Chapter 6 – Comprehensive Analysis of Mammalian Linker-Histone Variants and Their Mutants

Linker histone H1, the key chromatin structural protein facilitating higher order chromatin folding, is emerging as an important epigenetic mark and regulator for gene expression and cellular differentiation. Here we summarize a suite of analysis tools and applications we have employed to characterize H1 variants and mutations. In mammals, there are multiple H1 variants differentially regulated during development and cell differentiation. We first describe highly quantitative assays for gauging expression levels of H1 variants by random primer-based qRT-PCR and HPLC. Next, we introduce a set of genetic, cellular, and genomic approaches, including knockout and knock-in strategies, in vivo tagging, stem cell differentiation, and genome-wide mapping, for studying the functions of H1 variants. Finally, we illustrate the overexpression of H1 mutants in H1 knockouts to characterize H1 mutations in cancer. These methods should have wide applications in chromatin and epigenetic analysis.