HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/2001; 21(5):1854-65. DOI: 10.1128/MCB.21.5.1854-1865.2001
Source: PubMed


Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more
phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for
proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors
of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound
to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in
vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin
A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized
preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells
caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control
of the cell cycle.

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    • "The HIR complex functions in several chromatin-related processes including chromatin assembly (Sharp et al. 2001; Green et al. 2005; Prochasson et al. 2005), kinetochore function (Sharp et al. 2002), and transcription elongation (Formosa et al. 2002; Nourani et al. 2006). HIRA, the human homolog of yeast Hir1 and Hir2 (Hall et al. 2001), is also a histone chaperone (Ray-Gallet et al. 2002), and Hira of S. pombe is required for both heterochromatin formation and repression of antisense transcription (Blackwell et al. 2004; Anderson et al. 2009; Yamane et al. 2011). The CAF-1 complex was originally identified from Hela cells as an activity that assembles nucleosomes onto replicating DNA (Stillman 1986; Smith and Stillman 1989). "
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    ABSTRACT: Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field.
    Genetics 02/2012; 190(2):351-87. DOI:10.1534/genetics.111.132266 · 5.96 Impact Factor
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    • "No. 610001), Anti-beta actin (Abcam clone AC-15) were from the indicated suppliers. Anti-macroH2A and anti-HIRA antibodies were described previously [20,31]. SAHF (DAPI foci) were detected by staining with 0.13 μg/ml DAPI for 2 min at room temperature (as opposed to standard conditions of 1 μg/ml for 5 min). "
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    ABSTRACT: Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts. We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells. In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.
    Cell Division 06/2010; 5(1):16. DOI:10.1186/1747-1028-5-16 · 3.53 Impact Factor
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    • "The ingenuity pathway also showed that HIRA can bind with Histine H2 [62]. HIRA is a homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, whose expression blocks S-phase progression [63]. Although there is not much known on the role of PTTG in histone regulation, from the available literature it is evident that the overexpression of PTTG can induce apoptosis by up-regulating histone proteins. "
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    ABSTRACT: Pituitary tumor transforming gene (PTTG), also known as securin, is highly expressed in various tumors including pituitary, thyroid, colon, ovary, testis, lung, and breast. An overexpression of PTTG enhances cell proliferation, induces cellular transformation in vitro, and promotes tumor development in nude mice. PTTG also inhibits separation of sister chromatids leading to aneuploidy and genetic instability. A great amount of work has been undertaken to understand the biology of PTTG and its expression in various tumors. However, mechanisms by which PTTG mediates its tumorigenic function are not fully understood. To utilize this gene for cancer therapy, identification of the downstream signaling genes regulated by PTTG in mediation of its tumorigenic function is necessary. For this purpose, we expressed PTTG in human embryonic kidney (HEK293) cells that do not express PTTG and analyzed the downstream genes using microarray analysis. A total of 22,277 genes printed on an Affymetrix HG-U133A 2.0 GeneChip array were screened with labeled cRNA prepared from HEK293 cells infected with adenovirus vector expressing PTTG cDNA (AdPTTG cDNA) and compared with labeled cRNA prepared from HEK293 cells infected with control adenovirus (control Ad) or adenovirus vector expressing GFP (AdGFP). Out of 22,277 genes, 71 genes were down-regulated and 35 genes were up-regulated with an FDR corrected p-value of < or = 0.05 and a fold change of > or =2. Most of the altered genes identified are involved in the cell cycle and cell apoptosis; a few are involved in mRNA processing and nitrogen metabolism. Most of the up-regulated genes belong to the histone protein family. PTTG is a well-studied oncogene for its role in tumorigenesis. In addition to its importance in regulation of the cell cycle, this gene has also been recently shown to play a role in the induction of cell apoptosis. The microarray analysis in the present study demonstrated that PTTG may induce apoptosis by down-regulation of oncogenes such as v-Jun and v-maf and up-regulation of the histone family of genes.
    BMC Genomics 12/2009; 10(1):577. DOI:10.1186/1471-2164-10-577 · 3.99 Impact Factor
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