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

Abstract

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. We show that, in human cells, multiple ORC subunits associate with hetereochromatin protein 1 (HP1) alpha- and HP1beta-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 HP1alpha, and two domains of Orc3, a coiled-coil domain and a mod-interacting region domain, can independently bind to HP1alpha; 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 HP1alpha association to heterochromatin, loss of Orc1 and Orc5 caused aberrant HP1alpha distribution only to pericentric heterochromatin-surrounding nucleoli. Depletion of HP1alpha from human cells also shows loss of Orc2 binding to heterochromatin, suggesting that ORC and HP1 proteins are mutually required for each other to bind to heterochromatin. Similar to HP1alpha-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.

Fig. 1.

Multiple ORC subunits localize with HP1 to heterochromatic foci. YFP fusion constructs were transfected into MCF7 cells, and endogenous proteins were detected by indirect immunofluorescence. Orc3-YFP colocalizes with endogenous HP1α at heterochromatic foci (A, A’, and A”). Endogenous Orc2 and Orc3-YFP colocalize at the heterochromatic foci (B, B’, and B”). YFP-Orc1 colocalizes with endogenous Orc2 at the heterochromatic 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.)

Fig. 2.

ORC dynamics at the heterochromatin. (A) FRAP kinetics of various ORC subunits at the heterochromatic foci in MCF7 cells. Similar FRAP kinetics of ∼3–4 s t1/2 for YFP-HP1α, YFP-Orc2, and Orc3-YFP were observed at heterochromatic foci, whereas YFP-Orc1 showed >40 s t1/2 at heterochromatin. (B) GST pull-down assays using GST or GST-HP1α beads and incubating with in vitro transcribed and translated mRNA from individual ORC subunits shows direct binding of both human Orc1 and Orc3 to HP1α. The input is shown on Right.

Fig. 3.

Mapping the Orc3- and HP1α-interaction domain. (A) Schematic representation of predicted domains or motifs in Orc3 protein. (B) Various deletion and mutant Orc3 constructs were in vitro transcribed/translated and used in GST pull-down assays with GST alone or GST-HP1α in 100 mM NaCl. The MIR mutants are Mir2, V216D, I218E; Mir3, VV215,216DD, VI217,218EE, or a Mir-domain deletion. All Orc3 constructs were tagged with the HA epitope. Orc3 has two HP1α-interacting regions, one is the coiled-coil region at the N terminus and the second is the MIR-containing region. (C) Immunolocalization of Orc3-YFP and Orc3-YFP* (MIR mutants) in human cells after transfection into MCF7 cells. (Scale bar, 5 μm.)

Fig. 4.

Depletion of ORC results in abnormal distribution of HP1α protein. (A) Depletion of individual ORC subunits results in aberrant organization of HP1α proteins. Depletion of Orc2 (Ab) and Orc3 (Ac) from human HeLa cells results in loss of HP1 from heterochromatic foci and redistribution as homogenous pool. Depletion of Orc1 (Ad) and Orc5 (Ae) results in the redistribution of HP1 to the pericentric heterochromatin, mostly around the nucleolar periphery. (B) Luciferase siRNA-treated cells showing HP1α-positive foci (green) and CREST-labeling centromere (red). In contrast, the Orc1 siRNA-treated cells showed redistribution of HP1α foci and clustering of the centromeres around the nucleolar periphery. (C) Recruitment of Orc2 to heterochromatin is also HP1α-dependent. HP1α siRNA-treated cells show loss of Orc2 binding to prominent heterochromatic foci. Note that one HP1α-positive cell continues to show binding of Orc2 to the heterochromatic foci. Chromatin was stained with DAPI (blue). (Scale bar, 5 μm.)

Fig. 5.

ORC proteins are required for HP1-associated heterochromatin assembly. (A) Loss of ORC proteins results in abnormal compaction of satellite repeats. DNA FISH using a chromosome 9 satellite repeat probe in HeLa cells depleted of individual ORC subunits Orc3 (Ac) and Orc2 (Ad) results in loss of chromatin compaction at chromosome 9 satellite repeats. Similar observation was made for HP1α siRNA-treated cells (Ab). (Scale bar, 5 μm.) Higher-resolution images showing decondensed chromatin associated with chromosome 9 satellite repeat in cells depleted of HP1α (Af), Orc2 (Ag), and Orc3 (Ah) siRNA-treated cells. Note the single compacted DNA FISH signal in luciferase siRNA-treated cells (Aa and Ae). (B) ORC depletion does not affect Polycomb-associated heterochromatin. Localization of Polycomb-associated repressive mark; trimethylation of K27 on H3 was analyzed in cells depleted of HP1α, Orc1, Orc2, or Orc3. No change in the K27 trimethylation on H3 was observed, whereas the HP1α proteins continue to show abnormal distribution on ORC-depleted cells. (Scale bar, 10 μm.)