Human origin recognition complex is essential for HP1 binding to chromatin and heterochromatin organization Supriya G. Prasantha,b,1, Zhen Shenb, Kannanganattu V. Prasanthb, and Bruce Stillmana,1 aCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; and bDepartment of Cell and Developmental Biology, University of Illinois, Urbana- Champaign, IL 61801 Contributed by Bruce Stillman, July 9, 2010 (sent for review April 10, 2010) The origin recognition complex (ORC) is a DNA replication initiator (15, 22, 23), cytokinesis in human cells and Drosophila (24, 25), protein also known to be involved in diverse cellular functions regulation of dendrite development in postmitotic neurons (26), including gene silencing, sister chromatid cohesion, telomere bi- and neural transmission and synaptic function in Drosophila (27) ology, heterochromatin localization, centromere and centrosome (see reviews in refs. 28, 29). activity, and cytokinesis. We show that, in human cells, multiple ORC The human Orc2 subunit is associated with heterochromatin in subunits associate with hetereochromatin protein 1 (HP1) α-and a cell cycle-dependent manner, being present only at the cen- HP1β-containing heterochromatic foci. Fluorescent bleaching studies tromeres during late G2 and mitosis (15, 30). Furthermore, Orc2 indicate that multiple subcomplexes of ORC exist at heterochroma- biochemically interacts indirectly with the heterochromatin protein tin, with Orc1 stably associating with heterochromatin in G1 phase, 1 (HP1) in mammalian, Drosophila,andXenopus cells (14, 15, 31, whereas other ORC subunits have transient interactions throughout 32). Depletion of Orc2 or Orc3 in HeLa cells results in a mitotic the cell-division cycle. Both Orc1 and Orc3 directly bind to HP1α,and arrest with abnormally condensed chromosomes, lack of chromo- two domains of Orc3, a coiled-coil domain and a mod-interacting some alignment at the metaphase plate, and spindle defects (15). region domain, can independently bind to HP1α; however, both The interaction between ORC and heterochromatic proteins are essential for in vivo localization of Orc3 to heterochromatic foci. α β CELL BIOLOGY Direct binding of both Orc1 and Orc3 to HP1 suggests that, after the like HP1 and and ORC binding to centromeres suggests a di- rect role for ORC subunits in chromosome organization and for degradation of Orc1 at the G1/S boundary, Orc3 facilitates assembly – of ORC/HP1 proteins to chromatin. Although depletion of Orc2 and chromosome condensation and segregation during mitosis (33 Orc3 subunits by siRNA caused loss of HP1α association to hetero- 35). HP1 is a heterochromatin-associated protein that localizes to chromatin, loss of Orc1 and Orc5 caused aberrant HP1α distribution pericentromeres and telomeres, has a dose-dependent effect on only to pericentric heterochromatin-surrounding nucleoli. Depletion gene silencing, and has a critical role in heterochromatin forma- of HP1α from human cells also shows loss of Orc2 binding to het- tion and maintenance (36). To address the role of ORC/HP1 in erochromatin, suggesting that ORC and HP1 proteins are mutually heterochromatin organization and mitotic progression, we sys- required for each other to bind to heterochromatin. Similar to tematically analyzed the role of individual ORC subunits in HP1 HP1α-depleted cells, Orc2 and Orc3 siRNA-treated cells also show binding and recruitment to heterochromatic foci in human cells. loss of compaction at satellite repeats, suggesting that ORC to- We find that ORC and HP1 are required for maintenance of each gether with HP1 proteins may be involved in organizing higher- other’s association with heterochromatin and identify a role for order chromatin structure and centromere function. ORC in maintenance of higher-order chromatin structure at centromeres. Interestingly, different ORC subunits play differ- centromere | origin recognition complex | pericentric heterochromatin | ential roles in heterochromatin structure and function. chromatin structure Results he initiation of DNA replication in eukaryotic cells is a highly Multiple ORC Subunits Localize to Heterochromatin. Immunofluo- Tcoordinated process that is intimately linked to chromatin or- rescence studies showed that human and mouse Orc2 localizes in ganization. The accurate duplication of DNA is governed by many a cell cycle-regulated manner to many diffuse foci in the nucleus, proteins including the origin recognition complex (ORC) (1, 2). HP1-containing heterochromatic loci, and centrosomes and Binding of ORC to replication origins is a key step in initiation centromeres (Fig. 1 B’–D’) (11, 15, 20, 21, 30). In addition to Orc2, of DNA replication, leading to the recruitment of other prerep- other ORC subunits including Orc1, Orc3, and Orc5 localize at lication complex (pre-RC) proteins including Cdc6, Cdt1, and the Orc2- and HP1-associated heterochromatin in MCF7 cells 2-7 the MCM . Unlike yeast ORC, human ORC binds to chromatin (Fig. 1). In human cells, Orc2 coimmunoprecipitates with HP1α fi – in a nonsequence-speci c manner (3). In human cells, Orc2 5 and β (15), and in Drosophila embryonic nuclei, HP1 colocalizes forms the core ORC, and Orc1 is transiently associated with this – with Orc2 at heterochromatin (14). Because other ORC subunits complex (4 7). During the G1 to S phase transition, human Orc1 also colocalize with HP1α in human cells, it suggests an evolu- dissociates from chromatin, is ubiquitylated and degraded by tionary conserved function for ORC at heterochromatin. ubiquitin-mediated proteolysis, and is reloaded to the chromatin at the M-G1 transition when new pre-RCs are formed (5, 7–9). The remaining ORC subunits are dynamically associated with chro- Author contributions: S.G.P. and B.S. designed research; S.G.P., Z.S., and K.V.P. performed matin throughout the remainder of the cell cycle (5, 6). research; S.G.P. and B.S. contributed new reagents/analytic tools; S.G.P., Z.S., K.V.P., and In addition to their role in DNA replication, ORC subunits have B.S. analyzed data; and S.G.P. and B.S. wrote the paper. also been implicated in transcriptional gene silencing in Saccha- The authors declare no conflict of interest. romyces cerevisiae (10), heterochromatin formation in Drosophila, Freely available online through the PNAS open access option. – mouse, and humans (11 15), sister chromatid cohesion in S. cer- 1To whom correspondence may be addressed. E-mail: [email protected] or stillman@ evisiae, Schizosaccharomyces pombe, and Xenopus (16–18), ribo- cshl.edu. some biogenesis (19), centrosome biology in human and mouse This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (15, 20, 21), mitotic chromosome function in human and Xenopus 1073/pnas.1009945107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1009945107 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 A A’ A’’ A Orc3-YFP HP1 DAPI B B’ B’’ Orc3-YFP Orc2 pAb DAPI C C’ C’’ YFP-Orc1 Orc2 pAb DAPI D D’ D’’ D’’’ YFP-Orc5 Orc2 pAb HP1 DAPI Fig. 1. Multiple ORC subunits localize with HP1 to heterochromatic foci. YFP fusion constructs were transfected into MCF7 cells, and endogenous B Orc1 Orc2 Orc3 Orc4 Orc5 proteins were detected by indirect immunofluorescence. Orc3-YFP coloc- alizes with endogenous HP1α at heterochromatic foci (A, A’, and A”). En- Input dogenous Orc2 and Orc3-YFP colocalize at the heterochromatic foci (B, B’, Orc1 Orc2 Orc3 Orc4 Orc5 GST-HP1 GST-HP1 GST-HP1 GST-HP1 GST-HP1 and B”). YFP-Orc1 colocalizes with endogenous Orc2 at the heterochromatic GST GST GST GST GST foci (C, C’, and C”). YFP-Orc5 showed colocalization with both endogenous Orc2 and HP1α at the heterochromatic foci in MCF7 cells (D, D’, D”, and D”’). Chromatin was stained with DAPI in blue. (Scale bar, 5 μm.) Differential Binding of ORC Subunits to Heterochromatin. A large proportion of the HP1 protein pool displays a remarkably dynamic exchange at heterochromatin (37–39). Because the human Orc1, Orc2, Orc3, and Orc5 subunits localized to heterochromatin, we compared the mobility of YFP-tagged ORC subunits with YFP- α fl Fig. 2. ORC dynamics at the heterochromatin. (A) FRAP kinetics of various HP1 at heterochromatin by uorescence recovery after photo- ORC subunits at the heterochromatic foci in MCF7 cells. Similar FRAP kinetics bleaching (FRAP) analysis (Fig. 2A). A defined heterochromatic of ∼3–4 s t1/2 for YFP-HP1α, YFP-Orc2, and Orc3-YFP were observed at region in the nucleus was irreversibly bleached of the YFP signal, heterochromatic foci, whereas YFP-Orc1 showed >40 s t1/2 at heterochro- and the recovery kinetics of fluorescence intensity into the bleached matin. (B) GST pull-down assays using GST or GST-HP1α beads and incubating region were assessed. Orc2 and Orc3 had very similar and complete with in vitro transcribed and translated mRNA from individual ORC subunits kinetics of fluorescence recovery, as observed for HP1α. However, shows direct binding of both human Orc1 and Orc3 to HP1α. The input is Orc1 showed very different and incomplete recovery kinetics, shown on Right. suggesting that Orc1 chromatin association is unique among ORC fl subunits tested, perhaps re ecting different chromatin interaction (aa151-269) (32). To map the region of Orc3 that interacts with (Fig. 2A). Thus, by FRAP analysis, it is clear that ORC proteins, HP1α, various mutants of Orc3 were generated in a coupled in especially Orc2 and Orc3, assemble and disassemble from the vitro transcription-translation vector and tested for binding to GST heterochromatic foci in a dynamic fashion like the bulk of HP1α or GST-HP1α (Fig. 3 A and B and Fig. S2). Two independent protein, but Orc1 seems to exchange less frequently, if at all. domains in Orc3, a coiled-coil domain at the N terminus and α a second region containing a MOD1-interacting region (MIR; 213- Orc1 and Orc3 Directly Bind to HP1 . In Drosophila and Xenopus, fi α HP1 was found to interact with the N-terminal domain of the Orc1 218aa) (42), were identi ed, both of which directly bound to HP1 .