Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure

Abstract

We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone H1 can be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to ;20% of the level in wildtype larvae, lethality occurs in the late larval -pupal stages of development. Here we show that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me 2 and H4K20Me 2 . Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo.[Keywords: Linker histone H1; heterochromatin; histone methylation; polytene chromosomes; chromocenter; position effect variegation] Supplemental material is available at http://www.genesdev.org. The genomes of eukaryotes are packaged into a highly compact nucleoprotein complex called chromatin. The histones constitute a family of proteins that are intimately involved in organizing chromatin structure. There are five major classes of histones: the core histones H2A, H2B, H3, and H4, and the linker histones usually referred to as H1. The nucleosome core particle is the highly conserved repetitive unit of chromatin organization. It consists of an octamer of the four core histones around which ;145 base pairs (bp) of DNA are wrapped and protected from nuclease digestion (Van Holde 1988;Wolffe 1998). The linker histone H1 binds to core particles and protects an additional ;20 bp of DNA (linker DNA). In metazoans, the abundance of linker histones, although variable during development, approaches that of core histones (Woodcock et al. 2006), suggesting that they play an important role in establishing and maintaining the structure of the chromatin fiber.Much of our knowledge about the roles of linker histones comes from in vitro studies. These studies indicate that two principal functions of linker histones are to stabilize the DNA entering and exiting the core particle and to facilitate the folding of nucleosome arrays into more compact structures (Ramakrishnan 1997;Wolffe 1997). H1 also affects nucleosome core particle spacing and mobility. In vitro studies also suggest that H1 acts pri...

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

“…Flies carrying a GAL4-responsive, UAS-driven transgene (pINT-1-H1) encoding H1-specific hairpin RNA are mated to flies bearing the ubiquitous tubulin-GAL4 driver (see "Experimental Procedures"). Larvae carrying both transgenes express reduced levels of H1 protein (19). The extent of reduction depends upon the integration site of the pINT-1-H1 transgene and the temperature, which is due to higher transcriptional activity of GAL4 at elevated temperatures (20).…”

“…The pINT-1-H1 transgenes used in this study cause a depletion of H1 protein levels to ϳ10 -30% of normal. This reduction leads to marked changes in larval chromosome structure and gene expression and sharply reduces the rates of eclosion of adult flies (19).…”

“…Expression of the H1 proteins encoded by the transgenes was examined by immuno- anti-FLAG (19). B, expression levels of H1 transgenic proteins in third instar larvae.…”

“…To develop an in vivo approach for assessing the contributions of the histone H1 structural domains to the various biological functions of H1, we utilized a system that we described previously for depleting H1 in Drosophila by RNAi (19). Flies carrying a GAL4-responsive, UAS-driven transgene (pINT-1-H1) encoding H1-specific hairpin RNA are mated to flies bearing the ubiquitous tubulin-GAL4 driver (see "Experimental Procedures").…”

“…Drosophila melanogaster offers an attractive system for such studies because, in contrast to mammals, it expresses a single linker histone protein during much of its life cycle. H1 is essential for proper development of flies, and it plays key roles in several aspects of chromosome structure and gene regulation (7,19). Here we investigate the functional roles of the three H1 structural domains, the H1 NTD, GD, and CTD, in the roles of H1 in Drosophila development, chromosome structure, and epigenetic regulation.…”