![]() ![]() ![]() In addition, the UBN1/H3.3/H4/Asf1 crystal structure reveals striking structural similarity between UBN1/H3.3/H4 and DAXX/H3.3/H4. Determination of a crystal structure containing a UBN1-HRD peptide bound to H3.3/H4/Asf1 and mutational studies aided us in identification of residues within the UBN1-HRD and H3.3 that contribute to the specificity and strength of the UBN1/H3.3 interaction. We demonstrate that the UBN1-HRD specifically binds to H3.3/H4 over H3.1/H4 in a manner that is independent of the protein HIRA. In this study, we report that H3.3-specific binding by the HIRA complex is mediated by the UBN1-HRD. The molecular basis for how the HIRA complex selectively binds to H3.3, which differs from H3.1 by only 5 amino acids, is unknown. These data in combination with the observations of others 28 led us to the hypothesis that that the highly conserved UBN1-HRD may function to confer histone H3.3-binding specificity. Although the UBN1-HRD was initially proposed to mediate association with the WD repeats of HIRA 8, we more recently demonstrated that a less conserved region of UBN1 that resides sixty residues N terminal to the HRD is, in fact, responsible for HIRA-binding activity in UBN1 we named this domain the NHRD 29 ( Fig. 1, 23, 28) and UBN1 is homologous to Hpc2 with strong sequence conservation in the Hpc2-related domain (HRD) 8, 28. HIRA is homologous to Hir1 and Hir2 (ref. The human HIRA complex is orthologous to the Saccharomyces cerevisiae Hir histone chaperone complex, which regulates histone gene transcription 24, 25 and mediates replication-independent deposition of H3/H4 in yeast 26. CABIN1 and UBN1 were initially identified as a negative regulator for calcineurin signalling in T lymphocytes 21 and as a ubiquitously expressed nuclear protein that interacts with cellular and viral transcription factors 22, respectively, and later shown to be functional members of the HIRA histone chaperone complex 1, 8, 12, 23. While the H3/H4 histone chaperone ASF1a interacts with both CAF-1 and HIRA complexes and binds to both H3.3/H4 and H3.1/H4 for histone deposition 1, 18, 19, 20, CABIN1 and ubinuclein-1 (UBN1) are unique members of the HIRA complex 1. ![]() For example, DAXX promotes H3.3 deposition at gene regulatory regions of activated neurons and both HIRA and DAXX are involved in H3.3 deposition in non-proliferating senescent cells 17. However, DAXX/ATRX and HIRA likely also have some overlapping functions. In metazoan cells, there are two major H3 variants: H3.1 is deposited by the trimeric CAF-1 complex during DNA replication 1 and repair of ultraviolet-induced DNA damage 2, while histone H3.3 is deposited in a replication-independent manner 1, 3 by either DAXX/ATRX largely at heterochromatin, including pericentromeres, telomeres and endogenous retroviral elements 4, 5, 6, 7, or by the HIRA complex predominantly at gene regulatory regions, gene bodies, developmentally regulated genes 4, 8, 9, 10, 11, 12, 13, and sites of DNA and chromatin damage and repair 11, 14, 15, 16. This study points to UBN1 as the determinant of H3.3-specific binding and deposition by the HIRA complex. Comparison of the structure with the unrelated H3.3-specific chaperone DAXX reveals nearly identical points of contact between the chaperone and histone in the proximity of H3.3 G90, although the mechanism for H3.3 G90 recognition appears to be distinct. ![]() X-ray crystallographic and mutational studies reveal that conserved residues within the UBN1-HRD and H3.3 G90 as key determinants of UBN1–H3.3-binding specificity. Biochemical and biophysical studies show the UBN1-HRD preferentially binds H3.3/H4 over H3.1/H4. Here we demonstrate that the conserved UBN1 Hpc2-related domain (HRD) is a novel H3.3-specific-binding domain. The HIRA histone chaperone complex, composed of HIRA, ubinuclein-1 (UBN1) and CABIN1, cooperates with the histone chaperone ASF1a to mediate H3.3-specific binding and chromatin deposition. Histone chaperones bind specific histones to mediate their storage, eviction or deposition from/or into chromatin. ![]()
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