Identification and Functional Characterization of Polycomb Group Complexes in Drosophila

Polycomb group (PcG) and trithorax group (trxG) proteins were first discovered in Drosophila as repressors and activators of homeotic (HOX) genes, a set of transcription factors that specify cell identity along the anteroposterior axis of segmented animals. Subsequent work has shown that PcG and trxG proteins form multimeric complexes that are not required to initiate the regulation of HOX genes, but rather to maintain their expression state after the initial transcriptional regulators disappear from the embryo. The patterns of homeotic gene expression are initially set by the gap and pair-rule gene products. The early expressed gap and pair rule proteins disappear by about four hours of embryogenesis. Then, the PcG proteins maintain transcriptional repression of homeotic genes in cells where the initial expression state is off (McKeon et al., 1994; Simon et al., 1992; Struhl and Akam, 1985). In contrast, the trxG proteins maintain homeotic gene expression in cells where the initial expression state is on (reviewed in Kennison, 1993). By definition, an epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Epigenetic regulation of gene expression is necessary for the correct deployment of developmental programs and for the maintenance of cell fates. Since PcG proteins are responsible for the stable propagation of homeotic gene repression, after the initial decision has been made by other factors, they are referred as epigenetic regulators. Recent studies provide evidence that the PcG maintenance system regulates many other target genes in addition to homeotic genes, involved in development, cell proliferation, stem cell identity and cancer (Martinez and Cavalli, 2006; Ringrose and Paro 2004; Schwartz and Pirrotta 2007; Sparmann and van Lohuizen 2006)

dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing

Transcription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase dKDM2. In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression

Transcription-Independent Function of Polycomb Group Protein PSC in Cell Cycle Control

Polycomb group (PcG) proteins control development and cell proliferation through chromatin-mediated transcriptional repression. We describe a transcription-independent function for PcG protein Posterior sex combs (PSC) in regulating the destruction of cyclin B (CYC-B). A substantial portion of PSC was found outside canonical PcG complexes, instead associated with CYC-B and the anaphase-promoting complex (APC). Cell-based experiments and reconstituted reactions established that PSC and Lemming (LMG, also called APC11) associate and ubiquitylate CYC-B cooperatively, marking it for proteosomal degradation. Thus, PSC appears to mediate both developmental gene silencing and posttranslational control of mitosis. Direct regulation of cell cycle progression might be a crucial part of the PcG system's function in development and cancer

Transcription-independent function of polycomb group protein PSC in cell cycle control

Silence, Please Polycomb group (PcG) proteins play pivotal roles in epigenetic gene control, development, and disease. PcG proteins can control cell cycle progression, but the underlying mechanisms remain unclear. PcG proteins are thought to silence transcription of target genes through modulating chromatin structure. Mohd-Sarip et al. (p. 744 , published online 5 April) uncovered an unanticipated transcription-independent function for the canonical PcG protein “Posterior sex combs” in cell cycle control, through its direct regulation of cyclin B destruction. </jats:p

UV-sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-coupled repair

Transcription-coupled nucleotide-excision repair (TC-NER) is a subpathway of NER that efficiently removes the highly toxic RNA polymerase II blocking lesions in DNA. Defective TC-NER gives rise to the human disorders Cockayne syndrome and UV-sensitive syndrome (UV S S) . NER initiating factors are known to be regulated by ubiquitination 2 . Using a SILACbased proteomic approach, we identified UVSSA (formerly known as KIAA530) as part of a UV-induced ubiquitinated protein complex. Knockdown of UVSSA resulted in TC-NER deficiency. UVSSA was found to be the causative gene for UV S S, an unresolved NER deficiency disorder 3 . The UVSSA protein interacts with elongating RNA polymerase II, localizes specifically to UV-induced lesions, resides in chromatinassociated TC-NER complexes and is implicated in stabilizing the TC-NER master organizing protein ERCC6 (also known as CSB) by delivering the deubiquitinating enzyme USP7 to TC-NER complexes. Together, these findings indicate that UVSSA-USP7-mediated stabilization of ERCC6 represents a critical regulatory mechanism of TC-NER in restoring gene expression. Nucleotide-excision repair removes a wide range of DNA damage, including UV-induced lesions. Inherited NER defects lead to extreme cancer proneness (xeroderma pigmentosum) or dramatic premature aging (Cockayne syndrome), showing the clinical impact of NER 4 . NER is initiated by two damage recognition pathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER). DNA helix-distorting injuries located throughout the genome are repaired by GG-NER to avoid replication-induced mutations and resultant cancer. TC-NER targets transcription-blocking lesions to enable recovery of arrested transcription, thereby preventing damageinduced apoptosis and resultant aging. In addition, it was shown that TC-NER is also important in overcoming UV-induced transcriptionassociated mutations NER is regulated in response to UV irradiation by ubiquitination 2 of the process-initiating factors xeroderma pigmentosum group C (XPC) 2 ADVANCE ONLINE PUBLICATION Nature GeNetics l e t t e r s TC-NER-deficient cells To investigate the in vivo role of UVSSA, we measured the dynamic association of UVSSA with NER in living cells. Green fluorescent protein (GFP)-tagged UVSSA, which was shown to be biologically active ( Nature GeNetics ADVANCE ONLINE PUBLICATION 3 l e t t e r s with . In contrast, α-amanitin released RNA Pol II from DNA, which also resulted in increased mobility of UVSSA. The opposing effects by these transcription inhibitors on GFP-UVSSA mobility in living cells cannot be caused by an indirect UVSSA interaction with RNA Pol II via ERCC6 (ref. 25), as similar effects were found in CS-B cells To examine whether UVSSA is also present in active, chromatinbound TC-NER complexes via its interaction with RNA Pol IIo, we performed ChIP experiments with antibodies against HGMN1, a chromatin remodeler that is enriched in lesion-stalled TC-NER complexes ADVANCE ONLINE PUBLICATION Nature GeNetics l e t t e r s Whether this RNA Pol II recovery is a direct effect of UVSSA or an indirect consequence of rescued TC-NER remains to be determined. In order to examine how UVSSA influences ERCC6 protein stability, we immunoprecipitated GFP-UVSSA and analyzed UVSSAinteracting proteins by mass spectrometry. Of note, we identified the deubiquitinating enzyme ubiquitin carboxyl-terminal hydrolase 7 (USP7, also called HAUSP) that is known to have various functions in DDR In summary, within our mass spectrometry analysis of the UVinduced ubiquitinome, we identified an uncharacterized protein (UVSSA), which was highly enriched upon UV irradiation in immunopurified ubiquitinated protein complexes. Functional analysis indicated that UVSSA is a new factor implicated in TC-NER and, of note, is the causative gene in the unresolved UV S S-A NER disorder. Two accompanying studies aimed at finding the genetic defect in UV S S-A also identified UVSSA as the gene mutated in individuals with UV S S-A 20,21 . Their further functional analysis is consistent with our observation that UVSSA has an important role in TC-NER and is part of the protein complex containing the elongating form of RNA Pol II. Our data argue for a UV-independent UVSSA-RNA Pol IIo interaction. In contrast to GFP-UVSSA, we were not able to observe RNA Pol IIo accumulation at LUD with current technology. The transient or low-affinity interaction between UVSSA and RNA Pol IIo might be stabilized upon UV irradiation, explaining the UV dependency observed in native immunoprecipitations 20 and the UV independence of the fixed interactions by cross-linking in ChIP shown here. We propose a model Nature GeNetics ADVANCE ONLINE PUBLICATION 5 l e t t e r s Pol II after UV damage 21 seems to imply that UVSSA has functions in TC-NER beyond being a specific shuttle protein for USP7. The causative genes for the two TC-NER defective disorders Cockayne syndrome and UV S S (ERCC6, ERCC8 and UVSSA) are all cofactors of lesion-stalled RNA Pol II. However, the phenotypes of these two TC-NER deficiencies are strikingly different; Cockayne syndrome is characterized by severe neurological and developmental abnormalities in conjunction with UV sensitivity, whereas individuals with UV S S mainly exhibit sun sensitivity, without any clear additional complications METhoDS Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/. Note: Supplementary information is available on the Nature Genetics website. ACknoWLeDgMentS We thank R. Bernards and M. Epping (Nederlands Kanker Instituut) for the Myc-tagged USP7 expression construct and P. Verrijzer and A. Reddy (Erasmus Medical Centre) for shUSP7-expressing lentivirus. We thank H. Slor (Tel Aviv University) for the TA-24sv40 cell line and N.G.J. Jaspers and H. Lans for discussions and critical reading of the manuscript. This work was funded by the Netherlands Genomics Initiative NPCII (to P.S.), 935.19.021 and 935.11.042 (to W.V., C.L. and J.A.M.), the Dutch Organization for Scientific Research ZonMW Veni Grant (917.96.120 to J.A.M.) and TOP grant (912.08.031 to W.V.), Marie Curi