Histone H4


Modification of histones in gene activation and silencing

The functional organization of active and silent integrated luciferase transgenes were investigated in zebrafish, with the aim of accounting for the variegation of transgene expression in this species. The enrichment of transcriptionally active transgenes in acetylated histone H4 and the dynamic association of the transgenes with splicing factor SC35 and RNA Pol II are demonstrated. Analysis of interphase nuclei and extended chromatin fibers by immunofluorescence and in situ hybridization reveals a co-localization of transgenes with acetylated H4 in luciferase-expressing animals only. Enrichment of expressed transgenes in acetylated H4 is further demonstrated by their co-precipitation from chromatin using anti-acetylated H4 antibodies. Little correlation exists, however, between the level of histone acetylation and the degree of transgene expression. In transgene-expressing zebrafish, most transgenes co-localize with Pol II and SC35, whereas no such association occurs in non-expressing individuals. Inhibition of Pol II abolishes transgene expression and disrupts association of transgenes with SC35, although inactivated transgenes remains enriched in acetylated histones. Exposure of embryos to the histone deacetylation inhibitor TSA induces expression of most silent transgenes. Chromatin containing activated transgenes becomes enriched in acetylated histones and the transgenes recruit SC35 and Pol II. The results demonstrate a correlation between H4 acetylation and transgene activity, and argue that active transgenes dynamically recruit splicing factors and Pol II. The data also suggest that dissociation of splicing factors from transgenes upon Pol II inhibition is not a consequence of changes in H4 acetylation (Collas, 1999).

An important first step in the chromatin remodelling process is the initial binding of a transcriptional activator to a nucleosomal template. An investigation sought to determine the ability of AP-1, the Fos/Jun heterodimer (See Drosophila Jun), to interact with its cognate binding site located in the promoter region of the mouse fos-related antigen-2 ( the fra-2 promoter), when this site was reconstituted into a nucleosome. Two different nucleosome assembly systems were employed to assemble either principally non-acetylated or acetylated nucleosomes. Fos/Jun interactive capability with either an acetylated or an unacetylated nucleosome differs markedly: Fos/Jun binds to an unacetylated nucleosome with only a 4- to 5-fold reduction in DNA binding affinity as compared with naked DNA. Strikingly, the binding of Fos/Jun to a single high-affinity site incorporated into an acetylated nucleosome results in the complete disruption of nucleosomal structure without histone displacement. This disruption is sufficient to facilitate the subsequent binding of a second transcription factor. It is suggested that the disruption reported here, which is not energy dependent, involves a change in the conformation of a nucleosome produced by acetylated histones H3 and H4. This change of conformation alters the nucleosome structure sufficiently to modify the DNAseI sensitivity of the DNA segment (Ng, 1997).

The role of histone acetylation in X chromosome inactivation has been investigation, focusing on its possible involvement in the regulation of Xist, an essential gene expressed only from the inactive X (Xi). A region of H4 hyperacetylation extending up to 120 kb upstream from the Xist somatic promoter P1 has been identified. This domain includes the promoter P0, which gives rise to the unstable Xist transcript in undifferentiated cells. The hyperacetylated domain is not seen in male cells or in female XT67E1 cells, a mutant cell line heterozygous for a partially deleted Xist allele and in which an increased number of cells fail to undergo X inactivation. The hyperacetylation upstream of Xist is lost by day 7 of differentiation, when X inactivation is essentially complete. Wild-type cells that differentiate in the presence of the histone deacetylase inhibitor Trichostatin A are prevented from forming a normally inactivated X, as judged by the frequency of underacetylated X chromosomes detected by immunofluorescence microscopy. Mutant XT67E1 cells, lacking hyperacetylation upstream of Xist, are less affected. It is proposed that (1) hyperacetylation of chromatin upstream of Xist facilitates the promoter switch that leads to stabilization of the Xist transcript and (2) that the subsequent deacetylation of this region is essential for the further progression of X inactivation (O'Neill, 1999).

p300 and CREB-binding protein are functional homologs and global transcriptional coactivators that are involved in the regulation of various DNA-binding transcription factors. p300/CBP interacts with nuclear receptors, CREB, c-Jun, C-Myb, c-Fos, and MyoD. DNA-binding factors recruit p300/CBP by not only direct but also indirect interactions through cofactors. p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. The p300/CBP-histone acetyltransferase domain has no obvious sequence similarity to GCN5, another protein with known histone acetyltransferase activity, or to other previously described acetyltransferases. P300 acetylates all core histones in mononucleosomes and the four lysines in the Histone H4 N-terminal tail. These observations suggest that p300/CBP is not a simple adaptor between DNA binding factors and cellular p300/CBP associated factor (PCAF) or transcription factors; rather, p300/CBP per se may contribute directly to transcriptional regulation via targeted acetylation of chromatin (Ogryzko, 1996 and references).

RhoA (see Drosophila Rho1) and two other Rho-family proteins, Cdc42 and Rac1, regulate Serum Response Factor [SRF - see Drosophila Blistered)] activation of the c-fos serum response element. This pathway acts independently of known MAPK pathways and is regulated by agents such as serum and LPA, acting via heterotrimeric G protein-coupled receptors. Constitutively active forms of either of the small GTPases -- RhoA (RhoA.V14) or Cdc42 (Cdc42.V12) -- induces expression of extrachromosomal SRF reporter genes in microinjection experiments, but only Cdc42.V12 can efficiently activate a chromosomal template. Both SAPK/JNK-dependent or -independent signals can cooperate with RhoA.V14 to activate chromosomal SRF reporters; it is SAPK/JNK activation by Cdc42.V12 that allows SAPK/JNK to activate chromosomal templates. Cooperating signals can be bypassed by deacetylase inhibitors. Three findings show that histone H4 hyperacetylation is one target for cooperating signals, although it alone is not sufficient: (1) Cdc42.V12, but not RhoA.V14, induces H4 hyperacetylation; (2) cooperating signals use the same SAPK/JNK-dependent or -independent pathways to induce H4 hyperacetylation, and (3) growth factor and stress stimuli induce substantial H4 hyperacetylation, detectable in reporter gene chromatin. These data establish a link between signal-regulated acetylation events and gene transcription. Thus, in isolation, the SRF-controlled extrachromosomal reporter gene is a target for only a subset of signals that can activate the chromsomal c-fos promoter. This is thought to reflect differences in chromatin structure associated with the two types of templates (Alberts, 1998).

Members of the Mad family of bHLH-Zip proteins heterodimerize with Max to repress transcription in a sequence-specific manner (See Drosophila Myc, Evolutionary Homologs section). Transcriptional repression by Mad:Max heterodimers is mediated by ternary complex formation with either of the corepressors mSin3A or mSin3B (see Drosophila Sin3A). mSin3A is an in vivo component of large, heterogeneous multiprotein complexes and is tightly and specifically associated with at least seven polypeptides. Two of the mSin3A-associated proteins, p50 and p55, are highly related to the histone deacetylase HDAC1. The mSin3A immunocomplexes possess histone deacetylase activity that is sensitive to the specific deacetylase inhibitor trapoxin. mSin3A-targeted repression is reduced by trapoxin treatment, suggesting that histone deacetylation mediates transcriptional repression through Mad-Max-mSin3A multimeric complexes (Hassig, 1997).

Normal mammalian growth and development are highly dependent on the regulation of the expression and activity of the Myc family of transcription factors. Mxi1-mediated inhibition of Myc activity requires interaction with mammalian Sin3A or Sin3B proteins, which are purported to act as scaffolds for additional co-repressor factors. The identification of two such Sin3-associated factors, the nuclear receptor co-repressor (N-CoR) and histone deacetylase (HD1), provides a basis for Mxi1/Sin3-induced transcriptional repression and tumour suppression. The involvement of histone deacetylase suggests that the silencing function of Mxi1 involves a modification of chromatin involving deacetylation, converting chromatin into a form that impedes the interaction of the transcriptional apparatus with promoter regions (Alland, 1997).

Whereas liganded nuclear hormone receptors serve as transcriptional activators, unliganded nuclear receptors serve as repressors (For more information, See Ecdysone receptor, Evolutionary homologs section). How does the unliganded nuclear receptor transmit a repressive signal to the transcriptional apparatus and what is the nature of this signal? In fact, the target of the unliganded nuclear receptor is not RNA polymerase but chromatin, and repression is mediated by corepressors, proteins that associate with unliganded nuclear receptors that assemble a macromolecular complex that modifies chromatin so as to silence gene activity. The macromolecular complex acts to deacetylate histone. The transcriptional corepressors SMRT and N-CoR function as silencing mediators for retinoid and thyroid hormone receptors. SMRT and N-CoR directly interact with unliganded nuclear receptors, and these corepressors in turn directly interact with mSin3A, a corepressor for the Mad-Max heterodimer and a homolog of the yeast global-transcriptional repressor Sin3p. The recently characterized histone deacetylase 1 (HDAC1) interacts with Sin3A and SMRT to form a multisubunit, ternary repressor complex. Histone deacetylase in turn targets chromatin, converting it into a form that is unaccessable to the transcriptional apparatus. Consistent with this model, it is found that HDAC inhibitors synergize with retinoic acid to stimulate hormone-responsive genes and the differentiation of myeloid leukemia (HL-60) cells. Addition of a deacetylase inhibitor such as Trichostatin A relieves transcriptional repression resulting in a promoter that is sensitive to the addition of activating hormone. This work establishes a convergence of repression pathways for bHLH-Zip proteins and nuclear receptors and suggests that this type of regulation may be more widely conserved than previously suspected (Nagy, 1997).

A mouse histone deacetylase gene, HD1, is an interleukin-2-inducible gene in murine T cells. Sequence alignments reveal that murine HD1 is highly homologous to the yeast RPD3 pleiotropic transcriptional regulator. Indirect immunofluorescence microscopy proves that mouse HD1 is a nuclear protein. When expressed in yeast, murine HD1 is also detected in the nucleus, although it fails to complement the rpd3delta deletion phenotype. HD1 mRNA expression is low in G0 mouse cells but increases when the cells cross the G1/S boundary after growth stimulation. Immunoprecipitation experiments and functional in vitro assays show that HD1 protein is associated with histone deacetylase activity. Both HD1 protein levels and total histone deacetylase activity increase upon interleukin-2 stimulation of resting B6.1 cells. When coexpressed with a luciferase reporter construct, HD1 acts as a negative regulator of the Rous sarcoma virus enhancer/promoter. HD1 overexpression in stably transfected Swiss 3T3 cells causes a severe delay during the G2/M phases of the cell cycle. These results indicate that balanced histone acetylation/deacetylation is crucial for normal cell cycle progression of mammalian cells (Bartl, 1997).

The protein associations and enzymatic requirements were investigated for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuclear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear. Xenopus RbAp48 and SIN3 have been cloned and it has been shown that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase activity and transcriptional repression of the TRbetaA promoter. This repression requires deacetylase activity and nuclear import of RPD3 mediated by a carboxy-terminal nuclear localization signal. Exogenous RPD3 is not incorporated into oocyte deacetylase and ATPase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus. RPD3 associates with RbAp48 through N- and C-terminal contacts and RbAp48 also interacts with SIN3. Xenopus RbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo. Exogenous RPD3 may be targeted to histones through interaction with endogenous RbAp48 to direct transcriptional repression of the Xenopus TRbetaA promoter in the oocyte nucleus. However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors (Vermaak, 1999).

The Myc protein binds DNA and activates transcription by mechanisms that are still unclear. Chromatin immunoprecipitation (ChIP) was used to evaluate Myc-dependent changes in histone acetylation at seven target loci. Upon serum stimulation of Rat1 fibroblasts, Myc associates with chromatin, histone H4 becomes locally hyperacetylated, and gene expression is induced. These responses are lost or severely impaired in Myc-deficient cells, but are restored by adenoviral delivery of Myc simultaneous with mitogenic stimulation. When targeted to chromatin in the absence of mitogens, Myc directly induces H4 acetylation. In addition, Myc recruits TRRAP to chromatin, consistent with a role for this cofactor in histone acetylation. Finally, unlike serum, Myc alone is very inefficient in inducing expression of most target genes. Myc therefore governs a step, most likely H4 acetylation, that is required but not sufficient for transcriptional activation. It is proposed that Myc acts as a permissive factor, allowing additional signals to activate target promoters (Frank, 2001).

The N-terminal tails of the core histones play important roles in transcriptional regulation, but their mechanism(s) of action are poorly understood. Pure chromatin templates assembled with varied combinations of recombinant wild-type and mutant core histones have been employed to ascertain the role of individual histone tails, both in overall acetylation patterns and in transcription. In vitro assays show an indispensable role for H3 and H4 tails, especially major lysine substrates, in p300-dependent transcriptional activation, as well as activator-targeted acetylation of promoter-proximal histone tails by p300. These results indicate, first, that constraints to transcription are imposed by nucleosomal histone components other than histone N-terminal tails and, second, that the histone N-terminal tails have selective roles, which can be modulated by targeted acetylation, in transcriptional activation by p300 (An, 2002).

The first significant conclusion from these results is that the tails do not simply and uniquely impose constraints to the binding and function of either gene-specific transcriptional activators or components of the general transcriptional machinery. Instead, it seems clear that the globular domains themselves maintain a repressed state and that specific N-terminal tails and corresponding natural acetylatable lysine residues are actively required for the reversal of these effects. Another significant conclusion from the present study is that the H3 and H4 tails are selectively required for the observed derepression and net activation by Gal4-VP16 and p300 and, that these tails are not redundant for transcription. These results are consistent with differential effects of H3 versus H4 tail mutations on the transcriptional regulation of specific genes and differential functions for H3 and H4 tails versus H2A and H2B tails both in transcription and in higher-order chromatin structure (An, 2002 and references therein).

These results also establish a direct link between activator-dependent acetylation of histones by p300 and activator-dependent transcription. Beyond the fact that activator-dependent transcription requires activator- and p300-dependent histone tail acetylation, the selective requirement for H3 and H4 tails and corresponding acetylation sites for transcription correlates with the observations (1) that H3 is the preferred p300 substrate in chromatin, (2) that optimal H3 and H4 acetylation occurs independently of H2A and H2B tails, whereas maximal H2A and H2B acetylation is dependent upon H3 and H4 tails, and (3) that there is a strong activator-mediated targeting of acetylation to promoter-proximal H3 and H4 (An, 2002).

Transcriptional Regulation of Histone H4

The proximal promoter of the human H4 histone gene FO108 contains two regions of in vivo protein-DNA interaction: Sites I and II. Electrophoretic mobility shift assays using a radiolabeled DNA probe reveal that several proteins present in HeLa cell nuclear extracts bind specifically to Site I (nt-125 to nt-86). The most prominent complex, designated HiNF-C, and a complex of greater mobility, HiNF-C', are specifically compatable by an Sp1 consensus oligonucleotide. Fractionation of HiNF-C using wheat germ agglutinin affinity chromatography suggests that, like Sp1, HiNF-C contains N-acetylglucosamine moieties. Two minor complexes of even greater mobility, designated HiNF-E and F, are compatable by ATF consensus oligonucleotides. A DNA probe carrying a site-specific mutation in the distal portion of Site I fails to bind HiNF-E, indicating that this protein is associated specifically to this region. UV cross-linking analysis showed that several proteins of different molecular weights interact specifically with Site I. These data indicate that Site I possesses a bipartite structure and that multiple proteins present in HeLa cell nuclear extracts interact specifically with Site I sequences (Wright, 1995).

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B and H1 ( is activated at the G1/S phase transition of the cell cycle. The promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors, cyclin A, CDC2, and a member of the retinoblastoma protein family. The intrinsic DNA-binding subunit for HiNF-D is the ubiquitous protein CDP/cut (Drosophila homolog: Cut). The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and RB protein, whereas the CDP/cut complex with a nonhistone promoter reacts only with CDP and p107 (another member of the RB protein family) antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct RB-related proteins. CDP/cut can be shown to modulate H4 promoter activity via the HiNF-D binding site. Overexpression of CDP/cut represses H4 promoter activity via the HiNF-D(CDP/cut) site, suggesting that HiNF-D may repress or activat H4 transcription dependent on the availability of associated proteins. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclinA/CDC2/RB-related proteins (van Wijnen, 1996).

The histone H4 gene promoter provides a paradigm for defining transcriptional control operative at the G1/S phase transition point in the cell cycle. Transcription of the cell cycle-dependent histone H4 gene is upregulated at the onset of S phase; the cell cycle control element that mediates this activation has been functionally mapped to a proximal promoter domain designated Site II. Activity of Site II is regulated by an E2F-independent mechanism involving binding of the oncoprotein IRF2 and the multisubunit protein HiNF-D, which contains the following four subunits: the homeodomain protein CDP/cut, CDC2, cyclin A, and the tumor suppressor pRb (see Drosophila Retinoblastoma-family protein). To address mechanisms that define interactions of Site II regulatory factors with this cell cycle control element, these determinants of transcriptional regulation at the G1/S phase transition have been investigated in FDC-P1 hematopoietic progenitor cells. The representation and activities of histone gene regulatory factors were examined as a function of FDC-P1 growth stimulation. Striking differences in expression of the pRb-related growth regulatory proteins (pRb/p105, pRb2/p130, and p107) were found following the onset of proliferation. pRb2/p130 is present at elevated levels in quiescent cells and declines following growth stimulation. By contrast, pRb and p107 are minimally represented in quiescent FDC-P1 cells but are upregulated at the G1/S phase transition point. A dramatic upregulation of the cellular levels of pRb2/p130-associated protein kinase activity is observed when S phase is initiated. Selective interactions of pRb and p107 with CDP/cut are observed during the FDC-P1 cell cycle and suggest functional linkage to competency for DNA binding and/or transcriptional activity. These results are particularly significant in the context of hematopoietic differentiation where stringent control of the cell cycle program is requisite for expanding the stem cell population during development and tissue renewal (van Wijnen, 1997).

In female mammalian cells, dosage compensation for X-linked genes is achieved early in development by the transcriptional silencing of many genes on just one of the two X chromosomes. Several properties distinguish the inactive X (Xi) from its active counterpart (Xa). These include expression of Xist, a gene located in the X-inactivation center (Xic), late replication, differential methylation of selected CpG islands and underacetylation of histone H4. The relationship between these properties and transcriptional silencing remains unclear. Female mouse embryonic stem (ES) cells have two active X chromosomes, one of which is inactivated as cells differentiate in culture. These cells were used in studying the sequence of events leading to X-inactivation. By immunofluorescent labeling of metaphase chromosome spreads from ES cells with antibodies to acetylated H4, an underacetylated X chromosome can be shown to appear only after 4 days of differentiation, and only in female cells. The frequency of cells with an underacetylated X reaches a maximum by day 6. In undifferentiated cells, H4 in centric heterochromatin is acetylated to the same extent as that in euchromatin but has become relatively underacetylated, as in adult cells, by day 4 of differentiation (i.e. , when deacetylation of Xi is first seen). The overall deacetylation of Xi follows Xist expression and the first appearance of a single, late-replicating X, both of which occur on day 2. It also follows the silencing of X-linked genes. By days 2-4, levels of mRNA from four such genes (Hprt, G6pd, Rps4, and Pgk-1) have all fallen by approximately 50% (relative to the autosomal gene Aprt). The results show that properties that characterize Xi are put in place in a set order over several days. H4 deacetylation occupies a defined place within this sequence, suggesting that it is an intrinsic part of the X-inactivation process. The stage at which a completely deacetylated Xi is first seen suggests that deacetylation may be necessary for the maintenance of silencing but is not required for its initiation. Nor is it required for, or an immediate consequence of, late replication. However, it is noted that selective deacetylation of H4 on specific genes would not be detected by the microscopical approach used and that such selective deacetylation may still be part of the silencing process (Keohane, 1996).

Proteins of the ATF/CREB class of transcription factors stimulate gene expression of several cell growth-related genes through protein kinase A-related cAMP response elements. The promoter activity of cell cycle regulated histone H4 genes is regulated by at least four principal cis-acting elements that mediate G1/S phase control and/or enhancement of transcription during the cell cycle. Using protein-DNA interaction assays it has been shown that the H4 promoter contains two ATF/CREB recognition motifs that interact with CREB, ATF1, and ATF2 but not with ATF4/CREB2. One ATF/CRE motif is located in the distal promoter at the nuclear matrix-associated Site IV, and the second motif is present in the proximal promoter at Site I. Both ATF/CRE motifs overlap binding sequences for the multifunctional YY1 transcription factor, which has previously been shown to be nuclear matrix associated. Subnuclear fractionation reveals that there are two ATF1 isoforms that appear to differ with respect to DNA binding activity and partition selectively between nuclear matrix and nonmatrix compartments, consistent with the role of the nuclear matrix in regulating gene expression. Site-directed mutational studies demonstrate that Site I and Site IV together support ATF1- and CREB-induced trans-activation of the H4 promoter. Thus, these data establish that ATF/CREB factors functionally modulate histone H4 gene transcription at distal and proximal promoter elements (Guo, 1997).

The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. The epigenetic activator Ash1 is a multi-catalytic histone methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns (Beisel, 2002).

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