Research Article 5489 Single-cell analysis of Daxx and ATRX-dependent transcriptional repression Alyshia Newhart1, Ilona U. Rafalska-Metcalf1, Tian Yang2, Dmitri G. Negorev1 and Susan M. Janicki1,* 1Molecular and Cellular Oncogenesis Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA 2University of Pennsylvania, Roy and Diana Vagelos Scholars Program in Molecular Life Sciences, Philadelphia, PA 19104, USA *Author for correspondence ([email protected]) Accepted 6 August 2012 Journal of Cell Science 125, 5489–5501 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.110148 Summary Histone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0- expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function. Key words: Daxx, ATRX, Histone H3.3, ICP0, Transcription, Chromatin Journal of Cell Science Introduction 2004), and Daxx, together with the Snf2-type chromatin- For a gene to be silenced or expressed, the functions of multiple remodeling factor, ATRX, is required for H3.3 incorporation factors must be coordinated at the chromatin/DNA interface. into telomeres and pericentric heterochromatin (Drane´ et al., 2010; Since many of these factors were discovered using techniques, Goldberg et al., 2010; Lewis et al., 2010). The finding that Daxx/ which disrupt cellular architecture and average effects in cell ATRX and HIRA regulate mutually exclusive sites suggests that populations, it is not known how or when critical regulators are their targeting signals are critical regulators of replication selectively recruited to specific genomic sites in intact cells. One independent histone H3.3 chromatin assembly. of the pivotal advances to imaging chromatin in single cells has As histone H3.3 is incorporated into both transcriptionally been the development of reporter plasmids carrying sequence active and inactive chromatin, it is not known how these sites are elements from bacteria and bacteriophage that allow DNA and functionally linked by its deposition. Interestingly, disruption of RNA to be visualized in living cells (Rafalska-Metcalf and the HIRA and Daxx/ATRX pathways has markedly different Janicki, 2007). The genomic sites into which these plasmids effects on transcription and chromatin. Although histone H3.3 is integrate can be visualized when their binding proteins, fused to not incorporated into single-copy genes in HIRA2/2 embryonic auto-fluorescent proteins are expressed. As such, these types of stem cells (ESCs), large-scale changes in gene expression are not systems provide an unparalleled opportunity to observe the detected (Goldberg et al., 2010). However, knockdown of either temporal and spatial organization of gene regulatory events histone H3.3 or ATRX in ESCs induces a telomere dysfunction directly at chromatin (Darzacq et al., 2007; Shanbhag et al., 2010; phenotype, which indicates that this pathway is critical for Shav-Tal et al., 2004; Zhao et al., 2011). maintaining telomeric chromatin organization (Wong et al., Histone H3.3 is a constitutively expressed variant of histone H3, 2010; Wong et al., 2009). which is linked to the epigenetic inheritance of chromatin states Before the mechanistic role of Daxx and ATRX in histone (Elsaesser et al., 2010). Originally associated with sites of active H3.3 chromatin assembly was identified, both of these factors transcription (Ahmad and Henikoff, 2002), it is now known to also were known to be components of PML nuclear bodies (PML- be incorporated into heterochromatin with assembly at these NBs)/nuclear domain 10s (ND10s) and to function in the distinct sites regulated by different chaperones (Drane´ et al., 2010; transcriptional repression of genes and viruses. In fact, many Goldberg et al., 2010; Hake et al., 2005; Wong et al., 2009). HIRA viruses encode proteins that disorganize and/or degrade PML-NB is an H3.3-specific chaperone, which functions at single copy components, including Daxx, ATRX and PML, in the effort to genes (Goldberg et al., 2010; Ray-Gallet et al., 2002; Tagami et al., evade cellular resistance to infection (Geoffroy and Chelbi-Alix, 5490 Journal of Cell Science 125 (22) 2011). Specifically, the degradation of Daxx by the CMV viral translation can be confirmed by the appearance of the CFP-SKL tegument protein, pp71, is important for the initiation of protein in the peroxisomes in the cytoplasm. Therefore, this efficient HCMV gene expression (Cantrell and Bresnahan, ‘synthetic’ gene contains all of the elements required for RNA pol 2006; Hofmann et al., 2002; Hwang and Kalejta, 2007; Ishov II transcription, mRNA processing, and protein translation. As et al., 2002; Preston and Nicholl, 2006). Additionally, ICP0, the such, it is a powerful tool for defining the temporal and spatial HSV-1 E3 ubiquitin ligase required for viral replication and organization of gene regulatory events directly at chromatin activation of the latent genome, promotes the disaggregation of (Janicki et al., 2004). PML-NBs/ND10s and degradation of their components including To investigate replication independent histone H3.3 chromatin PML (Everett et al., 1998). assembly using this system, we evaluated the localization of HIRA In addition to viral infections, Daxx and ATRX are significantly and Daxx at a transgene array stably integrated into U2OS cells linked to several other diseases. ATRX is mutated in the intellectual (cell line, 2-6-3) (Janicki et al., 2004). Neither YFP-tagged nor disability syndrome, a-thalassemia/mental retardation, X-linked antibody labeled endogenous HIRA (Fig. 1B, panels a2h) (Picketts et al., 1996). Daxx and ATRX are mutated in pancreatic accumulated at the activated array marked by the activator, neuroendocrine tumors (PanNETs) (Jiao et al., 2011), ATRX is Cherry/CFP-tTA-ER. In contrast, both YFP-tagged and mutated in oligodendrogliomas and medulloblastomas (Heaphy endogenous Daxx were enriched at the inactive and activated et al., 2011; Molenaar et al., 2012), and Daxx, ATRX and H3.3 are arrays, marked by Cherry-lac repressor and Cherry-tTA-ER, all mutated in pediatric glioblastoma (Schwartzentruber et al., 2012; respectively (Fig. 1B, panels i–p; supplementary material Fig. Wu et al., 2012). As all PanNETs with inactivating mutations in S1). The enrichment of Daxx, but not HIRA, suggests that it, Daxx or ATRX are positive for the telomerase-independent together with ATRX, regulates histone H3.3 chromatin assembly telomere maintenance mechanism termed ALT (alternative at the array and is consistent with reports that Daxx and HIRA lengthening of telomeres) (Heaphy et al., 2011), this further function at mutually exclusive sites (Elsaesser and Allis, 2010; confirms the importance of these factors in telomere regulation. Goldberg et al., 2010). As ICP0-deficient HSV-1 replicates as well as wild-type virus in the ATRX-null and ALT-positive human osteosarcoma cell line, U2OS ATRX represses activation of the transgene array in U2OS (Heaphy et al., 2011; Lukashchuk and Everett, 2010; Yao and cells Schaffer, 1995), this also highlights the critical position of this As U2OS cells do not express ATRX (Heaphy et al., 2011; pathway at the interface between gene silencing and intrinsic Lukashchuk and Everett, 2010) (Fig. 2A), this suggests that the cellular defense. Daxx and ATRX pathway is not functional in this cell line. To We previously reported that an array of an inducible CMV- determine whether exogenously expressed ATRX is recruited to promoter regulated transgene stably integrated into U2OS cells the array, we co-expressed ATRX-YFP and Cherry-lac repressor. can be robustly activated and that histone H3.3 is strongly As they co-localized (Fig. 2B, panels a–d), this suggested that recruited to the active site (Janicki et al., 2004). Here, we report ATRX functions at the site. that Daxx, ATRX and PML are enriched at the CMV promoter of Previously, we reported that the transgene array in this cell line this transgene and that when it is integrated into HeLa cells, significantly decondenses during activation (Janicki et al., 2004; Journal of Cell Science which express both Daxx and ATRX, the array is refractory to Rafalska-Metcalf et al., 2010). To evaluate the effects of ATRX on activation. As ICP0 depletes Daxx, ATRX and PML from the array decondensation, we measured the pixel areas of the regions array, permits its activation, and prevents histone H3.3 from bound by Cherry-lac repressor (inactive array) and Cherry-tTA-ER being incorporated into its chromatin, this suggests that histone (activated arrays). In activated control cells, the array was ,56 H3.3 chromatin assembly is blocked during activation. larger than the inactive site (Fig.