Search PubMed⌕ Search

Biomedical subjects

C D Allis

Publications and source records attributed to C D Allis.

At least 91 records · Page 5Linked to original sources
PubMed1998

Roles of histone acetyltransferases and deacetylases in gene regulation.

Acetylation of internal lysine residues of core histone N-terminal domains has been found correlatively associated with transcriptional activation in eukaryotes for more than three decades. Recent discoveries showing that several transcriptional regulators possess intrinsic histone acetyltransferase (HAT) and deacetylase (HDAC) activities strongly suggest that histone acetylation and deacetylation each plays a causative role in regulating transcription. Intriguingly, several HATs have been shown an ability to acetylate nonhistone protein substrates (e.g., transcription factors) in vitro as well, suggesting the possibility that internal lysine acetylation of multiple proteins exists as a rapid and reversible regulatory mechanism much like protein phosphorylation. This article reviews recent developments in histone acetylation and transcriptional regulation. We also discuss several important, yet unanswered, questions.

Acetylation↗
PubMed1998

Linking histone acetylation to transcriptional regulation.

In eukaryotes, DNA is assembled with histones to form nucleosomes, the basic subunit of chromatin structure. The wrapping of DNA around histone octamers to form nucleosomal filaments and further folding of these filaments are necessary to contain eukaryotic genomes within nuclei. However, the dense packing of chromatin in nuclei and the association of DNA with histones restrict the access of proteins involved in gene transcription to DNA. Abundant biochemical data supports a long-standing correlation between histone acetylation and gene activation, suggesting that histone acetylation acts to enhance the access of transcription-associated proteins to DNA. However, despite this correlation, nuclear enzymes responsible for transcription-associated histone acetylation have been identified only recently. Here we review evidence suggesting that histone acetylation represents a major pathway for transcriptional regulation, and discuss possible roles for transcription-associated histone acetyltransferases in this regulation.

Acetylation↗
PubMed1998

Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation.

The temporal and spatial patterns of histone H3 phosphorylation implicate a specific role for this modification in mammalian chromosome condensation. Cells arrest in late G2 when H3 phosphorylation is competitively inhibited by microinjecting excess substrate at mid-S-phase, suggesting a requirement for activity of the kinase that phosphorylates H3 during the initiation of chromosome condensation and entry into mitosis. Basal levels of phosphorylated H3 increase primarily in late-replicating/early-condensing heterochromatin both during G2 and when premature chromosome condensation is induced. The prematurely condensed state induced by okadaic acid treatment during S-phase culminates with H3 phosphorylation throughout the chromatin, but in an absence of mitotic chromosome morphology, indicating that the phosphorylation of H3 is not sufficient for complete condensation. Mild hypotonic treatment of cells arrested in mitosis results in the dephosphorylation of H3 without a cytological loss of chromosome compaction. Hypotonic-treated cells, however, complete mitosis only when H3 is phosphorylated. These observations suggest that H3 phosphorylation is required for cell cycle progression and specifically for the changes in chromatin structure incurred during chromosome condensation.

Amino Acid Sequence↗
PubMed1997

Steroid receptor coactivator-1 is a histone acetyltransferase.

Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs) target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression. The steroid-receptor coactivator SRC-1 is a coactivator for many members of the steroid-hormone receptor superfamily of ligand-inducible transcription factors. Here we show that SRC-1 possesses intrinsic histone acetyltransferase activity and that it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates.

Acetylation↗
PubMed1997

Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.

The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).

Acetylation↗
PubMed1997

Histone H1b phosphorylation is dependent upon ongoing transcription and replication in normal and ras-transformed mouse fibroblasts.

We have previously shown that mouse phosphorylated histone H1b (pH1b) was localized near nuclear sites that contained splicing factors. This observation suggested to us that pH1b was associated with transcribing chromatin. Here we investigated the relationship between phosphorylation of H1b and transcription. We demonstrate that treatment of normal or ras-transformed mouse fibroblasts with the transcription inhibitor actinomycin D for 70 min results in a dramatic decrease in the level of pH1b. Similar results were observed when transcription was inhibited by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). When DRB was removed, the level of pH1b was restored after 2 h. Treatment of the cells with aphidicolin, a potent inhibitor of replication, resulted in a marked decrease in the level of pH1b after 30 min. This is the first report showing a dependence of histone modification upon ongoing transcription and replication. Inhibition of transcription or replication may hinder accessibility of H1b to the H1 kinase, supporting the idea that pH1b is associated with transcribing chromatin. Phosphorylation of H1b may be required to maintain a more decondensed chromatin structure to facilitate transcription and replication processes.

Animals↗
PubMed1997

Histone acetyltransferase activity and interaction with ADA2 are critical for GCN5 function in vivo.

Yeast GCN5 is one component of a putative adaptor complex that includes ADA2 and ADA3 and functionally connects DNA-bound transcriptional activators with general transcription factors. GCN5 possesses histone acetyltransferase (HAT) activity, conceptually linking transcriptional activation with enzymatic modification at chromatin. We have identified the minimal catalytic domain within GCN5 necessary to confer HAT activity and have shown that in vivo activity of GCN5 requires this domain. However, complementation of growth and transcriptional activation in gcn5- cells required not only the HAT domain of GCN5, but also interaction with ADA2. The bromodomain in GCN5 was dispensable for HAT activity and for transcriptional activation by strong activators; however, it was required for full complementation in other assays. Fusion of GCN5 to the bacterial lexA DNA binding domain activated transcription in vivo, and required both the HAT domain and the ADA2 interaction domain. These results suggest that both functions of GCN5, HAT activity and interaction with ADA2, are necessary for targeting and acetylation of nucleosomal histones.

Acetyltransferases↗
PubMed1997

Histones in transit: cytosolic histone complexes and diacetylation of H4 during nucleosome assembly in human cells.

The organization and acetylation of nascent histones prior to their stable incorporation into chromatin were examined. Through sedimentation and immunoprecipitation analyses of HeLa cytosolic extracts, two somatic non-nucleosomal histone complexes were detected: one containing nascent H3 and H4, and a second containing H2A (and probably H2B) in association with the nonhistone protein NAP-1. The H3/H4 complex has a sedimentation coefficient of 5-6S, consistent with the presence of one or more escort proteins. H4 in the cytosolic H3/H4 complex is diacetylated, fully in accord with the acetylation state of newly synthesized H4 in chromatin. The diacetylation of nascent human H4 is therefore completed prior to nucleosome assembly. As part of our studies of the nascent H3/H4 complex, the cytoplasmic histone acetyltransferase most likely responsible for acetylating newly synthesized H4 was also investigated. HeLa histone acetyltransferase B (HAT B) acetylates H4 but not H3 in vitro, and maximally diacetylates H4 even in the presence of sodium butyrate. Human HAT B acetylates H4 exclusively on the lysine residues at positions 5 and 12, in complete agreement with the highly conserved acetylation pattern of nascent nucleosomal H4 (Sobel et al., 1995), and has a native molecular weight of approximately 100 kDa. Based on our findings a model is presented for the involvement of histone acetylation and NAP-1 in H2A/H2B deposition and exchange, during nucleosome assembly and chromatin remodeling in vivo.

Acetylation↗
PubMed1997

Phosphorylation of linker histones by a protein kinase A-like activity in mitotic nuclei.

Micronuclear linker histones of the ciliated protozoan, Tetrahymena thermophila, are extensively phosphorylated in vivo. Each of these polypeptides, alpha, beta, gamma, and delta, contains sites for phosphorylation by cyclic-AMP dependent protein kinase (PKA) but not Cdc2 kinase, and some data have been presented implicating PKA kinase in their phosphorylation in vitro and in vivo (Sweet, M. T., and Allis, C. D. (1993) Chromosoma 102, 637-647; Sweet, M. T., Jones, K., and Allis, C. D. (1996) J. Cell Biol., in press). In this report we have extended these analyses by showing that Cdc2 and PKA kinase are not evenly distributed between micro- and macronuclei. Macronuclei, but not micronuclei, contain a 36-kDa polypeptide that is immunoreactive with p34Cdc2 antibodies. In contrast, a 40-kDa polypeptide is detected with PKA antibodies in micronuclei, that is not detected in macronuclei. In support, extracts from micronuclei, but not macronuclei, contain a kinase activity that resembles some, but not all, characteristics of PKA from other sources. Immunodepletion experiments using anti-PKA antibodies show that a 40-kDa polypeptide can be specifically removed from these extracts with a concomitant loss in kinase activity. Microsequence analyses of delta demonstrate that this linker histone is phosphorylated in vivo on two PKA consensus sequences located in its carboxyl-terminal domain, an optimum PKA consensus sequence, Arg-Lys-Asn-Ser, and a less optimal PKA sequence, Lys-Ser-Ser-Val. Collectively, these results suggest that PKA or a PKA-like kinase is responsible for the phosphorylation of linker histone in mitotically dividing micronuclei. In contrast, macronuclei, which divide amitotically, phosphorylate linker histone H1 using a distinct, Cdc2-like kinase.

Amino Acid Sequence↗
PubMed1997

Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.

We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that "drive" chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase.

Amino Acid Sequence↗
PubMed1997

An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

Amino Acid Sequence↗
PubMed1997

Histone acetyltransferase activity is conserved between yeast and human GCN5 and is required for complementation of growth and transcriptional activation.

Yeast and human ADA2 and GCN5 (y- and hADA2 and y- and hGCN5, respectively) have been shown to potentiate transcription in vivo and may function as adaptors to bridge physical interactions between DNA-bound activators and the basal transcriptional machinery. Recently it was shown that yGCN5 is a histone acetyltransferase (HAT), suggesting a link between enzymatic modification of nucleosomes and transcriptional activation. In this report, we demonstrate that hGCN5 is also an HAT and has the same substrate specificity as yGCN5. Since hGCN5 does not complement functional defects caused by deletion of yGCN5, we constructed a series of hGCN5-yGCN5 chimeras to identify human regions capable of activity in yeast. Interestingly, only the putative HAT domain of hGCN5, when fused to the remainder of yGCN5, complemented gcn5- cells for growth and transcriptional activation. Moreover, an amino acid substitution mutation within the HAT domain reduced both HAT activity in vitro and transcription in vivo. These findings directly link enzymatic histone acetylation and transcriptional activation and show evolutionary conservation of this potentially crucial pathway in gene regulation.

Acetylation↗
PubMed1997

Pdd1p associates with germline-restricted chromatin and a second novel anlagen-enriched protein in developmentally programmed DNA elimination structures.

Programmed DNA rearrangements, including DNA diminution, characterize the differentiation of somatic from germline nuclei in several developmental systems. Pdd1p (Programmed DNA degradation protein 1), a development-restricted polypeptide, has been implicated in heterochromatin assembly and DNA degradation during ciliate macronuclear development. Here, cross-linking and co-immunoprecipitation were used to verify that Pdd1p-associated chromatin is enriched in germline-restricted DNA. Pdd1p-associated proteins include general core histones and a second anlagen-enriched polypeptide (Pdd2p, formerly known as p43). Immunoblotting analyses demonstrate that, like Pdd1p, Pdd2p is developmentally regulated and present in conjugating cells during the time of germline DNA rearrangements and degradation. Pdd2p is post-translationally modified by phosphorylation at a time in development corresponding to dephosphorylation of Pdd1p and the formation of heterochromatic DNA elimination structures. Following gene cloning, the derived amino acid sequence of the PDD2 gene predicts a novel polypeptide containing multiple putative phosphorylation sites. In situ analyses, using both light and electron microscopy, demonstrate that Pdd1p and Pdd2p co-localize in DNA elimination structures within developing macronuclei. However, unlike Pdd1p, which also localizes to apoptotic macronuclei, Pdd2p appears to be restricted to a higher degree to germline DNA elimination structures. Taken together, the data presented here demonstrate a physical link between Pdd1p and germline-restricted chromatin and establish Pdd2p as the second member of a small group of developmentally restricted polypeptides implicated in programmed DNA elimination.

Amino Acid Sequence↗
PubMed1996

The TAF(II)250 subunit of TFIID has histone acetyltransferase activity.

The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.

Acetyltransferases↗
PubMed1996

Pdd1p, a novel chromodomain-containing protein, links heterochromatin assembly and DNA elimination in Tetrahymena.

During Tetrahymena conjugation, programmed DNA degradation occurs in two separate nuclei. Thousands of germline-specific deletion elements are removed from the genome of the developing somatic macronucleus, and the old parental macronucleus is degraded by an apoptotic mechanism. An abundant polypeptide, Pdd1p (formerly p65), localizes to both of these nuclei at the time of DNA degradation. Here we report that, in developing macronuclei, Pdd1p localizes to electron-dense, heterochromatic structures that contain germline-specific deletion elements. Pdd1p also associates with parental macronuclei during terminal stages of apoptosis. Sequencing of the PDD1 gene reveals it to be a member of the chromodomain family, suggesting a molecular link between heterochromatin assembly and programmed DNA degradation.

Amino Acid Sequence↗
PubMed1996

Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines.

The yeast transcriptional adaptor, Gcn5p, is a catalytic subunit of a nuclear (type A) histone acetyltransferase linking histone acetylation to gene activation. Here we report that Gcn5p acetylates histones H3 and H4 non-randomly at specific lysines in the amino-terminal domains. Lysine 14 of H3 and lysines 8 and 16 of H4 are highly preferred acetylation sites for Gcn5p. We also demonstrate that lysine 9 is the preferred position of acetylation in newly synthesized yeast H3 in vivo. This finding, along with the fact that lysines 5 and 12 in H4 are predominant acetylation sites during chromatin assembly of many organisms, indicates that Gcn5p acetylates a distinct set of lysines that do not overlap with those sites characteristically used by type B histone acetyltransferases for histone deposition and chromatin assembly.

Acetylation↗