RESUMEN
In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells.
Asunto(s)
Células Madre Embrionarias de Ratones , Animales , Ratones , Silenciador del Gen , Células Madre Embrionarias de Ratones/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Peptidasa Específica de Ubiquitina 7/genéticaRESUMEN
Histone post-translational modifications (PTMs) are key players in chromatin regulation. The identification of novel histone acylations raises important questions regarding their role in transcription. In this study, we characterize the role of an acylation on the lateral surface of the histone octamer, H3K122 succinylation (H3K122succ), in chromatin function and transcription. Using chromatin succinylated at H3K122 in in vitro transcription assays, we show that the presence of H3K122succ is sufficient to stimulate transcription. In line with this, we found in our ChIP assays H3K122succ enriched on promoters of active genes and H3K122succ enrichment scaling with gene expression levels. Furthermore, we show that the co-activators p300/CBP can succinylate H3K122 and identify sirtuin 5 (SIRT5) as a new desuccinylase. By applying single molecule FRET assays, we demonstrate a direct effect of H3K122succ on nucleosome stability, indicating an important role for histone succinylation in modulating chromatin dynamics. Together, these data provide the first insights into the mechanisms underlying transcriptional regulation by H3K122succ.
Asunto(s)
Histonas , Nucleosomas , Cromatina/genética , Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Nucleosomas/genética , Procesamiento Proteico-PostraduccionalRESUMEN
The DNA of each cell is wrapped around histone octamers, forming so-called 'nucleosomal core particles'. These histone proteins have tails that project from the nucleosome and many residues in these tails can be post-translationally modified, influencing all DNA-based processes, including chromatin compaction, nucleosome dynamics, and transcription. In contrast to those present in histone tails, modifications in the core regions of the histones had remained largely uncharacterised until recently, when some of these modifications began to be analysed in detail. Overall, recent work has shown that histone core modifications can not only directly regulate transcription, but also influence processes such as DNA repair, replication, stemness, and changes in cell state. In this review, we focus on the most recent developments in our understanding of histone modifications, particularly those on the lateral surface of the nucleosome. This region is in direct contact with the DNA and is formed by the histone cores. We suggest that these lateral surface modifications represent a key insight into chromatin regulation in the cell. Therefore, lateral surface modifications form a key area of interest and a focal point of ongoing study in epigenetics.
Asunto(s)
Cromatina/metabolismo , Metilación de ADN , Expresión Génica , Histonas/química , Histonas/metabolismo , Nucleosomas/genética , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Cromatina/genética , Silenciador del Gen , Genoma , Histonas/genética , Humanos , Dominios ProteicosRESUMEN
The linker histone H1 plays an essential role in maintaining and establishing higher-order chromatin structure. As with core histones, histone H1 is also extensively covalently modified. We showed previously that phosphorylation of S27 in human histone H1.4 (H1.4S27-P), prevents binding of heterochromatin protein 1 (HP1) family members (officially known as chromobox protein homologs) to the neighboring dimethylated K26. Here, we present the first functional characterization of H1.4S27-P in vivo and in vitro. We show that H1.4S27 phosphorylation is cell-cycle-regulated and its levels peak on metaphase chromosomes. We identify further Aurora B as the kinase phosphorylating H1.4S27. We demonstrate that histone H1.4 is the only somatic linker histone variant targeted by Aurora B and that Aurora B exclusively phosphorylates S27. Adjacent K26 dimethylation can regulate Aurora B activity towards S27, uncovering a crosstalk between these modifications. Finally, our fluorescence recovery after photobleaching (FRAP) analysis on histone H1.4 mutants suggests a role of S27 phosphorylation in the regulation of histone H1.4 mobility and chromatin binding in mitosis.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Aurora Quinasa B , Aurora Quinasas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Células HeLa , Heterocromatina/metabolismo , Histonas/química , Histonas/genética , Humanos , Metilación , Ratones , Mitosis/fisiología , Células 3T3 NIH , Fosforilación , Isoformas de Proteínas , Especificidad por SustratoRESUMEN
The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A C-terminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Secuencias de Aminoácidos , Línea Celular , Cromatina/genética , Histonas/genética , Humanos , Nucleosomas/genética , Nucleosomas/metabolismo , Unión ProteicaRESUMEN
The development of specific anti-modification antibodies as research tools has revolutionized the way histone methylation is studied. Lack of stringent quality controls, however, led to the development of nonspecific antibodies, compromising their use. In this chapter, we provide a series of protocols that collectively will help those studying histone methylation to develop and thoroughly validate high-end sequence-specific and methylation-dependent antibodies.
Asunto(s)
Histonas , Procesamiento Proteico-Postraduccional , Anticuerpos/metabolismo , Histonas/metabolismo , MetilaciónRESUMEN
Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme.
Asunto(s)
Proliferación Celular/fisiología , Citrulinación/fisiología , Glucólisis/fisiología , Neoplasias/metabolismo , Arginina Deiminasa Proteína-Tipo 1/metabolismo , Arginina Deiminasa Proteína-Tipo 3/metabolismo , Piruvato Quinasa/metabolismo , Regulación Alostérica , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Melanoma , Proteínas de la Membrana , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Neoplasias/genética , Arginina Deiminasa Proteína-Tipo 1/genética , Arginina Deiminasa Proteína-Tipo 3/genética , Hormonas Tiroideas , Regulación hacia Arriba , Proteínas de Unión a Hormona TiroideRESUMEN
In mammalian cells, the level of estrogen receptor alpha (ERalpha) is rapidly decreased upon estrogen treatment, and this regulation involves proteasome degradation. Using different approaches, we showed that the Mdm2 oncogenic ubiquitin-ligase directly interacts with ERalpha in a ternary complex with p53 and is involved in the regulation of ERalpha turnover (both in the absence or presence of estrogens). Several lines of evidence indicated that this effect of Mdm2 required its ubiquitin-ligase activity and involved the ubiquitin/proteasome pathway. Moreover, in MCF-7 human breast cancer cells, various p53-inducing agents (such as UV irradiation) or treatment with RITA (which inhibits the interaction of p53 with Mdm2) stabilized ERalpha and abolished its 17beta-estradiol-dependent turnover. Interestingly, our data indicated that ligand-dependent receptor turnover was not required for efficient transactivation. Altogether, our results indicate that the Mdm2 oncoprotein and stress-inducing agents complexly and differentially regulate ERalpha stability and transcriptional activity in human cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/farmacología , Estradiol/farmacología , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Células HeLa , Humanos , Inmunoprecipitación , Leupeptinas/farmacología , Proteínas de Neoplasias/farmacología , Proteínas Proto-Oncogénicas c-mdm2/genética , Transducción de Señal , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Rayos UltravioletaRESUMEN
In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best practices for figure preparation since itinadvertently included duplications from the Ponceau staining presented in Supplementary Fig. 1b (for which the same preparation ofnucleosomes from HeLa cells had been used). A new Fig. 1b is provided in the Author Correction.
RESUMEN
BACKGROUND: Dynamic changes in the modification pattern of histones, such as acetylation, phosphorylation, methylation, and ubiquitination, are thought to provide a code for the correct regulation of gene expression mostly by affecting chromatin structure and interactions of non-histone regulatory factors with chromatin. Recent studies have suggested the existence of an interplay between histone modifications during transcription. The CBP/p300 acetylase and the CARM1 methyltransferase can positively regulate the expression of estrogen-responsive genes, but the existence of a crosstalk between lysine acetylation and arginine methylation on chromatin has not yet been established in vivo. RESULTS: By following the in vivo pattern of modifications on histone H3, following estrogen stimulation of the pS2 promoter, we show that arginine methylation follows prior acetylation of H3. Within 15 min after estrogen stimulation, CBP is bound to chromatin, and acetylation of K18 takes place. Following these events, K23 is acetylated, CARM1 associates with chromatin, and methylation at R17 takes place. Exogenous expression of CBP is sufficient to drive the association of CARM1 with chromatin and methylation of R17 in vivo, whereas an acetylase-deficient CBP mutant is unable to induce these events. A mechanism for the observed cooperation between acetylation and arginine methylation comes from the finding that acetylation at K18 and K23, but not K14, tethers recombinant CARM1 to the H3 tail and allows it to act as a more efficient arginine methyltransferase. CONCLUSION: These results reveal an ordered and interdependent deposition of acetylation and arginine methylation during estrogen-regulated transcription and provide support for a combinatorial role of histone modifications in gene expression.
Asunto(s)
Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Transactivadores/metabolismo , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Arginina/metabolismo , Proteína de Unión a CREB , Células Cultivadas , Cromatina/metabolismo , Estrógenos/farmacología , Humanos , Lisina/metabolismo , Metilación , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transactivadores/genética , Transcripción Genética/efectos de los fármacosRESUMEN
The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin. Both events are under the control of Heat Shock Factor I (HSF1). Here we show that under heat shock, HSF1 recruits major cellular acetyltransferases, GCN5, TIP60 and p300 to pericentric heterochromatin leading to a targeted hyperacetylation of pericentric chromatin. Redistribution of histone acetylation toward pericentric region in turn directs the recruitment of Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, BRD4, which are required for satellite III transcription by RNAP II. Altogether we uncover here a critical role for HSF1 in stressed cells relying on the restricted use of histone acetylation signaling over pericentric heterochromatin (HC).
Asunto(s)
Respuesta al Choque Térmico , Heterocromatina/genética , Transducción de Señal/genética , Activación Transcripcional , Animales , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Células HeLa , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Heterocromatina/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Histones are highly covalently modified, but the functions of many of these modifications remain unknown. In particular, it is unclear how histone marks are coupled to cellular metabolism and how this coupling affects chromatin architecture. We identified histone H3 Lys14 (H3K14) as a site of propionylation and butyrylation in vivo and carried out the first systematic characterization of histone propionylation. We found that H3K14pr and H3K14bu are deposited by histone acetyltransferases, are preferentially enriched at promoters of active genes and are recognized by acylation-state-specific reader proteins. In agreement with these findings, propionyl-CoA was able to stimulate transcription in an in vitro transcription system. Notably, genome-wide H3 acylation profiles were redefined following changes to the metabolic state, and deletion of the metabolic enzyme propionyl-CoA carboxylase altered global histone propionylation levels. We propose that histone propionylation, acetylation and butyrylation may act in combination to promote high transcriptional output and to couple cellular metabolism with chromatin structure and function.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Transcripción Genética/genética , Acetilación , Acilcoenzima A/genética , Acilcoenzima A/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Células HeLa , Histona Acetiltransferasas/metabolismo , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Dominios Proteicos , Células RAW 264.7 , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of 3 key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all 3 acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Nucleosomas/metabolismo , Acetilación , Animales , Ensamble y Desensamble de Cromatina , Implantación del Embrión , Epigénesis Genética , Femenino , Código de Histonas , Histonas/genética , Ratones , Nucleosomas/genética , Procesamiento Proteico-PostraduccionalRESUMEN
N-terminal tails of histones are easily accessible outside of the nucleosomal core particle and post-translational modifications (PTMs) of these tails have been the focus of attention in the past 15-20 years. By recruiting (or excluding) specific readers, histone modifications can regulate chromatin dynamics and, by extension, DNA-dependent processes. However, until very recently, the direct impact of histone PTMs on nucleosome structure and thus on chromatin function has remained somewhat elusive. Recent findings of novel sites and types of histone PTMs located within the globular domain of histones and, in particular, on the lateral surface of the histone octamer have changed this. As a result of their structurally important location in close proximity to the DNA molecule, this new class of histone PTMs can have a direct impact on chromatin function. Depending on their precise position at the nucleosome lateral surface (e.g. near the DNA entry/exit sites or in the dyad region), histone PTMs can regulate nucleosome structure and/or stability differently. We review recent progress on how histone PTMs can influence DNA unwrapping and/or nucleosome disassembly and shed light on how these types of novel modifications contribute mechanistically to the regulation of transcriptional activity.
Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Transcripción Genética , ADN/genética , ADN/metabolismo , Histonas/química , Histonas/genética , Modelos MolecularesRESUMEN
Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001.
Asunto(s)
Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Transcripción Genética , Activación Transcripcional , Acetilación , Animales , Células Madre Embrionarias/metabolismo , Histonas/química , Humanos , Cinética , Lisina , Masculino , Metilación , Ratones , Células 3T3 NIH , Células-Madre Neurales/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Estabilidad Proteica , Transfección , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis , Factores de Transcripción p300-CBP/metabolismoRESUMEN
To ensure genome stability, pericentromeric regions are compacted in a dense heterochromatic structure through a combination of specific 'epigenetic' factors and modifications. A cascadal pathway is responsible for establishing pericentromeric chromatin involving chromatin modifiers and 'readers', such as H3K9 histone methyltransferases (Suv)39h and heterochromatin protein 1. Here we define how H3K64me3 on the lateral surface of the histone octamer integrates within the heterochromatinization cascade. Our data suggest that enrichment of H3K64me3 at pericentromeric chromatin foci is dependent on H3K9me3 but independent of a number of central factors such as heterochromatin protein 1, DNA methyltransferases and Suv4-20h histone methyltransferases. Our results support a model in which pericentromeric heterochromatin foci are formed along distinct pathways upon H3K9 trimethylation, involving H3K64me3 to potentially stabilize DNA-histone interactions, as well as sequential recruitment of repressive histone tail and DNA modifications. We hence suggest that multiple mechanisms ensure heterochromatin integrity at pericentromeres, with H3K64me3 as an important factor.
Asunto(s)
Centrómero/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animales , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN/genética , Técnica del Anticuerpo Fluorescente , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones , Células 3T3 NIH , Cigoto/metabolismoRESUMEN
BACKGROUND: The linker histone H1 has a key role in establishing and maintaining higher order chromatin structure and in regulating gene expression. Mammals express up to 11 different H1 variants, with H1.2 and H1.4 being the predominant ones in most somatic cells. Like core histones, H1 has high levels of covalent modifications; however, the full set of modifications and their biological role are largely unknown. RESULTS: In this study, we used a candidate screen to identify enzymes that methylate H1 and to map their corresponding methylation sites. We found that the histone lysine methyltransferases G9a/KMT1C and Glp1/KMT1D methylate H1.2 in vitro and in vivo, and we mapped this novel site to lysine 187 (H1.2K187) in the C-terminus of H1. This H1.2K187 methylation is variant-specific. The main target for methylation by G9a in H1.2, H1.3, H1.5 and H1.0 is in the C-terminus, whereas H1.4 is preferentially methylated at K26 (H1.4K26me) in the N-terminus. We found that the readout of these marks is different; H1.4K26me can recruit HP1, but H1.2K187me cannot. Likewise, JMJD2D/KDM4 only reverses H1.4K26 methylation, clearly distinguishing these two methylation sites. Further, in contrast to C-terminal H1 phosphorylation, H1.2K187 methylation level is steady throughout the cell cycle. CONCLUSIONS: We have characterised a novel methylation site in the C-terminus of H1 that is the target of G9a/Glp1 both in vitro and in vivo. To our knowledge, this is the first demonstration of variant-specific histone methylation by the same methyltransferases, but with differing downstream readers, thereby supporting the hypothesis of H1 variants having specific functions.
RESUMEN
Histone modifications are central to the regulation of all DNA-dependent processes. Lys64 of histone H3 (H3K64) lies within the globular domain at a structurally important position. We identify trimethylation of H3K64 (H3K64me3) as a modification that is enriched at pericentric heterochromatin and associated with repeat sequences and transcriptionally inactive genomic regions. We show that this new mark is dynamic during the two main epigenetic reprogramming events in mammals. In primordial germ cells, H3K64me3 is present at the time of specification, but it disappears transiently during reprogramming. In early mouse embryos, it is inherited exclusively maternally; subsequently, the modification is rapidly removed, suggesting an important role for H3K64me3 turnover in development. Taken together, our findings establish H3K64me3 as a previously uncharacterized histone modification that is preferentially localized to repressive chromatin. We hypothesize that H3K64me3 helps to 'secure' nucleosomes, and perhaps the surrounding chromatin, in an appropriately repressed state during development.
Asunto(s)
Epigénesis Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animales , Línea Celular , ADN/química , ADN/genética , ADN/metabolismo , Metilación de ADN , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/metabolismo , Heterocromatina/química , Heterocromatina/genética , Histonas/genética , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nucleosomas/química , Nucleosomas/metabolismo , Conformación Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Xenopus laevisRESUMEN
The Trithorax group (TrxG) is composed of diverse, evolutionary conserved proteins that form chromatin-associated complexes accounting for epigenetic transcriptional memory. However, the molecular mechanisms by which particular loci are marked for reactivation after mitosis are only partially understood. Here, based on genetic analyses in zebrafish, we identify the multidomain protein Brpf1 as a novel TrxG member with a central role during development. brpf1 mutants display anterior transformations of pharyngeal arches due to progressive loss of anterior Hox gene expression. Brpf1 functions in association with the histone acetyltransferase Moz (Myst3), an interaction mediated by the N-terminal domain of Brpf1, and promotes histone acetylation in vivo. Brpf1 recruits Moz to distinct sites of active chromatin and remains at chromosomes during mitosis, mediated by direct histone binding of its bromodomain, which has a preference for acetylated histones, and its PWWP domain, which binds histones independently of their acetylation status. This is the first demonstration of histone binding for PWWP domains. Mutant analyses further show that the PWWP domain is absolutely essential for Brpf1 function in vivo. We conclude that Brpf1, coordinated by its particular set of domains, acts by multiple mechanisms to mediate Moz-dependent histone acetylation and to mark Hox genes for maintained expression throughout vertebrate development.
Asunto(s)
Proteínas Portadoras/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Acetilación , Proteínas Adaptadoras Transductoras de Señales , Animales , Sitios de Unión/genética , Región Branquial/anatomía & histología , Región Branquial/crecimiento & desarrollo , Región Branquial/metabolismo , Proteínas Portadoras/genética , Línea Celular , Cromatina/metabolismo , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Inmunoprecipitación , Hibridación in Situ , Ratones , Proteínas Nucleares/genética , Unión Proteica , Proteínas Recombinantes/metabolismo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
Histone lysine methylation can have positive or negative effects on transcription, depending on the precise methylation site. According to the "histone code" hypothesis these methylation marks can be read by proteins that bind them specifically and then regulate downstream events. Hetero-chromatin protein 1 (HP1), an essential component of heterochromatin, binds specifically to methylated Lys(9) of histone H3 (K9/H3). The linker histone H1.4 is methylated on Lys(26) (K26/H1.4), but the role of this methylation in downstream events remains unknown. Here we identify HP1 as a protein specifically recognizing and binding to methylated K26/H1.4. We demonstrate that the Chromo domain of HP1 is mediating this binding and that phosphorylation of Ser(27) on H1.4 (S27/H1.4) prevents HP1 from binding. We suggest that methylation of K26/H1.4 could have a role in tethering HP1 to chromatin and that this could also explain how HP1 is targeted to those regions of chromatin where it does not colocalize with methylated K9/H3. Our results provide the first experimental evidence for a "phospho switch" model in which neighboring phosphorylation reverts the effect of histone lysine methylation.