Phosphorylation of SU(VAR)3-9 by the chromosomal kinase JIL-1

The histone methyltransferase SU(VAR)3-9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR)3-9's activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR)3-9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR)3-9 N-terminus. Among several other proteins known to bind Su(VAR)3-9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR)3-9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR)3-9 and JIL-1

The RNA Helicase Rm62 Cooperates with SU(VAR)3-9 to Re-Silence Active Transcription in Drosophila melanogaster

Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. We show that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase SU(VAR)3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of SU(VAR)3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range

Phosphorylation of SU(VAR)3–9 by the Chromosomal Kinase JIL-1

The histone methyltransferase SU(VAR)3–9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR)3–9's activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR)3–9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR)3–9 N-terminus. Among several other proteins known to bind Su(VAR)3–9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR)3–9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR)3–9 and JIL-1.This article is published as Boeke J, Regnard C, Cai W, Johansen J, Johansen KM, Becker PB, et al. (2010) Phosphorylation of SU(VAR)3–9 by the Chromosomal Kinase JIL-1. PLoS ONE 5(4): e10042. doi: 10.1371/journal.pone.0010042.</p

SU(VAR)3–9 and JIL-1 interact <i>in vivo</i>.

<p>(<b>A</b>) Coexpression of JIL-1 and SU(VAR)3–9 in SF9 cells using a baculoviral expression system. <i>left</i>: Western Blot of whole cell Sf9 extract. Proteins were detected using the indicated antibodies. <i>right:</i> Histone methyltransferase assay after co-infection of SF9 cells with flag-JIL-1 and his-SU(VAR)3–9 followed by affinity purification on a Talon™ metal affinity resin. (<b>B</b>) Precipitation of JIL-1 from an SF9 cell extract co-purifies active SU(VAR)3–9. <i>left</i>: Western Blot analysis of flag-coimmunoprecipitations using SF9 whole cell extract after co-infection with the indicated viruses. After the Co-IP flag M2-beads were washed and eluted with the flag peptide. 10% of the eluates were separated by SDS PAGE and analyzed by Western blotting using specific antibodies. <i>right:</i> Histone methyltransferase assay after co-infection of SF9 cells with flag-JIL-1, a flag-JIL^D392A and his-SU(VAR)3–9 followed by affinity purification on a flag M2 affinity resin.</p

SU(VAR)3–9 can repress transcription when tethered to a promoter.

<p>(<b>A</b>) SU(VAR)3–9 represses transcription in a TSA-dependent manner. (<b>B</b>) An N-terminal truncation of SU(VAR)3–9 lacking the domain that interacts with JIL-1 and RPD3 can no longer repress transcription. (<b>C</b>) SU(VAR)3–9's transcriptional repression capacity is 2 fold reduced in a S191A mutated SU(VAR)3–9. <i>Drosophila</i> SL2 cells were co-transfected with expression constructs for dorsal and twist and the reporter construct pG₅DE₅-tkluc together with the indicated plasmids coding for: GAL4-DBD or GAL4 fusion proteins of SU(VAR)3–9 (and deletions/mutations thereof) and RPD3 in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). <i>left:</i> luciferase assay of the activated reporter gene after transfection with the indicated plasmids. The repression capacity of GAL4-SU(VAR)3–9 was set to 100% and all other values were normalized accordingly. <i>right</i>: Western Blot analysis of the extracts used for the luciferase assay using an antibody specific against the GAL4-DBD [G: GAL4 DBD; R: GAL4-Rpd3; S: GAL4- SU(VAR)3–9].</p