Human origin recognition complex is essential for HP1 binding to chromatin and heterochromatin organization

The origin recognition complex (ORC) is a DNA replication initiator protein also known to be involved in diverse cellular functions including gene silencing, sister chromatid cohesion, telomere biology, heterochromatin localization, centromere and centrosome activity, and cytokinesis. Weshow that, in human cells, multiple ORC subunits associate with hetereochromatin protein 1 (HP1) α- and HP1β-containing heterochromatic foci. Fluorescent bleaching studies indicate that multiple subcomplexes of ORC exist at heterochromatin, with Orc1 stably associating with heterochromatin in G1 phase, whereas other ORC subunits have transient interactions throughout the cell-division cycle. Both Orc1 and Orc3 directly bind to HP1α, and two domains of Orc3, a coiled-coil domain and a mod-interacting region domain, can independently bind to HP1α; however, both are essential for in vivo localization of Orc3 to heterochromatic foci. Direct binding of both Orc1 and Orc3 to HP1 suggests that, after the degradation of Orc1 at the G1/S boundary, Orc3 facilitates assembly of ORC/HP1 proteins to chromatin. Although depletion of Orc2 and Orc3 subunits by siRNA caused loss of HP1α association to heterochromatin, loss of Orc1 and Orc5 caused aberrant HP1α distribution only to pericentric heterochromatin-surrounding nucleoli. Depletion of HP1α from human cells also shows loss of Orc2 binding to heterochromatin, suggesting that ORC and HP1 proteins aremutually required for each other to bind to heterochromatin. Similar to HP1α-depleted cells, Orc2 and Orc3 siRNA-treated cells also show loss of compaction at satellite repeats, suggesting that ORC together with HP1 proteins may be involved in organizing higher-order chromatin structure and centromere function.