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. 2001 Oct;2(10):915-9.
doi: 10.1093/embo-reports/kve210. Epub 2001 Sep 24.

Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila

B Czermin  1 , G Schotta, B B Hülsmann, A Brehm, P B Becker, G Reuter, A Imhof
Affiliations

Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila

B Czermin et al. EMBO Rep. 2001 Oct.

Abstract

Modification of histones can have a dramatic impact on chromatin structure and function. Acetylation of lysines within the N-terminal tail of the histone octamer marks transcriptionally active regions of the genome whereas deacetylation seems to play a role in transcriptional silencing. Recently, the methylation of the histone tails has also been shown to be important for transcriptional regulation and chromosome structure. Here we show by immunoaffinity purification that two activities important for chromatin-mediated gene silencing, the histone methyltransferase SU(VAR)3-9 and the histone deacetylase HDAC1, associate in vivo. The two activities cooperate to methylate pre-acetylated histones. Both enzymes are modifiers of position effect variegation and interact genetically in flies. We suggest a model in which the concerted histone deacetylation and methylation by a SU(VAR)3-9/HDAC1-containing complex leads to a permanent silencing of transcription in particular areas of the genome.

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Figures

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Fig. 1. Purification of a HIM from Drosophila embryo extracts. (A) Purification scheme for the purification of myc-tagged SU(VAR)3-9 from Drosophila embryonal extracts. (B) Purification of myc-SU(VAR)3-9 from nuclear extracts prepared from flies expressing a myc-tagged SU(VAR)3-9 (Aagaard, 1999) on a resource Q column. Western blot of fractions eluted from the resource Q column probed with an anti-c-myc antibody (9E10). (C) HIM activity assay with immunoprecipitates of SU(VAR)3-9 from an extract (myc-NE) or from partially purified myc-SU(VAR)3-9 fractions (fraction 26) prepared from flies expressing triple-myc-tagged SU(VAR)3-9 using an anti-myc antibody (9E10). As controls immunoprecipitations were also performed without the addition of antibody (mock) or from an extract prepared from wild-type embryos (wt).
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Fig. 2. Lysine specificity of purified SU(VAR)3-9. (A) Specificity assay of recombinant SUV3-9H1 (white bars) and SU(VAR)3-9 prepared as described in Figure 1A (dark bars) on various modified peptides. Modifications used were unmodified (wt), methylated on lysine 4 (K4me), methylated on lysine 9 (K9me) and acetylated on lysine 9 (K9ac). (B) Reactions were performed as in (A) with the exception that reactions contained either 50 nM Trichostatin A (+TSA) dissolved in ethanol or only ethanol (–TSA).
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Fig. 3. Association of SU(VAR)3-9 with HDAC1. (A) HDAC activity assay using immunoprecipitated material from two independently prepared nuclear extracts (myc-NE1 and myc-NE2) or a partially purified fraction (fraction 26) from the tagged fly strain precipitated with an anti-c-myc antibody (9E10) or no antibody (mock). (B) Western blot of immunoprecipitated material from (A) probed either with anti-HDAC1 (top panel), anti-HDAC3 antibodies (bottom panel, lanes 1–3) or anti-c-myc antibody (9E10) (bottom panel, lanes 5–6); the asterisk indicates the light chain of the antibody used for immunoprecipitation. The input lane (lane 1) contains ∼30% of the material used for immunoprecipitations. (C) HIM and HDAC activity assays of recombinantly expressed HDAC1 (left panel) and SUV3-91 (right panel). (D) HIM activity with immunoprecipitates using an anti-HDAC1 antibody from a partially purified fraction (Figure 1B, fraction 26). (E) HIM activity assay using material immunoprecipitated with an anti-flag antibody. Immunoprecipitates were purified from Drosophila control cells (SL-2) or cells stably expressing flag-tagged HDAC1 (fHDAC1).
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Fig. 4. The HDAC326 mutation dominates the PEV enhancer effect of an additional genomic copy of Su(var)3-9. The P[3–9] carries one additional genomic copy of Su(var)3-9 introduced by P[(ry+) 11kb SU(VAR)3-9] at 100E.
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References

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