Biomedical subjects

C David Allis

Publications and source records attributed to C David Allis.

At least 19 recordsLinked to original sources

PubMed2003

Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains.

On the histone H3 tail, Lys 9 and Lys 27 are both methylation sites associated with epigenetic repression, and reside within a highly related sequence motif ARKS. Here we show that the chromodomain proteins Polycomb (Pc) and HP1 (heterochromatin protein 1) are highly discriminatory for binding to these sites in vivo and in vitro. In Drosophila S2 cells, and on polytene chromosomes, methyl-Lys 27 and Pc are both excluded from areas that are enriched in methyl-Lys 9 and HP1. Swapping of the chromodomain regions of Pc and HP1 is sufficient for switching the nuclear localization patterns of these factors, indicating a role for their chromodomains in both target site binding and discrimination. To better understand the molecular basis for the selection of methyl-lysine binding sites, we solved the 1.8 A structure of the Pc chromodomain in complex with a H3 peptide bearing trimethyl-Lys 27, and compared it with our previously determined structure of the HP1 chromodomain in complex with a H3 peptide bearing trimethyl-Lys 9. The Pc chromodomain distinguishes its methylation target on the H3 tail via an extended recognition groove that binds five additional residues preceding the ARKS motif.

Amino Acid Motifs↗

PubMed2003

Apoptotic phosphorylation of histone H2B is mediated by mammalian sterile twenty kinase.

DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Here we report that phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. We identify a 34 kDa apoptosis-induced H2B kinase as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provide insights into a previously unrecognized physiological substrate for Mst1 kinase. Our data provide evidence for a potential apoptotic "histone code."

Apoptosis↗

PubMed2003

Histone and chromatin cross-talk.

Chromatin is the physiologically relevant substrate for all genetic processes inside the nuclei of eukaryotic cells. Dynamic changes in the local and global organization of chromatin are emerging as key regulators of genomic function. Indeed, a multitude of signals from outside and inside the cell converges on this gigantic signaling platform. Numerous post-translational modifications of histones, the main protein components of chromatin, have been documented and analyzed in detail. These 'marks' appear to crucially mediate the functional activity of the genome in response to upstream signaling pathways. Different layers of cross-talk between several components of this complex regulatory system are emerging, and these epigenetic circuits are the focus of this review.

Animals↗

PubMed2003

Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa.

Besides serving to package nuclear DNA, histones carry information in the form of a diverse array of post-translational modifications. Methylation of histones H3 and H4 has been implicated in long-term epigenetic 'memory'. Dimethylation or trimethylation of Lys4 of histone H3 (H3 Lys4) has been found in expressible euchromatin of yeasts and mammals. In contrast, methylation of Lys9 of histone H3 (H3 Lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin of mammals, yeasts, Drosophila melanogaster and plants. We have previously shown that a DNA methylation mutant of Neurospora crassa, dim-5 (defective in methylation), has a nonsense mutation in the SET domain of an H3-specific histone methyltransferase and that substitutions of H3 Lys9 cause gross hypomethylation of DNA. Similarly, the KRYPTONITE histone methyltransferase is required for full DNA methylation in Arabidopsis thaliana. We used biochemical, genetic and immunological methods to investigate the specific mark for DNA methylation in N. crassa. Here we show that trimethylated H3 Lys9, but not dimethylated H3 Lys9, marks chromatin regions for cytosine methylation and that DIM-5 specifically creates this mark.

DNA Methylation↗

PubMed2003

Modulation of histone acetylation by [4-(acetylamino)-N-(2-amino-phenyl) benzamide] in HCT-8 colon carcinoma.

CI-994 or N-acetyldinaline [4-(acetylamino)-N-(2-amino-phenyl) benzamide] is an antitumor cytostatic agent currently undergoing clinical trial. Although several changes in cellular metabolism induced by the drug have been characterized, the primary molecular mechanism of its antitumor activity has been previously unknown. Here, we show that CI-994 is a histone deacetylase (HDAC) inhibitor that causes histone hyperacetylation in living cells. In assays of isolated enzymes, CI-994 inhibited HDAC-1 and HDAC-2 in a concentration-dependent fashion but had no effect on the activity of the prototypical histone acetyltransferase GCN5. Acetylated histone H3-specific Western blots were used to monitor histone acetylation in HCT-8 colon carcinoma cells treated with CI-994 in vitro. CI-994 induced hyperacetylation of H3 in a time- and dose-dependent fashion. H3 hyperacetylation was detectable as early as 30 min after the addition of CI-994 to cells. These data demonstrate that inhibition of HDAC is an early event in cells treated with CI-994 and suggest that this inhibition is mechanistically related to the antitumor activity of this compound.

Acetylation↗

PubMed2002

Methylation of histone h3 at lysine 9 targets programmed DNA elimination in tetrahymena.

Histone H3 lysine 9 methylation [Me(Lys9)H3] is an epigenetic mark for heterochromatin-dependent gene silencing, mediated by direct binding to chromodomain-containing proteins such as Heterochromatin Protein 1. In the ciliate Tetrahymena, two chromodomain proteins, Pdd1p and Pdd3p, are involved in the massive programmed DNA elimination that accompanies macronuclear development. We report that both proteins bind H3(Lys9)Me in vitro. In vivo, H3(Lys9)Me is confined to the time period and location where DNA elimination occurs, and associates with eliminated sequences. Loss of parental Pdd1p expression drastically reduces H3(Lys9)Me. Finally, tethering Pdd1p is sufficient to promote DNA excision. These results extend the range of H3(Lys9)Me involvement in chromatin activities outside transcriptional regulation and also strengthen the link between heterochromatin formation and programmed DNA elimination.

Amino Acid Sequence↗

PubMed2002

Mitotic-specific methylation of histone H4 Lys 20 follows increased PR-Set7 expression and its localization to mitotic chromosomes.

We describe distinct patterns of histone methylation during human cell cycle progression. Histone H4 methyltransferase activity was found to be cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely followed the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. These data suggest that H4 Lys 20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16 acetylation and to establish a mechanism by which this mark is epigenetically transmitted.

Acetylation↗

PubMed2002

Induction and maintenance of nonsymmetrical DNA methylation in Neurospora.

One can imagine a variety of mechanisms that should result in self-perpetuating biological states. It is generally assumed that cytosine methylation is propagated in eukaryotes by enzymes that specifically methylate hemimethylated symmetrical sites (e.g., (5')CpGGpC(5') or (5')CpNpGGpNpC(5')). Although there is wide support for this model, we and others have found examples of methylation that must be propagated by a different mechanism. Most methylated regions of the Neurospora genome that have been examined are products of repeat-induced point mutation, a premeiotic genome defense system that litters duplicated sequences with C.G to T.A mutations and typically leaves them methylated at remaining cytosines. In general, such relics of repeat-induced point mutation are capable of triggering methylation de novo. Nevertheless, some reflect a mechanism that can propagate heterogeneous methylation at nonsymmetrical sites. We propose that de novo and maintenance methylation are manifestations of a single mechanism in Neurospora, catalyzed by the DIM-2 DNA methyltransferase. The action of DIM-2 is controlled by the DIM-5 histone H3 Lys-9 methyltransferase, which in turn is influenced by other modifications of histone H3. DNA methylation indirectly recruits histone deacetylases, providing the framework of a self-reinforcing system that could result in propagation of DNA methylation and the associated silenced chromatin state.

DNA Methylation↗

PubMed2002

Gene silencing: trans-histone regulatory pathway in chromatin.

The fundamental unit of eukaryotic chromatin, the nucleosome, consists of genomic DNA wrapped around the conserved histone proteins H3, H2B, H2A and H4, all of which are variously modified at their amino- and carboxy-terminal tails to influence the dynamics of chromatin structure and function -- for example, conjugation of histone H2B with ubiquitin controls the outcome of methylation at a specific lysine residue (Lys 4) on histone H3, which regulates gene silencing in the yeast Saccharomyces cerevisiae. Here we show that ubiquitination of H2B is also necessary for the methylation of Lys 79 in H3, the only modification known to occur away from the histone tails, but that not all methylated lysines in H3 are regulated by this 'trans-histone' pathway because the methylation of Lys 36 in H3 is unaffected. Given that gene silencing is regulated by the methylation of Lys 4 and Lys 79 in histone H3, we suggest that H2B ubiquitination acts as a master switch that controls the site-selective histone methylation patterns responsible for this silencing.

Chromatin↗

PubMed2002

Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast.

In eukaryotes, the DNA of the genome is packaged with histone proteins to form nucleosomal filaments, which are, in turn, folded into a series of less well understood chromatin structures. Post-translational modifications of histone tail domains modulate chromatin structure and gene expression. Of these, histone ubiquitination is poorly understood. Here we show that the ubiquitin-conjugating enzyme Rad6 (Ubc2) mediates methylation of histone H3 at lysine 4 (Lys 4) through ubiquitination of H2B at Lys 123 in yeast (Saccharomyces cerevisiae). Moreover, H3 (Lys 4) methylation is abolished in the H2B-K123R mutant, whereas H3-K4R retains H2B (Lys 123) ubiquitination. These data indicate a unidirectional regulatory pathway in which ubiquitination of H2B (Lys 123) is a prerequisite for H3 (Lys 4) methylation. We also show that an H2B-K123R mutation perturbs silencing at the telomere, providing functional links between Rad6-mediated H2B (Lys 123) ubiquitination, Set1-mediated H3 (Lys 4) methylation, and transcriptional silencing. Thus, these data reveal a pathway leading to gene regulation through concerted histone modifications on distinct histone tails. We refer to this as 'trans-tail' regulation of histone modification, a stated prediction of the histone code hypothesis.

Alleles↗

PubMed2002

Previously uncharacterized histone acetyltransferases implicated in mammalian spermatogenesis.

During spermiogenesis (the maturation of spermatids into spermatozoa) in many vertebrate species, protamines replace histones to become the primary DNA-packaging protein. It has long been thought that this process is facilitated by the hyperacetylation of histone H4. However, the responsible histone acetyltransferase enzymes are yet to be identified. CDY is a human Y-chromosomal gene family expressed exclusively in the testis and implicated in male infertility. Its mouse homolog Cdyl, which is autosomal, is expressed abundantly in the testis. Proteins encoded by CDY and its homologs bear the "chromodomain," a motif implicated in chromatin binding. Here, we show that (i) human CDY and mouse CDYL proteins exhibit histone acetyltransferase activity in vitro, with a strong preference for histone H4; (ii) expression of human CDY and mouse Cdyl genes during spermatogenesis correlates with the occurrence of H4 hyperacetylation; and (iii) CDY and CDYL proteins are localized to the nuclei of maturing spermatids where H4 hyperacetylation takes place. Taken together, these data link human CDY and mouse CDYL to the histone-to-protamine transition in mammalian spermiogenesis. This link offers a plausible mechanism to account for spermatogenic failure in patients bearing deletions of the CDY genes.

Acetylation↗

PubMed2002

Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation.

A novel histone methyltransferase, termed Set9, was isolated from human cells. Set9 contains a SET domain, but lacks the pre- and post-SET domains. Set9 methylates specifically lysine 4 (K4) of histone H3 (H3-K4) and potentiates transcription activation. The histone H3 tail interacts specifically with the histone deacetylase NuRD complex. Methylation of histone H3-K4 by Set9 precludes the association of NuRD with the H3 tail. Moreover, methylation of H3-K4 impairs Suv39h1-mediated methylation at K9 of H3 (H3-K9). The interplay between the Set9 and Suv39h1 histone methyltransferases is specific, as the methylation of H3-K9 by the histone methyltransferase G9a was not affected by Set9 methylation of H3-K4. Our studies suggest that Set9-mediated methylation of H3-K4 functions in transcription activation by competing with histone deacetylases and by precluding H3-K9 methylation by Suv39h1. Our results suggest that the methylation of histone tails can have distinct effects on transcription, depending on its chromosomal location, the combination of posttranslational modifications, and the enzyme (or protein complex) involved in the particular modification.

Amino Acid Sequence↗

PubMed2002

Evidence that Set1, a factor required for methylation of histone H3, regulates rDNA silencing in S. cerevisiae by a Sir2-independent mechanism.

Several types of histone modifications have been shown to control transcription. Recent evidence suggests that specific combinations of these modifications determine particular transcription patterns. The histone modifications most recently shown to play critical roles in transcription are arginine-specific and lysine-specific methylation. Lysine-specific histone methyltransferases all contain a SET domain, a conserved 130 amino acid motif originally identified in polycomb- and trithorax-group proteins from Drosophila. Members of the SU(VAR)3-9 family of SET-domain proteins methylate K9 of histone H3. Methylation of H3 has also been shown to occur at K4. Several studies have suggested a correlation between K4-methylated H3 and active transcription. In this paper, we provide evidence that K4-methylated H3 is required in a negative role, rDNA silencing in Saccharomyces cerevisiae. In a screen for rDNA silencing mutants, we identified a mutation in SET1, previously shown to regulate silencing at telomeres and HML. Recent work has shown that Set1 is a member of a complex and is required for methylation of K4 of H3 at several genomic locations. In addition, we demonstrate that a K4R change in H3, which prevents K4 methylation, impairs rDNA silencing, indicating that Set1 regulates rDNA silencing, directly or indirectly, via H3 methylation. Furthermore, we present several lines of evidence that the role of Set1 in rDNA silencing is distinct from that of the histone deacetylase Sir2. Together, these results suggest that Set1-dependent H3 methylation is required for rDNA silencing in a Sir2-independent fashion.

Acetylation↗

PubMed2002

PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin.

We have purified a human histone H4 lysine 20 methyltransferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Acetylation↗

PubMed2002

MLL targets SET domain methyltransferase activity to Hox gene promoters.

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. We show that MLL regulates target Hox gene expression through direct binding to promoter sequences. We further show that the MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins.

Animals↗

PubMed2002

Involvement of histone methylation and phosphorylation in regulation of transcription by thyroid hormone receptor.

Previous studies have established an important role of histone acetylation in transcriptional control by nuclear hormone receptors. With chromatin immunoprecipitation assays, we have now investigated whether histone methylation and phosphorylation are also involved in transcriptional regulation by thyroid hormone receptor (TR). We found that repression by unliganded TR is associated with a substantial increase in methylation of H3 lysine 9 (H3-K9) and a decrease in methylation of H3 lysine 4 (H3-K4), methylation of H3 arginine 17 (H3-R17), and a dual modification of phosphorylation of H3 serine 10 and acetylation of lysine 14 (pS10/acK14). On the other hand, transcriptional activation by liganded TR is coupled with a substantial decrease in both H3-K4 and H3-K9 methylation and a robust increase in H3-R17 methylation and the dual modification of pS10/acK14. Trichostatin A treatment results in not only histone hyperacetylation but also an increase in methylation of H3-K4, increase in dual modification of pS10/acK14, and reduction in methylation of H3-K9, revealing an extensive interplay between histone acetylation, methylation, and phosphorylation. In an effort to understand the underlying mechanism for an increase in H3-K9 methylation during repression by unliganded TR, we demonstrated that TR interacts in vitro with an H3-K9-specific histone methyltransferase (HMT), SUV39H1. Functional analysis indicates that SUV39H1 can facilitate repression by unliganded TR and in so doing requires its HMT activity. Together, our data uncover a novel role of H3-K9 methylation in repression by unliganded TR and provide strong evidence for the involvement of multiple distinct histone covalent modifications (acetylation, methylation, and phosphorylation) in transcriptional control by nuclear hormone receptors.

Animals↗

PubMed2002

Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases.

Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G(2) phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G(2)/M transition. During the G(2) phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system.

3T3 Cells↗

PubMed2002

Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression.

Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae. Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail. Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo. Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain. These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription.

Amino Acid Sequence↗