Position‐Effect Variegation Revisited

Prospects & Overviews Review essays Position-effect variegation revisited: HUSHing up heterochromatin in human cells Richard T. TimmsÃ, Iva A. TchasovnikarovaÃ and Paul J. LehnerÃ Much of what we understand about heterochromatin Introduction formation in mammals has been extrapolated from forward genetic screens for modifiers of position-effect variegation As a result of the many genome sequencing projects undertaken (PEV) in the fruit fly Drosophila melanogaster. The recent over the past two decades, we now have a detailed understand- identification of the HUSH (Human Silencing Hub) complex ing of the precise nature of the genetic code that holds the suggests that more recent evolutionary developments information required to build a biological machine as complex as the human body. However, merely cataloguing the exact contribute to the mechanisms underlying PEV in human sequence of the millions of the DNA bases A, C, G and T does cells. Although HUSH-mediated repression also involves not equate to being able to understand how multicellular heterochromatin spreading through the reading and organisms function, as this genetic information must clearly writing of the repressive H3K9me3 histone modification, be interpreted quite differently to enable the functions of all clear orthologues of HUSH subunits are not found in the different cell types. The DNA in the nucleus of eukaryotic cells is found in Drosophila but are conserved in vertebrates. Here we complex with proteins called histones, which together form a compare the insights into the mechanisms of PEV derived structure known as chromatin. The basic functional unit of from genetic screens in the fly, the mouse and in human chromatin is the nucleosome, consisting of 147 base pairs cells, review what is currently known about the HUSH of DNA wrapped around two copies of each of the histones complex and discuss the implications of HUSH-mediated H2A, H2B, H3 and H4. Histones serve a dual role. First, they silencing for viral latency. Future studies will provide provide an essential role in the compaction of DNA, enabling approximately two metres of DNA to fit inside the nucleus of a mechanistic insight into HUSH complex function and human cell which has a diameter of around just ten microns reveal the relationship between HUSH and other epige- across. Second, the structure of the chromatin fibre regulates netic silencing complexes. the accessibility of the DNA to the plethora of factors that regulate all aspects of DNA metabolism, including gene Keywords: expression, DNA replication and the repair of DNA damage. .haploid genetic screen; heterochromatin; HUSH Therefore, the expression of genetic information does not complex; position-effect variegation; retroviral silencing depend solely on the DNA sequence itself, but also upon the DOI 10.1002/bies.201500184 Department of Medicine, Cambridge Institute for Medical Research, Abbreviations: Addenbrooke’s Hospital, Cambridge, UK ES cells, embryonic stem cells; E(var), enhancer of variegation; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; *Corresponding authors: H3K9me3, tri-methylated lysine-9 of histone H3; HIV-1, human immunodefi- Richard T. Timms ciency virus type 1; HP1, heterochromatin protein 1; HUSH, Human Silencing E-mail: rtt20@cam.ac.uk Hub; KRAB-ZFP, Kruppel-associated€ box domain-containing zinc finger Iva A. Tchasovnikarova protein; LTR, long terminal repeat; Momme, modifier of murine metastable E-mail: it257@cam.ac.uk epiallele; PEV, position-effect variegation; Su(var), suppressor of variegation. Paul J. Lehner E-mail: pjl30@cam.ac.uk Bioessays 38: 333–343, ß 2016 WILEY Periodicals, Inc. www.bioessays-journal.com 333 R. T. Timms et al. Prospects & Overviews .... composition and activity of a myriad of chromatin-associated HUSH, thereby suggesting a novel route to heterochromatin proteins. formationinmammaliancells. Histone proteins can be post-translationally modified, and the exact nature and position of these modifications has dramatic consequences for chromatin function [1]. Forward genetics screens in Drosophila These marks either exert their effects directly, altering histone-histone or histone-DNA interactions to produce a have identified modifiers of position- change in nucleosomal architecture [2], or indirectly by effect variegation serving as a docking site for chromatin remodelling enzymes [3]. The various histone modifications are recog- Our understanding of the mechanisms of heterochromatin nised by distinct protein machineries, and hence define formation in higher eukaryotes is predominantly derived from distinct biological entities [4]. A variety of protein domains classic forward genetic screens in the fruit fly Drosophila have now been characterised that act as epigenetic ‘readers’ melanogaster for modifiers of position-effect variegation. Review essays by specifically recognising modified histones, such as the In Drosophila this has been studied extensively using the chromodomain that binds methylated lysine residues [5], white gene. The white gene encodes a transporter which and the bromodomain that binds acetylated lysine resi- is required for the correct deposition of the pigments that dues [6]. Chromatin binding proteins can possess multiple give the Drosophila eye its characteristic red colour. The such domains, leading to the attractive idea of a ‘histone white gene is normally housed in a euchromatic environ- code’ in which a particular combination of histone marks ment, facilitating its expression and hence red pigment could be interpreted to produce a unique biological deposition. When placed into a heterochromatic environ- outcome [7]. ment, however, epigenetic silencing of the white gene leads In all higher eukaryotic organisms, a large fraction of to loss of pigment deposition and a mutant white eye the genome is packaged into an inactive form known as phenotype [14]. The silencing observed is variable and only heterochromatin. In contrast with active euchromatin, occurs in a proportion of the cells of the eye, resulting in heterochromatin is typically characterised as being highly patches of red and white colour – the so-called ‘variegated’ condensed, gene-poor and less transcriptionally active. These phenotype (Fig. 1). two chromatin states were originally distinguished on the A particularly well-studied example is the Inversion(1)- basis of differential cytological staining [8], but it is now clear white-mottled-4 (wm4) allele, where the white gene is that they represent two distinct biochemical entities. Euchro- subject to silencing as a result of an inversion that places matin is normally associated with high levels of histone it into the vicinity of heterochromatin formed at the border acetylation, as well as marks such as methylated lysine-4 of of the nucleolus organiser [15]. This and other such histone H3 which are found across active chromatin [9]. In indicator strains allowed the development of forward contrast, heterochromatin is associated with low levels of genetic screens to identify dominant mutations in the histone acetylation and high levels of tri-methylated lysine-9 genes that were required for heterochromatin-induced of histone H3 (H3K9me3). These modifications are dynami- epigenetic silencing. Flies can be screened for mutations cally deposited and removed by histone-modifying enzymes. that either suppress silencing of the white gene, resulting in In the case of H3K9me3, SET domain-containing proteins such a reversion to a red eye phenotype, or for mutations that as SUV39H1 and SETDB1 function as epigenetic writers by enhance silencing of the white gene, resulting in a more depositing the methyl mark [10], while the Jumonji-domain complete white eye phenotype (Fig. 1). Overall, around containing family of demethylases act as epigenetic erasers by 140 such suppressors of variegation (termed Su(var))and removing the methyl mark [11]. 230 enhancers of variegation (E(var)) have been identified Our knowledge of the mechanisms that regulate to date, with the molecular identities of approximately 30 heterochromatin through the repressive H3K9me3 histone genes characterised [16]. modification has come from the study of so-called ‘chromo- The study of two Su(var) genes in particular has formed somal position effects’ [12], which are the main focus of the foundation for much of our present understanding of this review. Position effects refer to the differences in heterochromatin. Su(var) 2-5 encodes the heterochromatin- expression observed when an identical gene is positioned associated protein HP1a (heterochromatin protein 1a) which at different sites in the genome. The expression level of contains an N-terminal chromodomain and a C-terminal reporter genes varies widely depending on the specific site chromoshadow domain [17]. Su(var) 3-9 encodes the SU(VAR) of integration, with – broadly speaking – integration into 3-9 protein which contains an N-terminal chromodomain and euchromatin resulting in strong expression and integration a C-terminal SET domain [18]. The latter endows the protein into heterochromatin resulting in transcriptional repression with histone lysine methyltransferase (HKMT) activity, which [13]. The identities of the genes responsible for heterochro- is specifically targeted towards lysine-9 of histone H3 (H3K9) matin-mediated position effects have been largely studied [19, 20]. Both HP1a and SU(VAR) 3-9 localise to pericentro- through forward genetic screens in lower organisms. In meric heterochromatin and associate with each other [20, 21], the

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