We identified a human multiprotein complex (WINAC) that directly interacts with the vitamin D receptor (VDR) through the Williams syndrome transcription factor (WSTF). WINAC has ATP-dependent chromatin-remodeling activity and contains both SWI/SNF components and DNA replication-related factors. The latter might explain a WINAC requirement for normal S phase progression. WINAC mediates the recruitment of unliganded VDR to VDR target sites in promoters, while subsequent binding of coregulators requires ligand binding. This recruitment order exemplifies that an interaction of a sequence-specific regulator with a chromatin-remodeling complex can organize nucleosomal arrays at specific local sites in order to make promoters accessible for coregulators. Furthermore, overexpression of WSTF could restore the impaired recruitment of VDR to vitamin D regulated promoters in fibroblasts from Williams syndrome patients. This suggests that WINAC dysfunction contributes to Williams syndrome, which could therefore be considered, at least in part, a chromatin-remodeling factor disease.

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di-and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di-and trimethylation of H3K4, resulting in regulation of transcriptional initiation.[Keywords: Nucleosome; ubiquitylation; histone code; transcription; USP21; H2A] Supplemental material is available at http://www.genesdev.org.

Posttranslational histone modifications are important for the regulation of many biological phenomena. Here, we show the purification and characterization of nucleosomal histone kinase-1 (NHK-1). NHK-1 has a high affinity for chromatin and phosphorylates a novel site, Thr 119, at the C terminus of H2A. Notably, NHK-1 specifically phosphorylates nucleosomal H2A, but not free H2A in solution. In Drosophila embryos, phosphorylated H2A Thr 119 is found in chromatin, but not in the soluble core histone pool. Immunostaining of NHK-1 revealed that it goes to chromatin during mitosis and is excluded from chromatin during S phase. Consistent with the shuttling of NHK-1 between chromatin and cytoplasm, H2A Thr 119 is phosphorylated during mitosis but not in S phase. These studies reveal that NHK-1-catalyzed phosphorylation of a conserved serine/threonine residue in H2A is a new component of the histone code that might be related to cell cycle progression.[Keywords: Nucleosome; phosphorylation; histone code; transcription; NHK-1] Supplemental material is available at http://genesdev.org.

In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.

We examine in vitro nucleosome assembly by nucleosome assembly protein-1 (NAP-1) and ATP-utilizing chromatin assembly and remodeling factor (ACF). In contrast to previous studies that used relaxed, circular plasmids as templates, we have found that negatively supercoiled templates reveal the distinct roles of NAP-1 and ACF in histone deposition and the formation of an ordered nucleosomal array. NAP-1 can efficiently deposit histones onto supercoiled plasmids. Furthermore, NAP-1 exhibits a greater affinity for histones H2A-H2B than does naked DNA, but in the presence of H3-H4, H2A-H2B are transferred from NAP-1 to the plasmid templates. These observations underscore the importance of a high affinity between H2A-H2B and NAP-1 for ordered transfer of core histones onto DNA. In addition, recombinant ACF composed of imitation switch and Acf1 can extend closely packed nucleosomes, which suggests that recombinant ACF can mobilize nucleosomes. In the assembly reaction with a supercoiled template, ACF need not be added simultaneously with NAP-1. Regularly spaced nucleosomes are generated even when recombinant ACF is added after core histones are transferred completely onto the DNA. Atomic force microscopy, however, suggests that NAP-1 alone fails to accomplish the formation of fine nucleosomal core particles, which are only formed in the presence of ACF. These results suggest a model for the ordered deposition of histones and the arrangement of nucleosomes during chromatin assembly in vivo.The assembly of genomic DNA and histones into chromatin is a fundamental process of eukaryotes that affects a broad range of biological phenomena, including DNA replication, DNA repair, gene expression, and progression through the cell cycle (for reviews, see Refs. 1-7). The basic structural unit of chromatin is the nucleosome, consisting of 145 base pairs (bp) 1 of DNA wrapped around an octamer of the core histones H2A, H2B, H3, and H4. Crystallographic studies have shown that an H3-H4 tetramer occupies the central region of the nucleosome, and the H2A-H2B dimers bind to the peripheral region (8, 9). Thus, it is implicit in the structure of the nucleosome that H3 and H4 must be deposited prior to the incorporation of H2A and H2B. In agreement with this, several studies of histone transfer and of chromatin assembly during DNA replication have indicated that H3-H4 deposition occurs first, then H2A-H2B, followed by linker histones (such as histone H1) and the establishment of the ordered nucleosomal arrays that are characteristic of bulk chromatin in vivo (for reviews, see Refs. 10 -13).Biochemical studies have led to the identification of proteins that mediate the reconstitution of core histones into nucleosomes. Nearly all of these factors are core histone-binding proteins that contain stretches of acidic amino acid residues. Some of these histone chaperones, such as nucleoplasmin and nucleosome assembly protein-1 (NAP-1), exhibit a preference for binding to histones H2A and H2B relative to histones H3 and H4. Other histone chaperones,...