Independent Biological and Biochemical Functions for Individual Structural Domains of Drosophila Linker Histone H1

Abstract

Linker histone H1 is among the most abundant components of chromatin. H1 has profound effects on chromosome architecture. H1 also helps to tether DNA-and histone-modifying enzymes to chromatin. Metazoan linker histones have a conserved tripartite structure comprising N-terminal, globular, and long, unstructured C-terminal domains. Here we utilize truncated Drosophila H1 polypeptides in vitro and H1 mutant transgenes in vivo to interrogate the roles of these domains in multiple biochemical and biological activities of H1. We demonstrate that the globular domain and the proximal part of the C-terminal domain are essential for H1 deposition into chromosomes and for the stability of H1-chromatin binding. The two domains are also essential for fly viability and the establishment of a normal polytene chromosome structure. Additionally, through interaction with the heterochromatin-specific histone H3 Lys-9 methyltransferase Su(var)3-9, the H1 C-terminal domain makes important contributions to formation and H3K9 methylation of heterochromatin as well as silencing of transposons in heterochromatin. Surprisingly, the N-terminal domain does not appear to be required for any of these functions. However, it is involved in the formation of a single chromocenter in polytene chromosomes. In summary, we have discovered that linker histone H1, similar to core histones, exerts its multiple biological functions through independent, biochemically separable activities of its individual structural domains.DNA in the eukaryotic nucleus is packaged into a compact nucleoprotein complex called chromatin (1, 2). The histones constitute a family of basic proteins that play a vital role in organizing chromatin. There are five major classes of histones: the core histones H2A, H2B, H3, and H4 and the linker histone H1. The nucleosome core particle, the fundamental unit of chromatin, consists of an octamer of two copies of each of the core histones around which about 146 bp of DNA is wrapped. H1 binds to the nucleosome core particle and the linker DNA between adjacent core particles. The stoichiometry of H1 to nucleosome core particles in cells of complex eukaryotes ranges from about 0.5 to approximately equimolar (3). Given this abundance, the linker histone H1 is expected to play important roles in chromatin structure. Indeed, numerous in vitro studies, as well as experiments in vivo, have shown that H1 has a profound effect on chromatin structure (reviewed in Refs. 4 and 5). For example, incorporation of H1 into chromatin leads to an increase in the spacing between nucleosome core particles. H1 also facilitates the folding of chromatin into more compact structures. In addition to these architectural roles, there are now an increasing number of reports describing interactions between H1 linker histones and a variety of other nuclear proteins (6), including functional interactions between H1, histone-modifying enzymes, and DNA methyltransferases that lead to modulation of epigenetic marking of chromatin (7,8). In this context, it is notable th...

Emerging roles of linker histones in regulating chromatin structure and function

“…5b). Whereas the globular domain of H1 is required for its deposition into chromatin and for physical interactions with HP1 (REFS 36,137), different regions of the C-terminal domain bind to linker DNA 10 , promote oligonucleosome condensation 138,139 and mediate interactions of H1 with DNMTs, STAT, Su(var)3-9 and SUUR 86,103,132,137 .…”

Regulatory functions and chromatin loading dynamics of linker histone H1 during endoreplication in Drosophila

“…We demonstrated previously that H1 exerts its multiple biological functions through independent biochemically separable activities of its three structural domains (Kavi et al 2016). Moreover, different segments of the H1 protein are required for direct physical interactions with its several different biochemical partners.…”

“…The tripartite structure of H1 provides multiple binding interfaces for interacting proteins and thus allows H1 to mediate several biochemically separable functions in vivo (Kavi et al 2016). For instance, the globular domain and proximal 25% of the CTD are required for H1 loading into chromatin, while the proximal 75% of the CTD is needed for normal polytene morphology, H3K9 methylation, and physical interactions with Su(var)3-9.…”

“…To test this hypothesis, we compared polytene chromosome localization of SUUR in wild-type animals with that in animals depleted of H1 by RNAi. To avoid a complete disruption of polytene morphology (Lu et al 2009;Kavi et al 2016), H1 was only moderately depleted (to ∼30% of normal) ( Fig. 2C; see the Supplemental Material).…”

“…Alternatively, GST pull-downs were performed with recombinant H1 polypeptides (full-length and C-terminally truncated). The expression constructs and purification protocol have been described (Kavi et al 2016). See the Supplemental Material for details of cloning, protein purification, and pull-down protocols.…”