The mammalian HIRA gene encodes a histone-interacting protein whose homolog in Xenopus laevis is characterized here. In vitro, recombinant Xenopus HIRA bound purified core histones and promoted their deposition onto plasmid DNA. The Xenopus HIRA protein, tightly associated with nuclear structures in somatic cells, was found in a soluble maternal pool in early embryos. Xenopus egg extracts, known for their chromatin assembly efficiency, were specifically immunodepleted for HIRA. These depleted extracts were severely impaired in their ability to assemble nucleosomes on nonreplicated DNA, although nucleosome formation associated with DNA synthesis remained efficient. Furthermore, this defect was largely corrected by reintroduction of HIRA along with (H3-H4)(2) tetramers. We thus delineate a nucleosome assembly pathway that depends on HIRA.

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B-and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second ␣ helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development.Developmental anomalies are frequently observed in humans in association with deletions affecting the proximal region of the long arm of chromosome 22. These 22q deletion disorders (22DD) include the DiGeorge syndrome (Mendelion inheritance in man [MIM] 188400) and the velocardiofacial syndrome (MIM 192430), whose phenotypes overlap partially. Main clinical features associated with 22DD comprise abnormalities of the face and palate, hypoplastic parathyroid glands, and conotruncal malformations (38), all probably resulting from anomalies of neural crest cells in the embryological region of the pharyngeal arches and pouches (26). Genetically, 90% of all patients have a large (approximately 3-Mb-long) 22q deletion. Although most deletions occur de novo, up to 28% could be inherited (38). In these familial cases whose transmission is autosomal dominant, the phenotypic expression of the same chromosomal defect is largely variable. The additional lack of correlation between the extent of the deletion and the intensity of the phenotype seems to argue against different contiguous genes being each responsible for distinct clinical features. Recently, however, the hypothesis that two causative genes each mapping to the same 22q region may together be...

No abstract

The HIRA gene encodes a nuclear protein with histone-binding properties that have been conserved from yeast to humans. Hir1p and Hir2p, the two HIRA homologues in Saccharomyces cerevisiae, are transcriptional co-repressors whose action resides at the chromatin level and occurs in a cell-cycle-regulated fashion. In mammals, HIRA is an essential gene early during development, possibly through the control of specific gene-transcription programmes, but its exact function remains to be deciphered. Here we report on the subnuclear distribution and cell-cycle behaviour of the HIRA protein. Using both biochemical and immunofluorescence techniques, a minor fraction of HIRA was found tightly associated with the nuclear matrix, the material that remains after nuclease treatment and high-salt extraction. However, most HIRA molecules proved extractable. In non-synchronized cell populations, extraction from chromatin necessitated 300 mM NaCl whereas 150 mM was sufficient in mitotic cells. Immunofluorescence staining and microscopic examination of mitotic cells revealed HIRA as excluded from condensed chromosomes, confirming a lack of association with chromatin during mitosis. Western-blot analysis indicated that HIRA molecules were hyper-phosphorylated at this point in the cell cycle. Metabolic labelling and pulse-chase experiments characterized HIRA as a stable protein with a half-life of approx. 12 h. The mitotic phosphorylation of HIRA could provide the dividing cell with a way to retarget HIRA-containing multi-protein complexes to different chromatin regions in daughter compared with parental cells.