The Differences BetweenCis- andTrans-Gene Inactivation Caused by Heterochromatin inDrosophila

Abstract

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo-formed euchromatinheterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2. KEYWORDS heterochromatin; Drosophila; PEV; trans-inactivation; nuclear compartmentalization P osition-effect variegation (PEV) is an epigenetic phenomenon of inactivation of a gene in a portion of cells caused by relocation of a gene into or very close to the heterochromatin. Heterochromatin has a distinct chromatin structure that includes specific histone modifications, associated proteins, and a condensed nucleosome package. This structure can spread from the euchromatin-heterochromatic border into the euchromatin by self-assembly and propagation of a complex containing SU(VAR)3-9 histone methyltransferase, HP1a, and SU(VAR)3-7 proteins, thus affecting the expression of euchromatic genes near the border (Grewal and Elgin 2002; Schotta et al. 2003;Hines et al. 2009;Elgin and Reuter 2013). Analysis of the spreading of heterochromatin using high-throughput approaches has been performed in a single paper aimed at the analysis of white-mottled X-chromosomal inversions, demonstrating PEV of the white gene (Vogel et al. 2009). It was found that HP1a propagates up to 175 kb into euchromatin from the heterochromatin border and demonstrates uneven distribution over the spreading area. Only the white gene among 20 measured genes in this region demonstrates decreased expression as a result of PEV.Here we present a detailed study of the genetic system (inversion In(2)A4) demonstrating cis-effects of heterochromatin on gene expression as well as inversion-induced transinactivation of the transgenes located on the homologous nonrearranged chromosome. RNA-Seq analysis shows that only a few euc...

Trans-inactivation: Repression in a wrong place

“…In(2)A4 induces robust and widespread trans-inactivation of mini-white reporter genes located on the homologous, non-rearranged chromosome as well as of genes in cis, located near the new eu-heterochromatin border. [18][19][20] Trans-inactivation effects in In (2)A4 and bw D are similar in many aspects, apparently reflecting common properties of trans-inactivation process in Drosophila. FISH data show that, like in the bw D case, the normal chromosome and the inversion are somatically paired, and the region of the inversion near the new eu-heterochromatin boundary and the homologous region of the normal chromosome are dragged into the heterochromatic nuclear compartment.…”

“…These include rearrangements with breakpoints near or inside the brown gene, such as the brown Dominant (bw D ) allele, 15,16 mini-white-containing transgenes over the PEV-inducing rearrangements 17 and the In(2)A4 inversion. [18][19][20] The beststudied trans-inactivation-inducing rearrangement is bw D allele, which emerged as a result of insertion of 1.6 Mb of AAGAG satellite into the coding region of the brown gene. 21 This allele can repress the wild-type brown gene on the opposite chromosome, as well as reporter genes inserted near the brown.…”

“…It appears that the HP1a participates in the repression independently of Su(var)3-9-mediated H3K9me2 modification. 18 In contrast to bw D transposition of satellite sequences into the otherwise unrearranged chromosome, In (2)A4 affects the linear order of genes in a region of more than one megabase. This indicates that somatic pairing between the In(2)A4 carrying chromosome and the normal chromosome proceeds via the formation of loop-like structure (Fig.…”

“…The loop formed by rearrangement and the normal chromosome is visible on polytene chromosome spreads (our unpublished observations) and FISH signals from the putatively paired regions in transheterozygous In(2) A4/C individuals revealed a single spot in a majority of the cells, confirming strong somatic pairing. 18 We suppose that the pairing is preserved due to a relatively small size of the inverted euchromatin block (approximately 1 Mb of DNA) and a large size of the separated heterochromatin block (sections h35-h37 of 2Lh), sufficient to promote effective sticking to the main bulk of the pericentromeric heterochromatin. These peculiarities of the In(2)A4 structure should stabilize the loop formed by the paired chromosomes.…”

“…E is the region near the histone genes cluster, no repression observed here, and F is the "island" of trans-inactivation after the histone genes cluster. Below the linear map, the putative organization of the loop of paired chromosomes in the nuclear space is shown according to a combination of FISH data [18][19][20] and polytene chromosomes arrangement. The light green is euchromatin; the brown gradient zone is HP1a-stained pericentromeric heterochromatin compartment.…”