Downloaded from http://cshperspectives.cshlp.org/ on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Position-Effect Variegation, Heterochromatin Formation, and Gene Silencing in Drosophila Sarah C.R. Elgin1 and Gunter Reuter2 1Department of Biology, CB-1137, Washington University, St. Louis, Missouri 63130; 2Institute of Biology, Developmental Genetics, Martin Luther University Halle, D-06120 Halle, Germany Correspondence: [email protected] SUMMARY Position-effect variegation (PEV) results when a gene normally in euchromatin is juxtaposed with hetero- chromatin by rearrangement or transposition. When heterochromatin packaging spreads across the het- erochromatin/euchromatin border, it causes transcriptional silencing in a stochastic pattern. PEV is intensely studied in Drosophila using the white gene. Screens for dominant mutations that suppress or enhance white variegation have identified many conserved epigenetic factors, including the histone H3 lysine 9 methyltransferase SU(VAR)3-9.Heterochromatin protein HP1a binds H3K9me2/3 and interacts with SU(VAR)3-9,creating a core memory system. Genetic, molecular, and biochemical analysis of PEVin Drosophila has contributed many key findings concerning establishment and maintenance of hetero- chromatin with concomitant gene silencing. Outline 1 Genes abnormally juxtaposed with 6 Not all heterochromatin is identical: Spatial heterochromatin show a variegating phenotype organization matters 2 Screens for suppressors and enhancers of PEV 7 How is heterochromatin formation targeted in have identified chromosomal proteins and Drosophila? modifiers of chromosomal proteins 8 PEV, heterochromatin formation, and gene 3 Distribution and association patterns of silencing in different organisms chromosomal proteins 9 Summing up: There is much that we do not know 4 Histone modification plays a key role in about heterochromatin heterochromatin silencing References 5 Chromosomal proteins form mutually dependent complexes to maintain and spread heterochromatic structure Editors: C. David Allis, Marie-Laure Caparros, Thomas Jenuwein, and Danny Reinberg Additional Perspectives on Epigenetics available at www.cshperspectives.org Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a017780 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a017780 1 Downloaded from http://cshperspectives.cshlp.org/ on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press S.C.R. Elgin and G. Reuter OVERVIEW Genes that are abnormally juxtaposed with heterochromatin, comitant association of heterochromatin protein 1 (HP1a) either by rearrangement or transposition, show a variegating and other interacting proteins, including H3K9 methyltrans- phenotype. This is a result of the gene being silenced in some ferases (HKMTs); the multiple interactions of these proteins of the cells in which it is normally active. Because the change are required for the spreading and maintenance of hetero- is caused by a change in the position of the gene in the chromatin. Targeting of heterochromatin formation to partic- genome, rather than a change in the gene itself, this phenom- ular regions of the genome appears to involve multiple enon is termed “position-effect variegation” (PEV). The silenc- mechanisms, from satellite DNA-specific binding proteins ing that occurs in PEV can be attributed to the packaging of the to utilization of the RNA interference (RNAi) machinery. reporter gene in a heterochromatic form, indicating that en- Although heterochromatic regions (pericentric regions, telo- dogenous heterochromatin formation, once initiated, can meres, the Y chromosome, and the small fourth chromosome) spread to encompass nearby genes. Genetic, cytological, share a common biochemistry, each is distinct, and each is and biochemical analyses are all possible in Drosophila me- complex in different ways. Heterochromatin in Drosophila is lanogaster. In this article we will show how these different gene poor, but it is not devoid of genes, and counterintuitive- approaches have converged to identify many contributors to ly, those genes that reside in heterochromatin are often de- this system, leading to characterization of both structural pro- pendent on this environment for full expression. A complete teins and modifying enzymes that play key roles in establish- understanding of heterochromatin formation and mainte- ing and maintaining heterochromatin. nance (including targeting and spreading) will need to in- Heterochromatin formation depends critically on meth- clude an explanation for the varying responses of different ylation of histone H3 at lysine 9 (H3K9me2/3), with con- genes to this chromatin environment. 2 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a017780 Downloaded from http://cshperspectives.cshlp.org/ on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press PEV, Heterochromatin Formation, and Gene Silencing in Drosophila 1 GENES ABNORMALLY JUXTAPOSED WITH lead to PEV (Girton and Johansen, 2008). PEV has subse- HETEROCHROMATIN SHOW A VARIEGATING quently been observed in a variety of organisms, including PHENOTYPE yeasts (see Allshire and Ekwall 2014), flies, and mammals Large segments of the eukaryotic genome are packaged in a (see Blewitt and Whitelaw 2013; Brockdorff and Turner permanently inactive form of chromatin termed constitu- 2014), but has been used as a tool to study heterochromatin tive heterochromatin. This chromatin fraction was origi- formation primarily in Drosophila. nally identified as that portion of the genome that remains PEV indicates that such rearrangements allow packag- condensed and deeply staining (heteropycnotic) in inter- ing of the newly positioned gene into a heterochromatic phase; such material is generally associated with the telo- configuration, and suggests that this is the consequence of meres and pericentric regions of the chromosomes. Het- heterochromatin “spreading” along the chromosome from erochromatic regions tend to be late replicating and show the adjacent constitutive heterochromatin region. Appar- little orno meioticrecombination.These domainsaredom- ently, the rearrangement has removed a normally existing inated by repetitious DNA sequences (30%–80%), both barrier or buffer zone. The consequence is an altered pack- tandem repeats of short motifs (known as “satellite” DNA), aging with concomitant silencing of genes normally pack- and remnants of transposable elements (TEs), including aged in a euchromatic form. Visual inspection of the poly- DNA transposons and retroviruses. Although gene poor, tene chromosomes of larvae carrying such a rearrangement these domains are not devoid of genes, and intriguingly, shows that the region carrying the reporter gene is pack- those genes that are present frequently are dependent on aged in a dense block of heterochromatin only in the cells in that environment for optimal expression. About one third which the gene is inactive (Zhimulev et al. 1986). Patterns of the Drosophila genome is considered heterochromatic, of variegated expression, observed as a consequence of re- including the entire Y chromosome, most of the small arrangement of white, vary in the number of pigmented fourth chromosome, the pericentric region that covers 40% cells, the size of the pigmented patches, and the level of of the X chromosome, and pericentric regions that cover pigment in the two different cell types observed, one with 20% of the large autosomes (Smith et al. 2007). During a high level of expression, and one with a low level or no the last few decades we have learned a great deal about expression (Fig. 1A). In a system using an inducible lac-Z the biochemistry of heterochromatin, and much of that gene as a reporter, investigators observed that silencing understanding derives from our studies with Drosophila occurs in early embryogenesis, just after heterochromatin (see Schotta et al. 2003; Schulze and Wallrath 2007; Girton is first observed cytologically, and is epigenetically inherit- and Johansen 2008; Eissenberg and Reuter 2009 for prior ed in both somatic and germline lineages; the mosaic phe- reviews). notype is determined during differentiation by variegated One of the first mutations identified in D. melanogaster relaxation of silencing in third instar larvae (Lu et al. 1996). was white, a mutation that results in a fly with a white eye, However, not all variegating genes remain silent until af- rather than the characteristic red pigmentation. Using X ter differentiation, and the balance of factors leading to rays as a mutagen, Muller (1930) observed an unusual phe- the on/off decision no doubt differs for different genes. notype, in which the eye was variegating, with some patches (See Ashburner et al. 2005, Chapter 28 for a more detailed of red and some patches of white facets (Fig. 1A). This discussion.) phenotype suggested that the white gene itself was not dam- A fly line showing a PEV phenotype can be used to aged—after all, some facets remained red, and flies with screen for dominant second site mutations that are either entirely red eyes could be recovered as revertants, again us- suppressors or enhancers of PEV. These second site muta- ing X rays as the mutagen. However, the white gene had tions can be induced by chemical mutagens that cause clearly been silenced in some of the cells in which it is nor- point mutations or small insertions/deletions, but do not mally expressed. Subsequent examination of the polytene impact the chromosome