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) 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 are mutually 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 higherorder chromatin structure and centromere function.centromere | origin recognition complex | pericentric heterochromatin | chromatin structure

The Polycomb Repressive Complex 1 Protein BMI1 Is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells

“…Association of SUMOylated HP1a and HP1b to a non-coding RNA results in increased binding affinity for H3K9 me3 and this is further enhanced and stabilized by the suppressor of variegation protein DEK1 (11,12). The DEK1-HP1-SUV39 complex then propagates the H3K9 me3 marks on the chromatin, resulting in heterochromatin formation (13)(14)(15)(16)(17). Interestingly, ATRx localizes at both telomeric and PCH, and germline mutations in ATRx are associated with the ␣-thalassemia with mental retardation X-linked syndrome (18 -20).…”

Transcriptional silencing functions of the yeast protein Orc1/Sir3 subfunctionalized after gene duplication

“…For example, human and Drosophila ORC bind to HP1 (heterochromatin protein 1) (1)(2)(3)(4) and human ORC associates with telomeric and pericentromeric heterochromatin (5)(6)(7). Orc1 and the deacetylase Sir2 also repress genes near telomeres in the evolutionarily distant organism Plasmodium falciparum, which causes malaria (8).…”

Nonreplicative functions of the origin recognition complex

“…Nevertheless, it is still unclear whether ORC interact with HP1 on the same regions of heterochomatin, because depletion of the individual ORC subunits in human cells has different outcomes on HP1 localization on heterochromatin. For instance, unlike ORC1 and ORC5, ORC2 and ORC3 participate in HP1 recruiting to the centromeric regions (Figure 1b) [46]. …”

“…Hossain and Stillman demonstrated how the ORC1 protein acts in the repression of the E2F1-dependent transcription of the CCNE1 gene, which encodes the Cyclin E protein [46,59] (Figure 1a). According to this study, ORC1 binds to the Retinoblastoma (RB) protein at the CCNE1 promoter and recruits the SUV39H1 histone-methyltransferase, which introduces the H3K9me3 mark, and thus, facilitates HP1 binding.…”

“…This study not only uncovered the role of ORC1 in the expression of a single gene, but also highlighted its regulatory function in the cell cycle progression, since Cyclin E is one of the key factors that regulate the cell cycle. Moreover, since ORC1 is known to bind both SUV39H1 and HP1 [46], it may participate in the regulation of transcription of other E2F1-dependent genes during G1 phase.…”