Effect of chromatin structure modifiers on the trans-acting heterochromatin position effect in Drosophila melanogaster

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Abstract

The heterochromatin position effect is manifested in the inactivation of euchromatin genes transferred to heterochromatin. In chromosomal rearrangements, genes located near the new eu-heterochromatin boundary in the rearrangement (cis-inactivation) and, in rare cases, genes of a region of the normal chromosome homologous to the region of the eu-heterochromatin boundary of the chromosome with the rearrangement (trans-inactivation) are subject to inactivation. The In(2)A4 inversion is able to trans-inactivate the UAS-eGFP reporter gene located on the normal chromosome. We knockdown a number of chromatin proteins using temperature-controlled RNA interference and investigated the effect of knockdown on trans-inactivation of the reporter. We found suppression of trans-inactivation by knockdowns of Su(var)2-HP2, a protein that binds to the key heterochromatin protein HP1a, SAYP, a subunit of the chromatin remodelling complex, and Eggless histone methyltransferase (SETDB1), which introduces a H3K9me3 histone mark, recognized by the HP1a protein. The method of studying the effects of gene knockdown on heterochromatin position effects presented in this work is of independent methodological interest.

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About the authors

А. А. Solodovnikov

National Research Centre “Kurchatov Institute”

Email: slavrov.defy@gmail.com
Russian Federation, Moscow

S. А. Lavrov

National Research Centre “Kurchatov Institute”

Author for correspondence.
Email: slavrov.defy@gmail.com
Russian Federation, Moscow

A. S. Shatskikh

National Research Centre “Kurchatov Institute”

Email: slavrov.defy@gmail.com
Russian Federation, Moscow

V. A. Gvozdev

National Research Centre “Kurchatov Institute”

Email: slavrov.defy@gmail.com

Academician

Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. System for studying the effect of knockdown of position effect modifier genes on trans-inactivation of the UAS-eGFP reporter caused by In(2)A4 inversion: In(2)A4 inversion causes mosaic inactivation of the UAS-eGFP reporter on the normal chromosome in Malpighian vessels. The position of the UAS-eGFP reporter on the normal chromosome relative to the A4 break site is shown (dashed vertical line). Below is the structure of inversion A4, Gx - blocks of heterochromatin, in A4 the block is divided into large and separated small fragments c - centromere. The cis-acting position effect extends to euchromatin from the main and separated blocks heterochromatin (blue arrows), trans-inactivation affects the reporter on the homologous normal chromosome. Photographs show mosaic eGFP expression (lack of fluorescence in individual cells) during In(2)A4-induced trans-inactivation (eGFP/A4), compared to eGFP expression at the same temperature but in a wild-type chromosome background (eGFP/+) (A); system for studying the knockdown of genes – modifiers of EP for trans-inactivation (b). The genotype contains: chromosomes 2 – A4 inversion in a heterozygote with a normal chromosome with a trans-inactivable UAS-eGFP reporter; chromosome 3 – a chromosome with the GAL4 and GAL80ts genes under tubulin promoters in a heterozygote with the chromosome, containing a UAS-RNAi transgene – a source of dsRNA to one of the modifier genes under study (egg, SAYP, Su(var)2-HP2, XNP, CG2116, E(var)3-9 and Polybromo). GAL4 activates transcription of UAS-eGFP as well as UAS-RNAi, causing knockdown of the EP modifier. GAL80ts inactivates GAL4 by suppressing transcription from UAS-dependent promoters, and the degree of suppression decreases with increasing temperature. Knockdown of the EP modifier can be expressed in increased or suppressed trans-inactivation (illustrations of the effects are shown in the photo on the right).

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3. Fig. 2. Scheme of crosses to obtain genotypes used to study the effects of knockdowns of chromatin components on trans-inactivation, a table of the genes studied and the effect of knockdown of varying degrees on the viability of flies: the course of crosses and the genotypes used in the work. Four genotypes were obtained, representing all possible combinations of “presence – absence of trans-inactivation” and “presence – absence of knockdown”. All genotypes contain the GAL4 and GAL80ts genes. The ratio of reporter fluorescence in eGFP/A4 to eGFP/+ without UAS-RNAi (UAS-eGFP/A4; tubGAL4 tubGAL80ts/TM6 to UAS-eGFP/CyO; tubGAL4 tubGAL80ts/TM6) shows the level of trans-inactivation without modifier gene knockdown. Reporter fluorescence ratio in eGFP/A4; UAS-RNAi to eGFP/+; UAS-RNAi (UAS-eGFP/A4; tubGAL4 tubGAL80ts/UAS-RNAi to UAS-eGFP/CyO; tubGAL4 tubGAL80ts/UAS-RNAi) shows the level of trans-inactivation upon knockdown of the gene under study. The ratio of trans-inactivation levels “knockdown/without knockdown” determines the degree of influence of the modifier gene on trans-inactivation (a); the effect of knockdown of the studied genes on the viability of individuals. The extent of knockdown increases with increasing temperature when inactivation of the GAL80ts repressor increases the concentration of active GAL4). Developmental stages are designated as L (larva), K (pupa), V (adults). Accordingly, LKV denotes survival at all stages at a given temperature, LK - larvae and pupae survive, L - only larvae. In the case of XNP knockdown, males survived (b).

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4. Fig. 3. Pairwise comparison of the fluorescence intensity of Malpighian vessel cells in flies with UAS-eGFP under normal conditions and with trans-inactivation without knockdown (–RNAi) and with knockdown of one of the proteins (–Xnp, –SAYP, –CG2116, –olybromo, –eggless , –E(var)3-9, –HP2).

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