The origin recognition complex (ORC) is an initiator protein for DNA replication, but also effects transcriptional silencing in Saccharomyces cerevisiae and heterochromatin function in Drosophila. It is not known, however, whether any of these functions of ORC is conserved in mammals. A novel protein, HBO1 (histone acetyltransferase binding to ORC), has been identified that interacts with human ORC1 protein, the largest subunit of ORC. HBO1 exists as part of a multisubunit complex that possesses histone H3 and H4 acetyltransferase activities. A fraction of the relatively abundant HBO1 protein associates with ORC1 in human cell extracts. HBO1 is a member of the MYST domain family that includes S. cerevisiae Sas2p, a protein involved in control of transcriptional silencing that also has been genetically linked to ORC function. Thus the interaction between ORC and a MYST domain acetyltransferase is widely conserved. Roles for ORC-mediated acetylation of chromatin in control of both DNA replication and gene expression are suggested (Iizuka, 1999).
The minichromosome maintenance (MCM) proteins, together with the origin recognition complex (ORC) proteins and Cdc6, play an essential role in eukaryotic DNA replication through the formation of a pre-replication complex at origins of replication. A yeast two-hybrid screen was used to identify MCM2-interacting proteins. One of the proteins identified is identical to the ORC1-interacting protein termed HBO1. HBO1 belongs to the MYST family, characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain. Biochemical studies confirmed the interaction between MCM2 and HBO1 in vitro and in vivo. An N-terminal domain of MCM2 is necessary for binding to HBO1, and a C2HC zinc finger of HBO1 is essential for binding to MCM2. A reverse yeast two-hybrid selection was performed to isolate an allele of MCM2 that is defective for interaction with HBO1; this allele was then used to isolate a suppressor mutant of HBO1 that restores the interaction with the mutant MCM2. This suppressor mutation was located in the HBO1 zinc finger. Taken together, these findings strongly suggest that the interaction between MCM2 and HBO1 is direct and mediated by the C2HC zinc finger of HBO1. The biochemical and genetic interactions of MYST family protein HBO1 with two components of the replication apparatus, MCM2 and ORC1, suggest that HBO1-associated HAT activity may play a direct role in the process of DNA replication (Burke, 2001).
The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) that involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Hbo1, a member of the MYST histone acetyltransferase family, is a positive regulatory factor for pre-RC assembly. When Hbo1 expression is inhibited in human cells, Mcm2-7 fails to associate with chromatin even though ORC and Cdc6 loading is normal. When Xenopus egg extracts are immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 is lost, and DNA replication is abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts can be restored by the addition of recombinant Cdt1 (Iizuka, 2006).
The androgen receptor (AR), a member of the nuclear receptor superfamily, plays a central role in male sexual differentiation and prostate cell proliferation. Results of treating prostate cancer by androgen ablation indicate that signals mediated through AR are critical for the growth of these tumors. Like other nuclear receptors, AR exerts its transcriptional function by binding to cis-elements upstream of promoters and interacting with other transcriptional factors (e.g. activators, repressors and modulators). To determine the mechanism of AR-regulated transcription, the yeast two-hybrid system was used to identify AR-associated proteins. One of the proteins identified is identical to the human origin recognition complex-interacting protein termed HBO1. A ligand-enhanced interaction between AR and HBO1 was further confirmed in vivo and in vitro. Immunofluorescence experiments showed that HBO1 is a nuclear protein, and Northern blot analysis revealed that it is ubiquitously expressed, with the highest levels present in human testis. HBO1 belongs to the MYST family, which is characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain. Surprisingly, two yeast members of the MYST family, SAS2 and SAS3, have been shown to function as transcription silencers, despite the presence of the histone acetyltransferase domain. Using a GAL4 DNA-binding domain assay, a transcriptional repression domain was mapped within the N-terminal region of HBO1. Transient transfection experiments reveal that HBO1 specifically represses AR-mediated transcription in both CV-1 and PC-3 cells. These results indicate that HBO1 is a new AR-interacting protein capable of modulating AR activity. It could play a significant role in regulating AR-dependent genes in normal and prostate cancer cells (Sharma, 2000).
The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR). Chromatin immunoprecipitation assays were used to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR is also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and is enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, binds TR in late-G(1)/S-arrested cells; this coincides with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase. ORC binding to TR is LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bind TR specifically, and the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) was identified as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats (Stedman, 2004)
CDK11(p58), a 58kDa protein of the PITSLRE kinase family, plays an important role in cell cycle progression, and is closely related to cell apoptosis. To gain further insight into the function of CDK11(p58), a human fetal liver cDNA library was screened for its interacting proteins using the yeast two-hybrid system. Histone acetyltransferase (HAT) HBO1, a MYST family protein, interacts with CDK11(p58) in vitro and in vivo. CDK11(p58) and HBO1 colocalize in the cell nucleus. Recombinant CDK11(p58) enhances the HAT activity of HBO1 significantly in vitro. Meanwhile, overexpression of CDK11(p58) in mammalian cells leads to the enhanced HAT activity of HBO1 towards free histones. Thus, it is concluded that CDK11(p58) is a new interacting protein and a novel regulator of HBO1. Both of the proteins may be involved in the regulation of eukaryotic transcription (Zong, 2005).
Members of the ING family of tumor suppressors regulate cell cycle progression, apoptosis, and DNA repair as important cofactors of p53. ING1 and ING3 are stable components of the mSin3A HDAC (see Drosophila Sin3A) and Tip60/NuA4 HAT complexes, respectively. The three remaining human ING proteins have been purified. While ING2 is in an HDAC complex similar to ING1, ING4 associates with the HBO1 HAT required for normal progression through S phase and the majority of histone H4 acetylation in vivo. ING5 fractionates with two distinct complexes containing HBO1 or nucleosomal H3-specific MOZ/MORF HATs. These ING5 HAT complexes interact with the MCM helicase and are essential for DNA replication to occur during S phase. The data also indicate that ING subunits are crucial for acetylation of chromatin substrates. Since INGs, HBO1, and MOZ/MORF contribute to oncogenic transformation, the multisubunit assemblies characterized here underscore the critical role of epigenetic regulation in cancer development (Doyon, 2006).
Modulators of cofactor recruitment by nuclear receptors are expected to play an important role in the coordination of hormone-induced transactivation processes. To identify such factors interacting with the N-terminal domain of the progesterone receptor (PR), this domain was used as bait in the yeast Sos-Ras two-hybrid system. cDNAs encoding the C-terminal MYST domain of HBO1, a member of the MYST acetylase family, were thus selected from a human testis cDNA library. In transiently transfected CV1 cells, the wild type HBO1 (611 aa) enhances transcription mediated by steroid receptors, notably PR, MR (mineralocorticoids) and GR (glucocorticoids), and strongly induced PR and ER (estrogens) co-activation by Steroid Receptor Coactivator 1a (SRC-1a). As assessed by two-hybrid and GST pull-down assays, the HBO1 MYST acetylase domain (aa 340-611) interacts mainly with the N-terminal domain (NTD), and also contacts the DNA binding (DBD) and the hinge domains of hormone-bound PR. The HBO1 N-terminal region (aa 1-340) associates additionally with PR ligand binding domain (LBD). HBO1 interact through its N-terminal domain with SRC-1a in the absence of steroid receptor. The latter co-association enhances specifically AF2 transactivating function encompassed in the LBD. Conversely, the MYST acetylase domain specifically enhances SRC-1 coupling with PR NTD, through a hormone-dependent mechanism. In HEK293 cells expressing human PRA or PRB, HBO1 selectively raises a SRC-1-dependent response of PRB but fails to regulate PRA activity. HBO1 acts through modification of a LBD-controlled structure present in the N-terminus of PRB leading to the modulation of SRC-1 functional coupling with AF3-mediated transcription. Importantly, real-time RT-PCR analysis also revealed that HBO1 enhances SRC-1 co-activation of PR-dependent transcription of human endogenous genes such as alpha-6 integrin and 11beta-hydroxydehydrogenase 2 but not that of amphiregulin. Immunofluorescence and confocal microscopy of HEK-PRB cells demonstrate that the hormone induces the colocalization of HBO1 with PR-SRC-1 complex into nuclear speckles characteristic of PR-mediated chromatin remodeling. These results suggest that HBO1 might play an important physiological role in human PR signaling (Georgiakaki, 2006).
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