Biomedical subjects

C D Allis

Publications and source records attributed to C D Allis.

At least 55 records · Page 3Linked to original sources

PubMed2000

Light induces chromatin modification in cells of the mammalian circadian clock.

The mammalian circadian clock resides in neurons of the hypothalamic suprachiasmatic nucleus (SCN). Light entrains phase resetting of the clock using the retino-hypothalamic tract, via release of glutamate. Nighttime light exposure causes rapid, transient induction of clock and immediate-early genes implicated in phase-shifting the pacemaker. Here we show that a nighttime light pulse caused phosphorylation of Ser10 in histone H3's tail, in SCN clock cells. The effect of light was specific, and the kinetics of H3 phosphorylation were characteristic of the early response, paralleling c-fos and Per1 induction. Using fos-lacZ transgenic mice, we found that H3 phosphorylation and Fos induction occurRed in the same SCN neurons. Systemic treatment with the GABAB receptor agonist baclofen prevented light-induced c-fos and Per1 expression and H3 phosphorylation, indicating that one signaling pathway governs both events. Our results suggest that dynamic chromatin remodeling in the SCN occurs in response to a physiological stimulus in vivo.

Animals↗

PubMed2000

The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation.

In Drosophila, dosage compensation-the equalization of most X-linked gene products in males and females-is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1 and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2 RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophila males.

Animals↗

PubMed2000

A novel chromodomain protein, pdd3p, associates with internal eliminated sequences during macronuclear development in Tetrahymena thermophila.

Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

Amino Acid Sequence↗

PubMed2000

Developmentally regulated rpd3p homolog specific to the transcriptionally active macronucleus of vegetative Tetrahymena thermophila.

A clear relationship exists between histone acetylation and transcriptional output, the balance of which is conferred by opposing histone acetyltransferases (HATs) and histone deacetylases (HDACs). To explore the role of HDAC activity in determining the transcriptional competency of chromatin, we have exploited the biological features of Tetrahymena as a model. Each vegetative cell contains two nuclei: a somatic, transcriptionally active macronucleus containing hyperacetylated chromatin and a transcriptionally silent, germ line micronucleus containing hypoacetylated histones. Using a PCR-based strategy, a deacetylase gene (named THD1) encoding a homolog of the yeast HDAC Rpd3p was cloned. Thd1p deacetylates all four core histones in vitro. It resides exclusively in the macronucleus during vegetative growth and is asymmetrically distributed to developing new macronuclei early in their differentiation during the sexual pathway. Together, these data are most consistent with a potential role for Thd1p in transcriptional regulation and suggest that histone deacetylation may be important for the differentiation of micronuclei into macronuclei during development.

Amino Acid Sequence↗

PubMed2000

Somatic linker histone H1 is present throughout mouse embryogenesis and is not replaced by variant H1 degrees.

A striking feature of early embryogenesis in a number of organisms is the use of embryonic linker histones or high mobility group proteins in place of somatic histone H1. The transition in chromatin composition towards somatic H1 appears to be correlated with a major increase in transcription at the activation of the zygotic genome. Previous studies have supported the idea that the mouse embryo essentially follows this pattern, with the significant difference that the substitute linker histone might be the differentiation variant H1 degrees, rather than an embryonic variant. We show that histone H1 degrees is not a major linker histone during early mouse development. Instead, somatic H1 was present throughout this period. Though present in mature oocytes, somatic H1 was not found on maternal metaphase II chromatin. Upon formation of pronuclear envelopes, somatic H1 was rapidly incorporated onto maternal and paternal chromatin, and the amount of somatic H1 steadily increased on embryonic chromatin through to the 8-cell stage. Microinjection of somatic H1 into oocytes, and nuclear transfer experiments, demonstrated that factors in the oocyte cytoplasm and the nuclear envelope, played central roles in regulating the loading of H1 onto chromatin. Exchange of H1 from transferred nuclei onto maternal chromatin required breakdown of the nuclear envelope and the extent of exchange was inversely correlated with the developmental advancement of the donor nucleus.

Animals↗

PubMed1999

Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena.

Studies into posttranslational modifications of histones, notably acetylation, have yielded important insights into the dynamic nature of chromatin structure and its fundamental role in gene expression. The roles of other covalent histone modifications remain poorly understood. To gain further insight into histone methylation, we investigated its occurrence and pattern of site utilization in Tetrahymena, yeast, and human HeLa cells. In Tetrahymena, transcriptionally active macronuclei, but not transcriptionally inert micronuclei, contain a robust histone methyltransferase activity that is highly selective for H3. Microsequence analyses of H3 from Tetrahymena, yeast, and HeLa cells indicate that lysine 4 is a highly conserved site of methylation, which to date, is the major site detected in Tetrahymena and yeast. These data document a nonrandom pattern of H3 methylation that does not overlap with known acetylation sites in this histone. In as much as H3 methylation at lysine 4 appears to be specific to macronuclei in Tetrahymena, we suggest that this modification pattern plays a facilitatory role in the transcription process in a manner that remains to be determined. Consistent with this possibility, H3 methylation in yeast occurs preferentially in a subpopulation of H3 that is preferentially acetylated.

Acetyltransferases↗

PubMed1999

Excision of micronuclear-specific DNA requires parental expression of pdd2p and occurs independently from DNA replication in Tetrahymena thermophila.

Elimination of germ-line DNA segments is an essential step in the somatic development of many organisms and in the terminal differentiation of several specialized cell types. In binuclear ciliates, including Tetrahymena thermophila, DNA elimination occurs during the conversion of the germ-line micronuclear genome into the somatic genome of the new macronucleus. Little is known about molecular determinants and regulatory mechanisms involved in this process. Pdd2p is one of a small set of Tetrahymena polypeptides whose time of synthesis, nuclear localization, and physical association with sequences destined for elimination suggest an involvement in the DNA elimination process. In this study, we report that loss of parental expression of Pdd2p leads to the perturbation of several DNA rearrangement processes in developing zygotic macronuclei, including excision of internal eliminated sequences, excision of chromosome breakage sequences, and endoreplication of the new macronuclear genome and eventually results in lethality of the progeny. We demonstrate that excision and elimination of micronuclear-specific DNA occurs independently of endoreplication of the new macronuclear genome that takes place during the same period of time. Thus, our data indicate that parental expression of Pdd2p is required for successful DNA elimination and development of somatic nuclei.

Animals↗

PubMed1999

Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide.

Gene activation is a highly regulated process that requires the coordinated action of proteins to relieve chromatin repression and to promote transcriptional activation. Nuclear histone acetyltransferase (HAT) enzymes provide a mechanistic link between chromatin destabilization and gene activation by acetylating the epsilon-amino group of specific lysine residues within the aminoterminal tails of core histones to facilitate access to DNA by transcriptional activators. Here we report the high-resolution crystal structure of the HAT domain of Tetrahymena GCN5 (tGCN5) bound with both its physiologically relevant ligands, coenzyme A (CoA) and a histone H3 peptide, and the structures of nascent tGCN5 and a tGCN5/acetyl-CoA complex. Our structural data reveal histone-binding specificity for a random-coil structure containing a G-K-X-P recognition sequence, and show that CoA is essential for reorienting the enzyme for histone binding. Catalysis appears to involve water-mediated proton extraction from the substrate lysine by a glutamic acid general base and a backbone amide that stabilizes the transition-state reaction intermediate. Comparison with related N-acetyltransferases indicates a conserved structural framework for CoA binding and catalysis, and structural variability in regions associated with substrate-specific binding.

Acetyltransferases↗

PubMed1999

Increased Ser-10 phosphorylation of histone H3 in mitogen-stimulated and oncogene-transformed mouse fibroblasts.

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

3T3 Cells↗

PubMed1999

Requirement of Rsk-2 for epidermal growth factor-activated phosphorylation of histone H3.

During the immediate-early response of mammalian cells to mitogens, histone H3 is rapidly and transiently phosphorylated by one or more unidentified kinases. Rsk-2, a member of the pp90rsk family of kinases implicated in growth control, was required for epidermal growth factor (EGF)-stimulated phosphorylation of H3. RSK-2 mutations in humans are linked to Coffin-Lowry syndrome (CLS). Fibroblasts derived from a CLS patient failed to exhibit EGF-stimulated phosphorylation of H3, although H3 was phosphorylated during mitosis. Introduction of the wild-type RSK-2 gene restored EGF-stimulated phosphorylation of H3 in CLS cells. In addition, disruption of the RSK-2 gene by homologous recombination in murine embryonic stem cells abolished EGF-stimulated phosphorylation of H3. H3 appears to be a direct or indirect target of Rsk-2, suggesting that chromatin remodeling might contribute to mitogen-activated protein kinase-regulated gene expression.

3T3 Cells↗

PubMed1999

Crystal structure and mechanism of histone acetylation of the yeast GCN5 transcriptional coactivator.

The yeast GCN5 (yGCN5) transcriptional coactivator functions as a histone acetyltransferase (HAT) to promote transcriptional activation. Here, we present the high resolution crystal structure of the HAT domain of yGCN5 and probe the functional importance of a conserved glutamate residue. The structure reveals a central protein core associated with AcCoA binding that appears to be structurally conserved among a superfamily of N-acetyltransferases, including yeast histone acetyltransferase 1 and Serratia marcescens aminoglycoside 3-N-acetyltransferase. A pronounced cleft lying above this core, and flanked by N- and C-terminal regions that show no sequence conservation within N-acetyltransferase enzymes, is implicated by cross-species conservation and mutagenesis studies to be a site for histone substrate binding and catalysis. Located at the bottom of this cleft is a conserved glutamate residue (E173) that is in position to play an important catalytic role in histone acetylation. Functional analysis of an E173Q mutant yGCN5 protein implicates glutamate 173 to function as a general base for catalysis. Together, a correlation of the yGCN5 structure with functionally debilitating yGCN5 mutations provides a paradigm for understanding the structure/function relationships of the growing number of transcriptional regulators that function as histone acetyltransferase enzymes.

Acetylation↗

PubMed1999

Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A.

Gene transcription requires the release of inactive DNA from its packaging of histone proteins. Following the discovery of the first transcription-associated histone acetyltransferase, tetrahymena GCN5, it was shown that yeast GCN5 is recruited to the promoter and causes hyper-acetylation of histones and transcriptional activation of target genes, establishing a direct connection between histone acetylation and transcriptional activation. Many other important transcription regulators have been found to have histone acetyltransferase activity, including TAFII230/250, p300/CBP and its associated factor PCAF. Here we present the solution structure of the catalytic domain of tGCN5 (residues 47-210) in complex with coenzyme A. The structure contains two domains; the amino-terminal domain is similar to those of other GCN5-related N-acetyltransferases but the carboxy-terminal domain is not. Coenzyme A binds in a deep hydrophobic pocket between the two domains. Chemical shift changes upon titration with histone H3 peptides indicate a binding site at the domain boundary opposite to the coenzyme A site. The structural data indicate a single-step acetyl-transfer reaction mechanism catalysed by a hydrogen bond to the backbone amide group of leucine 126 and the side-chain carboxyl group of a conserved acidic residue.

Acetyltransferases↗

PubMed1999

Catalytic mechanism and function of invariant glutamic acid 173 from the histone acetyltransferase GCN5 transcriptional coactivator.

Within chromatin, reversible acetylation of core histones is critical for transcriptional activation of eukaryotic target genes. The recent identification of intrinsic histone acetyltransferase (HAT) catalytic activity from a number of transcriptional co-activators (including yeast GCN5, p300/CBP, P/CAF, and TAFII250), has underscored the importance of protein acetylation in transcriptional control. The GCN5 family is the prototype for a diverse group of at least four distinct human HATs families. Although there is now a clear link between in vivo HAT catalytic activity and gene activation, little is known about the molecular mechanisms of histone acetylation. Herein, we report the first detailed biochemical study that probes the catalytic mechanism and the function of invariant glutamic acid 173 within the GCN5 family of HATs. Our results suggest that the HAT reaction involves the formation of a ternary complex (histones, acetyl-CoA, and enzyme) where the epsilon-amino group of histone lysine residues directly attacks the bound acetyl-CoA. The acetylation reaction requires deprotonation of the epsilon-amino group prior to nucleophilic attack. Employing site-directed mutagenesis, chemical modification, steady-state, and pH-dependent rate analysis, it is demonstrated that glutamic acid 173 is an essential catalytic residue, acting as a general base catalyst by deprotonating the histone substrate.

Acetyl Coenzyme A↗

PubMed1999

Cell cycle-regulated histone acetylation required for expression of the yeast HO gene.

Expression of the yeast HO gene in late G1 of the cell cycle requires the SWI/SNF chromatin remodeling complex, the Gcn5p histone acetyltransferase, and two different sequence-specific transcriptional activators, Swi5p and Swi4p/Swi6p. We have used chromatin immunoprecipitation assays to investigate the role of each of these trans-acting factors in establishing a cell cycle-regulated domain of histone acetylation surrounding the HO upstream regulatory region. We detect a approximately 1-kb domain of H3 and H4 acetylation that is established in mid-G1, prior to and independent of HO transcription, which then declines with kinetics similar to inactivation of HO. This cell cycle burst of histone acetylation requires Gcn5p, SWI/SNF, and the Swi5p activator, but occurs in the absence of the Swi4p activator. We also find that inactivation of the Sin3p/Rpd3p deacetylase complex leads to a high level of acetylation at the HO locus throughout the cell cycle. We propose a sequential model for activation of HO in which the Swi5p-dependent recruitment of the Gcn5p acetyltransferase requires chromatin remodeling events by the SWI/SNF complex.

Acetylation↗

PubMed1999

Identification and mutation of phosphorylation sites in a linker histone. Phosphorylation of macronuclear H1 is not essential for viability in tetrahymena.

Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kinase consensus sequences. Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34(cdc2) site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena.

Amino Acid Sequence↗

PubMed1999

Phosphorylation of histone H3 is required for proper chromosome condensation and segregation.

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.

Animals↗

PubMed1999

Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates.

A number of transcriptional coactivators possess intrinsic histone acetylase activity, providing a direct link between hyperacetylated chromatin and transcriptional activation. We have determined the core histone residues acetylated in vitro by recombinant p300 and PCAF within mononucleosomes. p300 specifically acetylates all sites of histones H2A and H2B known to be acetylated in bulk chromatin in vivo but preferentially acetylates lysines 14 and 18 of histone H3 and lysines 5 and 8 of histone H4. PCAF primarily acetylates lysine 14 of H3 but also less efficiently acetylates lysine 8 of H4. PCAF in its native form, which is present in a stable multimeric protein complex lacking p300/CBP, primarily acetylates H3 to a monoacetylated form, suggesting that PCAF-associated polypeptides do not alter the substrate specificity. These distinct patterns of acetylation by the p300 and PCAF may contribute to their differential roles in transcriptional regulation.

Acetylation↗

PubMed1999

In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment.

Chromatin structure plays important roles in regulating many DNA-templated processes, such as transcription, replication, and recombination. Considerable progress has recently been made in the identification of large, multisubunit complexes dedicated to these nuclear processes, all of which occur on nucleosomal templates. Mapping specific genomic loci relative to the position of selectively modified or unique histone variants or nonhistone components provides valuable insights into how these proteins (and their modifications) function in their normal chromatin context. Here we describe a versatile and high-resolution method which involves two basic steps: (1) in vivo formaldehyde cross-linking of intact cells followed by (2) selective immunoprecipitation of protein-DNA complexes with specific antibodies. This method allows for detailed analyses of protein-DNA interactions in a native chromatin environment. Recently, this technique has been successfully employed to map the boundaries of specifically modified (e.g., acetylated) histones along target genes, to define the cell cycle-regulated assembly of origin-dependent replication and centromere-specific complexes with remarkable precision, and to map the in vivo position of reasonably rare transcription factors on cognate DNA sites. Thus, the basic chromatin immunoprecipitation technique is remarkably versatile and has now been used in a wide range of cell types, including budding yeast, fly, and human cells. As such, it seems likely that many more studies, centered around chromatin structure and protein-DNA interactions in its native setting, will benefit from this technique. In this article, a brief review of the history of this powerful approach and a discussion of the basic method are provided. Procedures for protein recovery as well as limitations and extensions of the method are also presented.

Cell Nucleus↗