The proteasome inhibitors, lactacystin and N-acetyl-leucyl-leucyl-norlucinal, caused a rapid and near-complete loss of approximately 22-23-kDa ubiquitinated nucleoproteins, which we have identified as monoubiquitinated nucleosomal histones H2A and H2B by immunological and two-dimensional electrophoretic techniques. In human SKBr3 breast tumor cells, depletion of monoubiquitinated histones by the proteasome inhibitors coincided with the accumulation of high molecular weight ubiquitinated proteins in both nucleoprotein and cytosolic fractions and decreased unconjugated ubiquitin in the cytosol, without changes in the nonubiquitinated core histones. Unconjugated ubiquitin was not detected in isolated tumor cell nuclei. A similar loss in monoubiquitinated histones occurred in cells harboring a defective, temperature-sensitive mutation of the ubiquitin-activating E1 enzyme, after these cells were elevated from 33 degrees C to the non-permissive temperature of 39 degrees C. DNA replication and RNA transcription were decreased by the proteasome inhibitors most strongly after 90% of the ubiquitin had been removed from ubiquitinated histones H2A and H2B, suggesting a relationship between the nucleosomal histone ubiquitin status and the processing of genetic information. Interestingly, although both proteasome inhibitors caused a generalized decrease in methionine incorporation into proteins, they strongly induced the synthesis of the hsp72 and hsp90 stress proteins. Finally, treating cells with heat-shock at 43 degrees C, with stress response-provoking chemicals or with several other proteasome inhibitors caused ubiquitinated proteins to accumulate, depleted free ubiquitin, and concomitantly decreased nucleosomal monoubiquitinated histones. These results suggest that deubiquitination of nucleosomal histones H2A and H2B may play a previously unrecognized role in the cellular stress response, as well as in the processing of chromatin, and emphasize the important role of the proteasome in cellular homeostasis.

Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that function as activators or repressors. Although both transcription factors share a common domain involved in forming multimers, we demonstrate that Sp1 and Sp3 form separate complexes in estrogen-dependent human breast cancer cells. Sp1 and Sp3 complexes associate with histone deacetylases (HDACs) 1 and 2. Although most HDAC2 is not phosphorylated in the breast cancer cells, HDAC2 bound to Sp1 and Sp3 and cross-linked to chromatin in situ is highly enriched in a phosphorylated form that has a reduced mobility in SDS-polyacrylamide gels. We show that protein kinase CK2 is associated with and phosphorylates HDAC2. Alkaline phosphatase treatment of HDAC2 and Sp1 and Sp3 complexes reduced the associated HDAC activity. Protein kinase CK2 is up-regulated in several cancers including breast cancer, and Sp1 and Sp3 have key roles in estrogen-induced proliferation and gene expression in estrogen-dependent breast cancer cells. CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities and dynamic chromatin remodeling of estrogenregulated genes.Remodeling of chromatin structure mediated by ATP-driven chromatin-remodeling complexes and histone-modifying enzymes has a crucial role in gene expression. Acetylation of the core histones favors decondensation of the chromatin fiber by preventing interfiber interactions, whereas the unacetylated histone state contributes to chromatin condensation (1, 2). Dynamic histone acetylation catalyzed by histone deacetylases (HDAC) 1 and histone acetyltransferases allows the chromatin fiber to rapidly oscillate from the condensed and decondensed states (3, 4). In mammalian cells three classes of HDACs are identified. Class I HDACs, such as HDAC1 and HDAC2, are homologous to yeast RPD3, whereas class II HDACs are similar to yeast HDA1. Class III HDACs are related to yeast SIR2 (5, 6). HDAC1 and -2 are components of large multisubunit complexes called Sin3 or NuRD, which are recruited by transcriptional factors such as Mad, YY1, and Rb (5, 7-9).Mammalian cells ubiquitously express Sp1 and Sp3. Sp3 has three isoforms, a long (L-Sp3) and two short forms (M1-Sp3, M2-Sp3) that are the products of differential translational initiation (10). Sp3 may act as a repressor or an activator, with the short forms acting only as repressors (10). The protein structure of L-Sp3 is very similar to that of Sp1, except that Sp3 has a repression domain located N-terminal to the zinc finger DNAbinding domain (11). It has been reported that the relative levels of Sp3 forms change with differentiation, with the differentiated Caco-2 cells expressing more long than short forms (12). Further, alterations in the relative levels of Sp1 to Sp3 have been recorded, with Sp3 levels being greater than Sp1 in primary keratinocytes (13).In this study we investigated the association of histone deacetylase with Sp3 in human breast cancer cells. We ...

Because of the potential role of histone ubiquitination in altering chromatin structure, we characterized the levels of ubiquitination of specific histones in meiotic and postmeiotic germ cells in rat testes by two-dimensional gel electrophoresis. The levels of the major ubiquitinated histone forms, mono-and poly-ubiquitinated H2A, were highest in the pachytene spermatocyte stage, declined thereafter through the round spermatid stage, and reached their lowest levels in elongating spermatids. Three additional ubiquitinated histone species, besides H2A, were detected using anti-ubiquitin antibodies specifically in the fraction enriched in elongating spermatids. Based on their electrophoretic mobilities, they corresponded to uH3, uTH3, and uH2B. Polyubiquitinated forms of these proteins were also observed. The identity of these proteins was confirmed by immunoblotting with anti-H3 antisera and by differential extraction of the proteins from the nucleus with increasing salt concentrations. This is the first report of ubiquitination of H3 in vivo. We speculate that its ubiquitination could loosen the nucleosome structure in preparation for histone removal, be a consequence of nucleosome relaxation or disruption caused by other means, or target H3 for degradation.

Nucleosomes associated with transcribing chromatin of mammalian cells have an unfolded structure in which the normally buried cysteinyl-thiol group of histone H3 is exposed. In this study we analyzed transcriptionally active/competent DNA-enriched chromatin fractions from chicken mature and immature erythrocytes for the presence of thiol-reactive nucleosomes using organomercury-agarose column chromatography and hydroxylapatite dissociation chromatography of chromatin fractions labeled with [ 3 H]iodoacetate. In mature and immature erythrocytes, the active DNA-enriched chromatin fractions are associated with histones that are rapidly highly acetylated and rapidly deacetylated. When histone deacetylation was prevented by incubating cells with histone deacetylase inhibitors, sodium butyrate or trichostatin A, thiol-reactive H3 of unfolded nucleosomes was detected in the soluble chromatin and nuclear skeleton-associated chromatin of immature, but not mature, erythrocytes. We did not find thiol-reactive nucleosomes in active DNA-enriched chromatin fractions of untreated immature erythrocytes that had low levels of highly acetylated histones H3 and H4 or in chromatin of immature cells incubated with inhibitors of transcription elongation. This study shows that transcription elongation is required to form, and histone acetylation is needed to maintain, the unfolded structure of transcribing nucleosomes.Acetylation of the core histones (H2A, H2B, H3, and H4) is a dynamic process catalyzed by histone acetyltransferases and histone deacetylases (1, 2). In chicken immature erythrocytes, 4% of the modifiable lysine sites participate in dynamic histone acetylation. These core histones are rapidly acetylated (t1 ⁄2 ϭ 12 min for monoacetylated H4) and rapidly deacetylated (t1 ⁄2 ϭ 5 min for the tetraacetylated isoform of H4) (3, 4). Histones undergoing rapid acetylation and deacetylation are associated with transcriptionally active chromatin (5-7). The recent findings that histone acetyltransferases and deacetylases are transcriptional coactivators and corepressors have increased our understanding of how the process of dynamic histone acetylation is established on transcriptionally active chromatin (2, 8).Transcriptionally active chromatin has a soluble and insoluble nature (9). Transcribed DNA is found in chromatin fragments that are soluble in 0.15 M NaCl and/or 2 mM MgCl 2 and in chromatin fragments associated with the low salt-insoluble residual nuclear material (nuclear skeletons) (for review, see Davie (10)). Chromatin engaged in transcription is thought to be retained by the nuclear skeleton by multiple dynamic attachments between the nuclear matrix and transcribed chromatin; hence rendering the transcribing chromatin insoluble (11, 12). As histone acetyltransferase and deacetylase activities are associated with the nuclear matrix (7, 13), we proposed that these nuclear matrix-bound enzymes may mediate some of the dynamic attachments between active chromatin and nuclear matrix (13,14). Most information on the structure ...

Histone deacetylase 2 (HDAC2) is one of the histone-modifying enzymes that regulate gene expression by remodeling chromatin structure. Along with HDAC1, HDAC2 is found in the Sin3 and NuRD multiprotein complexes, which are recruited to promoters by DNA-binding proteins. In this study, we show that the majority of HDAC2 in human breast cancer cells is not phosphorylated. However, the minor population of HDAC2, preferentially cross-linked to DNA by cisplatin, is mono-, di-, or triphosphorylated. Furthermore, HDAC2 phosphorylation is required for formation of Sin3 and NuRD complexes and recruitment to promoters by transcription factors including p53, Rb, YY1, NF-B, Sp1, and Sp3. Unmodified HDAC2 requires linker DNA to associate with chromatin but is not cross-linked to DNA by formaldehyde. We provide evidence that unmodified HDAC2 is associated with the coding region of transcribed genes, whereas phosphorylated HDAC2 is primarily recruited to promoters.Histone-modifying enzymes and ATP-dependent chromatin-remodeling complexes affect gene expression by altering the compaction level of chromatin and the accessibility of DNA to binding proteins. Histone hyperacetylation is generally associated with chromatin decondensation and increased transcriptional activity, whereas histone hypoacetylation contributes to chromatin condensation and transcriptional repression (1, 2). Dynamic histone acetylation at transcriptionally active genes, resulting from the opposing activities of histone deacetylases (HDACs) 2 and histone acetyltransferases, leads to a rapid oscillation between condensed and decondensed chromatin states (3, 4). Four classes of HDACs have been identified in mammalian cells (5). HDAC1 and 2 belong to class I and are homologous to yeast RPD3. Both HDACs are core components of multi-protein corepressor complexes like Sin3 and NuRD, in which their activities are modulated through interactions with other proteins while being recruited by transcription factors to specific promoters (6). HDAC1 and 2 are phosphoproteins, and this post-translational modification enhances their enzymatic activity (7-9). In studies with exogenously expressed tagged HDAC1 and 2, HDAC phosphorylation appeared to be a prerequisite to form the corepressor complexes. However, studies characterizing endogenous HDAC corepressor complexes are lacking.The role of phosphorylation in the recruitment of HDAC2 by transcription factors has not yet been investigated. Several transcription factors repress gene expression by recruiting HDAC1/2 corepressor complexes to the promoters that they affect. Further, pending the promoter context, transcription factors recruit HDAC1 and 2 corepressor complexes to mediate dynamic deacetylation of histones and non-histone chromosomal proteins associated with or close to the promoter. We have previously reported that the Sp1 and Sp3 transcription factors are associated with phosphorylated HDAC2 in breast cancer cells. Although most HDAC2 in breast cancer cells was not phosphorylated, it was the phosphorylated form of...