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. 2017 Dec 1;10(1):56.
doi: 10.1186/s13072-017-0163-z.

Functional Dissection of Drosophila Melanogaster SUUR Protein Influence on H3K27me3 Profile

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Free PMC article

Functional Dissection of Drosophila Melanogaster SUUR Protein Influence on H3K27me3 Profile

Olga V Posukh et al. Epigenetics Chromatin. .
Free PMC article

Abstract

Background: In eukaryotes, heterochromatin replicates late in S phase of the cell cycle and contains specific covalent modifications of histones. SuUR mutation found in Drosophila makes heterochromatin replicate earlier than in wild type and reduces the level of repressive histone modifications. SUUR protein was shown to be associated with moving replication forks, apparently through the interaction with PCNA. The biological process underlying the effects of SUUR on replication and composition of heterochromatin remains unknown.

Results: Here we performed a functional dissection of SUUR protein effects on H3K27me3 level. Using hidden Markow model-based algorithm we revealed SuUR-sensitive chromosomal regions that demonstrated unusual characteristics: They do not contain Polycomb and require SUUR function to sustain H3K27me3 level. We tested the role of SUUR protein in the mechanisms that could affect H3K27me3 histone levels in these regions. We found that SUUR does not affect the initial H3K27me3 pattern formation in embryogenesis or Polycomb distribution in the chromosomes. We also ruled out the possible effect of SUUR on histone genes expression and its involvement in DSB repair.

Conclusions: Obtained results support the idea that SUUR protein contributes to the heterochromatin maintenance during the chromosome replication. A model that explains major SUUR-associated phenotypes is proposed.

Keywords: Drosophila; Epigenetic inheritance; H3K27me3; Heterochromatin; Polycomb; Replication.

Figures

Fig. 1
Fig. 1
SuUR mutation affects H3K27me3 level independently from local under-replication. a Examples of chromosomal regions that display decrease in H3K27me3 levels upon SuUR mutation, but are fully polytenized in wild type. Compared to under-replicated regions 71C and 64C. Polytenization data were taken from [13]. Arrowheads show the positions of qPCR primers that were used to validate polytenization levels in these regions. Blue shading demarks SSRs identified with HMM. b SSRs identified with HMM display systematic decrease in H3K27me3 levels upon SuUR mutation. Compared to 1000 random chromosomal regions. c qPCR analysis of the polytenization levels in SSRs, shown in (a)
Fig. 2
Fig. 2
H3K27me3 profile is unaffected by SuUR mutation in early embryos. Examples of H3K27me3 profiles in some SSRs, in 0–4-h embryos and in salivary glands of wild type strain and SuUR mutants. No major changes occur upon SuUR mutation in embryos as compared to salivary glands. The arrow shows the position of Pc-binding site in 75C that is unchanged between wild type and SuUR mutants (see below)
Fig. 3
Fig. 3
SSRs are devoid of Pc protein while SNRs represent PRC1 domains. a SSRs (shaded in blue) are devoid of Pc binding as exemplified by 10 Mb span of chromosome 2L. SNRs (see text) match the Pc-bound domains (arrows). H3K27me3 profiles are presented as quantile normalized log2(IP/inp) values. Pc DamID profiles are presented as log10(P) units, where P—significance level assessed with Fisher’s exact test. Dotted lines designate FDR = 0.5 levels for Pc enrichment. Blue shading shows positions of SSRs, arrows point at SNRs. b Scatter plot of Polycomb DamID data in salivary glands of wild type strain and in SuUR mutants. SNRs are shown in blue while SSRs—in red. Pc DamID signal is presented as log10(P) units, where P—significance level assessed with Fisher’s exact test. c Pc peak density in SSRs, SNRs and in 1000 random regions across the genome. SuUR mutation does not affect Pc distribution nor in SSRs nor in SNRs. d Scatter plot of quantile-normalized H3K27me3 ChIP-chip data in salivary glands of wild type strain and in SuUR mutants. SNRs are shown in blue while SSRs—in red. The scales are log2(IP/inp) values. e Genomic regions corresponding to SSRs and SNRs in Kc167 cells show characteristic distribution of chromatin types according to the classification of [5]
Fig. 4
Fig. 4
Both SSRs and SNRs contain SUUR protein. a DamID profiling detects SUUR protein in SSR (shaded in blue) and in SNRs (black frames). The 600-kb fragment of chromosome 3R is shown. H3K27me3 profiles are presented as quantile normalized log2(IP/inp) values. SUUR and Pc DamID profiles are presented as log10(P) units, where P—significance level assessed with Fisher’s exact test. Dotted lines designate FDR = 0.5 levels for Pc enrichment. b SUUR peak density in SSRs, SNRs and in 1000 random regions across the genome
Fig. 5
Fig. 5
mxc G43 mutants increase polytenization in under-replicated regions. a, b Polytenization profiles in two under-replicated regions 25A and 75C were built using qPCR in mxc G43 mutants (red) and their wild type siblings (blue). c Polytenization levels of the tags from the pericentric heterochromatin were assessed with qPCR. Although located in pericentric heterochromatin, Gbp5 gene is 100% polytenized in all genotypes and is shown for comparison. mxc G43 mutation affects under-replication in both pericentric heterochromatin and in the late replicating regions scattered along chromosomal arms
Fig. 6
Fig. 6
Scheme explaining the effects of SUUR on H3K27me3 maintenance and under-replication in polytene chromosomes. a Possible mechanisms that were checked in this study that could affect H3K27me3 in SuUR mutants. b In wild type, SUUR contributes to chromatin maintenance in a way that replication forks are delayed to allow the H3K27me3-enriched chromatin context to be re-established. This would result in under-replication in the most extended SSRs and SNRs. PRCs also act in SNRs and produce H3K27 methylation. c In SuUR mutants, SUUR-dependent chromatin maintenance is inactivated, so the replication is more efficient while H3K27me3 is lost in SSRs. In SNRs, H3K27me3 level is reconstituted by the resident PRCs. Below the schematic pictures of polytene chromosomes are shown. Red dots—H3K27me3 histones

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