNA-templated processes in eukaryotes. Access of regulatory factors for DNA replication, repair and gene transcription, to binding sites in chromatin is influenced by histone binding to DNA in nucleosomes, and by higher order folding of nucleosomal filaments that is mediated, in part, by histone H1. Non-allelic amino acid sequence variants of histone H1 are co-expressed in most eukaryotes, and tissue-specific patterns of H1 variant expression in metazoans have long suggested that H1 variants may be functionally distinct. However, direct support for this hypothesis is lacking, in part, due to the inability of current chromatographic procedures to adequately resolve H1 variants and post-translationally modified forms. To facilitate investigation of the functional significance of H1 heterogeneity, a novel HPLC method, cation-exchange-hydrophilic-interaction chromatography (CX-HILIC), was developed that resolved 14 major and numerous minor peaks for H1 from pooled chicken blood. This was unexpected because only six genes for H1 variants are known in this species. Electrophoretic analyses revealed that each CX-HILIC peak represented one of the six non-allelic variants of H1 described previously, but that four variants were represented by multiple peaks. Mass spectrometry revealed that HI recovered from CX-HILIC peaks was more heterogenous than was apparent in electrophoretic analyses and that the molecular mass of most molecules was smaller than predicted by the corresponding gene sequences. This was not attributable to proteolysis during handling since CX-HILIC peaks of H5 processed in parallel contained molecules that matched the expected value or exceeded it due to covalent modification. Analyses of H1 prepared from blood of individual chickens revealed that allelic variation was responsible for much of the CX-HILIC heterogeneity of H1 from pooled blood. Analyses of peptides from pooled erythrocyte H1 and liver H1 suggested that heterogeneity also arises from limited proteolysis of N-termini of H1 in vivo. Amino acid sequencing revealed that the genes encoding two non-allelic variants have been identified incorrectly in the literature. Together, these analyses demonstrate an unexpectedly high degree of heterogeneity in avian H1 and suggest the novel hypothesis that limited proteolysis may regulate H1 function in vivo.Ph.D