Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei

Rudenko G (2012) Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei. PLoS Pathog 8(11): e1003010. doi:10.1371/journal.ppat.1003010 Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei Megan L. Povelones 0 Eva Gluenz 0 Marcin Dembek 0 Keith Gull 0 Gloria Rudenko 0 Elisabetta Ullu, Yale University, United States of America 0 1 Division of Cell and Molecular Biology, Imperial College London , South Kensington, London , United Kingdom , 2 The Sir William Dunn School of Pathology, University of Oxford , Oxford , United Kingdom The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ,1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6-8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1mediated chromatin functions in antigenic variation in T. brucei. - Funding: This research was funded by the Wellcome Trust. G.R. is a Wellcome Senior Fellow in the Basic Biomedical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The African trypanosome Trypanosoma brucei is a unicellular parasite causing African sleeping sickness, which is transmitted by tsetse flies in sub-Saharan Africa. As an extracellular parasite of the mammalian bloodstream, T. brucei has evolved a sophisticated strategy to antigenically vary its major surface coat protein, variant surface glycoprotein (VSG) [1,2]. The T. brucei genome contains a vast repertoire of silent VSG genes and pseudogenes, most of which are located in tandem arrays at subtelomeric locations [3,4]. The VSG repertoire varies in both size and composition between different T. brucei strains, with the exact sizes still unclear due to the technical complications of cloning, sequencing and assembling these subtelomeric sequences [5]. However, a conservative estimate proposes that the T. brucei 927 strain contains more than 1500 VSGs, of which only one VSG is expressed at a time [6,7]. The active VSG is located in one of about 15 telomeric VSG expression sites (ES). ESs are transcribed by RNA polymerase I (Pol I) [8,9], which normally exclusively transcribes ribosomal DNA (rDNA) [10]. For antigenic variation to work, it is key that only one VSG is expressed at a time, and the extensive repertoire of VSGs is kept transcriptionally silent. These restrictions need to operate within the context of a T. brucei genome which is primarily organised as very extensive polycistronic transcription units constitutively expressed by Pol II [6,11]. Although it is unclear how ESs are controlled, it has recently been shown that chromatin remodeling must play a key role in their regulation [1214]. In eukaryotes DNA is packaged into nucleosomes, whereby ,146 bp of DNA is wrapped around a histone octamer consisting of two histone H2A/H2B dimers and two histone H3/H4 dimers. A linker histone H1 (H1) typically interacts with both the nucleosome and the linker DNA to stabilize higher order chromatin structure [15]. H1 has been shown to be dispensable in several unicellular eukaryotes including yeast and Tetrahymena [1618]. The exact role of H1 has been surprisingly hard to discern despite its association with heterochromatin and proposed function as a general transcriptional repressor [1923]. Knock-out of H1 in S. cerevisiae, Tetrahymena or mammalian cells affects transcription of a relatively small subset of genes in these different organisms, and does not have a major effect on global transcription [2426]. In addition, yeast cells lacking histone H1 demonstrate genomic instability, most likely due to increased homologous recombination (HR) in its absence [27]. Trypanosoma brucei causes African sleeping sickness, endemic to sub-Saharan Africa. Bloodstream form T. brucei is covered with a dense coat of variant surface glycoprotein (VSG). Only one VSG is expressed at a time out of a vast repertoire of ,1500 VSGs. The active VSG is transcribed in a telomeric VSG expression site (ES), and VSG switching allows immune evasion. Exactly how monoallelic exclusion of VSG ESs operates, and how switching between ESs is mediated remains mysterious, although epigenetics and chromatin structure clearly play a major role. The linker histone H1 is thought to orchestrate higher order chromatin structure in eukaryotes, but its exact function is unclear. We investigated the role of histone H1 in the regulation of antigenic variation in T. brucei. We show that histone H1 is associated with chromatin and is required for higher order chromatin structure. Depletion of histone H1 results in derepression of silent VSG ES promoters, indicating that H1-mediated chromatin functions in antigenic variation in T. brucei. The chromatin of T. brucei has several unusual properties. The core histones of T. brucei are divergent compared with those of higher eukaryotes, particularly at the N-termini which can be posttranslationally modified [2830]. In addition, T. brucei chromatin has a more open conformation, does not form 30-nm fibres in vitro, and chromosomes fail to condense prior to nuclear mitosis [31]. These characteristic features of T. brucei chromatin are typically influenced by the linker histone H1 in other eukaryotes arguing that T. brucei H1 could play a different role [15]. Histone H1 proteins in T. brucei are distinct from those in other eukaryotes, in that they lack the central globular domain thought (...truncated)