RESUMEN
Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening, and therefore, HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of Hdac1 and Hdac2 in embryonic stem cells (ESCs) reduces expression of the pluripotency-associated transcription factors Pou5f1, Sox2, and Nanog (PSN). By shaping global histone acetylation patterns, HDACs indirectly regulate the activity of acetyl-lysine readers, such as the transcriptional activator BRD4. Here, we use inhibitors of HDACs and BRD4 (LBH589 and JQ1, respectively) in combination with precision nuclear run-on and sequencing (PRO-seq) to examine their roles in defining the ESC transcriptome. Both LBH589 and JQ1 cause a marked reduction in the pluripotent gene network. However, although JQ1 treatment induces widespread transcriptional pausing, HDAC inhibition causes a reduction in both paused and elongating polymerase, suggesting an overall reduction in polymerase recruitment. Using enhancer RNA (eRNA) expression to measure enhancer activity, we find that LBH589-sensitive eRNAs are preferentially associated with superenhancers and PSN binding sites. These findings suggest that HDAC activity is required to maintain pluripotency by regulating the PSN enhancer network via the recruitment of RNA polymerase II.
Asunto(s)
Histonas , Factores de Transcripción , Histonas/metabolismo , Factores de Transcripción/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas Nucleares/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Redes Reguladoras de Genes , Panobinostat , Histona Acetiltransferasas/genética , Acetilación , Inhibidores de Histona DesacetilasasRESUMEN
In this Letter, the western blot for LSD1 in the right panel of Fig. 2b ('TCP +') was inadvertently duplicated from the tubulin blot immediately below. The actual tubulin western blot shows the same result, with no significant change to the levels of tubulin (see Fig. 1 of this Amendment). In addition, the western blots for LSD1 and HDAC1 of Fig. 3b and c have been corrected to include vertical black lines to delineate the juxtaposition of lanes that were non-adjacent in the original blotting experiment (see Fig. 2 of this Amendment). Supplementary Figs. 4a, 6b and 9b have also been corrected to delineate non-adjacent lanes with vertical black lines (see Supplementary Information of this Amendment). The complete raw data images from these western blotting experiments can also be found in the Supplementary Information of this Amendment. The original Letter has not been corrected.
RESUMEN
Cell type specification relies on the capacity of undifferentiated cells to properly respond to specific differentiation-inducing signals. Using genomic approaches along with loss- and gain-of-function genetic models, we identified OCT4-dependent mechanisms that provide embryonic stem cells with the means to customize their response to external cues. OCT4 binds a large set of low-accessible genomic regions. At these sites, OCT4 is required for proper enhancer and gene activation by recruiting co-regulators and RAR:RXR or ß-catenin, suggesting an unexpected collaboration between the lineage-determining transcription factor and these differentiation-initiating, signal-dependent transcription factors. As a proof of concept, we demonstrate that overexpression of OCT4 in a kidney cell line is sufficient for signal-dependent activation of otherwise unresponsive genes in these cells. Our results uncover OCT4 as an integral and necessary component of signal-regulated transcriptional processes required for tissue-specific responses.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Embrionarias/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Vía de Señalización Wnt , Animales , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Reprogramación Celular , Células Madre Embrionarias/efectos de los fármacos , Regulación de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/efectos de los fármacos , Regiones Promotoras Genéticas , Interferencia de ARN , Receptor alfa de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico/metabolismo , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Transcripción Genética , Transfección , Tretinoina/farmacología , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
The class I histone deacetylase (HDAC) enzymes;HDAC1,2 and 3 form the catalytic engine of at least seven structurally distinct multiprotein complexes in cells. These molecular machines play a vital role in the regulation of chromatin accessibility and gene activity via the removal of acetyl moieties from lysine residues within histone tails. Their inhibition via small molecule inhibitors has beneficial effects in a number of disease types, including the clinical treatment of hematological cancers. We have previously reported a library of proteolysis targeting chimeras (PROTACs) incorporating a benzamide-based HDAC ligand (from CI-994), with an alkyl linker and ligand for the von Hippel-Lindau (VHL) E3 ubiquitin ligase that degrade HDAC1-3 at submicromolar concentrations. Here we report the addition of two novel PROTACs (JPS026 and JPS027), which utilize a ligand for the cellular inhibitor of apoptosis (IAP) family of E3 ligases. We found that both VHL (JPS004)- and IAP (JPS026)-based PROTACs degrade HDAC1-3 and induce histone acetylation to a similar degree. However, JPS026 is significantly more potent at inducing cell death in HCT116 cells than is JPS004. RNA sequencing analysis of PROTAC-treated HCT116 cells showed a distinct gene expression signature in which cell cycle and DNA replication machinery are repressed. Components of the mTORC1 and -2 complexes were also reduced, leading to an increase in FOXO3 and downstream target genes that regulate autophagy and apoptosis. In summary, a novel combination of HDAC and IAP ligands generates a PROTAC with a potent ability to stimulate apoptosis and differential gene expression in human cancer cells.
Asunto(s)
Quimera Dirigida a la Proteólisis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Humanos , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Proteolisis , Histonas/metabolismo , Ligandos , Transcriptoma , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Class I histone deacetylases (HDAC1, HDAC2, and HDAC3) are recruited by cognate corepressor proteins into specific transcriptional repression complexes that target HDAC activity to chromatin resulting in chromatin condensation and transcriptional silencing. We previously reported the structure of HDAC3 in complex with the SMRT corepressor. This structure revealed the presence of inositol-tetraphosphate [Ins(1,4,5,6)P4] at the interface of the two proteins. It was previously unclear whether the role of Ins(1,4,5,6)P4 is to act as a structural cofactor or a regulator of HDAC3 activity. Here we report the structure of HDAC1 in complex with MTA1 from the NuRD complex. The ELM2-SANT domains from MTA1 wrap completely around HDAC1 occupying both sides of the active site such that the adjacent BAH domain is ideally positioned to recruit nucleosomes to the active site of the enzyme. Functional assays of both the HDAC1 and HDAC3 complexes reveal that Ins(1,4,5,6)P4 is a bona fide conserved regulator of class I HDAC complexes.
Asunto(s)
Histona Desacetilasa 1/química , Histona Desacetilasas/química , Fosfatos de Inositol/fisiología , Proteínas Represoras/química , Secuencia de Aminoácidos , Dimerización , Células HEK293 , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 1/fisiología , Histona Desacetilasas/metabolismo , Histona Desacetilasas/fisiología , Humanos , Fosfatos de Inositol/química , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Especificidad por Sustrato , TransactivadoresRESUMEN
Histone deacetylase (HDAC) enzymes play crucial roles in epigenetic gene expression and are an attractive therapeutic target. Five HDAC inhibitors have been approved for cancer treatment to date, however, clinical applications have been limited due to poor single-agent drug efficacy and side effects associated with a lack of HDAC isoform or complex selectivity. An emerging strategy aiming to address these limitations is the development of bifunctional HDAC therapeutics-single molecules comprising a HDAC inhibitor conjugated to another specificity targeting moiety. This review summarises the recent advancements in novel types of dual-targeting HDAC modulators, including proteolysis-targeting chimeras (PROTACs), with a focus on HDAC isoform and complex selectivity, and the future potential of such bifunctional molecules in achieving enhanced drug efficacy and therapeutic benefits in treating disease.
Asunto(s)
Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/metabolismo , Terapia Molecular Dirigida , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismoRESUMEN
At face value, the Sin3 histone deacetylase (HDAC) complex appears to be a prototypical co-repressor complex, that is, a multi-protein complex recruited to chromatin by DNA bound repressor proteins to facilitate local histone deacetylation and transcriptional repression. While this is almost certainly part of its role, Sin3 stubbornly refuses to be pigeon-holed in quite this way. Genome-wide mapping studies have found that Sin3 localises predominantly to the promoters of actively transcribed genes. While Sin3 knockout studies in various species result in a combination of both up- and down-regulated genes. Furthermore, genes such as the stem cell factor, Nanog, are dependent on the direct association of Sin3 for active transcription to occur. Sin3 appears to have properties of a co-repressor, co-activator and general transcription factor, and has thus been termed a co-regulator complex. Through a series of unique domains, Sin3 is able to assemble HDAC1/2, chromatin adaptors and transcription factors in a series of functionally and compositionally distinct complexes to modify chromatin at both gene-specific and global levels. Unsurprisingly, therefore, Sin3/HDAC1 have been implicated in the regulation of numerous cellular processes, including mammalian development, maintenance of pluripotency, cell cycle regulation and diseases such as cancer.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Animales , Proteínas Co-Represoras/química , Proteínas Co-Represoras/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Organogénesis/fisiología , Estructura Secundaria de Proteína , Complejo Correpresor Histona Desacetilasa y Sin3/química , Complejo Correpresor Histona Desacetilasa y Sin3/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced, but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5), which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9), is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However, LSD1 is not essential for the maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to differentiate fully, and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers, LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation, which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Silenciador del Gen , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Fibroblastos , Histona Demetilasas , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Regiones Promotoras Genéticas/genéticaRESUMEN
Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic components of corepressor complexes that modulate gene expression. In most cell types, deletion of both Hdac1 and Hdac2 is required to generate a discernible phenotype, suggesting their activity is largely redundant. We have therefore generated an ES cell line in which Hdac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon cell cycle progression, because differentiated, nonproliferating cells retain their viability. Furthermore, we observe increased mitotic defects, chromatin bridges, and micronuclei, suggesting HDAC1/2 are necessary for accurate chromosome segregation. Consistent with a critical role in the regulation of gene expression, microarray analysis of Hdac1/2-deleted cells reveals 1,708 differentially expressed genes. Significantly for the maintenance of stem cell self-renewal, we detected a reduction in the expression of the pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is regulated through binding of an inositol tetraphosphate molecule (IP4) sandwiched between the HDAC and its cognate corepressor. This raises the important question of whether IP4 regulates the activity of the complex in cells. By rescuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. Our data indicate that HDAC1/2 have essential and pleiotropic roles in cellular proliferation and regulate stem cell self-renewal by maintaining expression of key pluripotent transcription factors.
Asunto(s)
División Celular/fisiología , Células Madre Embrionarias/enzimología , Histona Desacetilasa 1/fisiología , Histona Desacetilasa 2/fisiología , Células Madre Pluripotentes/enzimología , Acetilación , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Ratones , Ratones Noqueados , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Histone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T-cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 misregulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T-cell receptor (TCR) signaling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signaling, thus blocking development. Furthermore, mice with reduced HDAC1/2 activity (Hdac1 deleted and a single Hdac2 allele) develop a lethal pathology by 3-months of age, caused by neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T-cell development and the maintenance of genomic stability.
Asunto(s)
Transformación Celular Neoplásica/genética , Inestabilidad Genómica/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Linfocitos T/enzimología , Animales , Animales Recién Nacidos , Transformación Celular Neoplásica/inmunología , Cromatina/genética , Aberraciones Cromosómicas , Daño del ADN/genética , Daño del ADN/inmunología , Activación Enzimática/genética , Activación Enzimática/inmunología , Femenino , Inestabilidad Genómica/inmunología , Haploinsuficiencia/genética , Haploinsuficiencia/inmunología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Masculino , Ratones , Ratones Noqueados , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/citología , Timo/citología , Transcriptoma/inmunologíaRESUMEN
Histone deacetylases 1-3 (HDAC1, HDAC2, and HDAC3) and their associated corepressor complexes play important roles in regulating chromatin structure and gene transcription. HDAC enzymes are also validated drug targets for oncology and offer promise toward new drugs for neurodegenerative diseases and cardiovascular diseases. We synthesized four novel heterobifunctional molecules designed to recruit the mouse double minute 2 homologue (MDM2) E3 ligase to degrade HDAC1-3 utilizing the MDM2 inhibitor idasanutlin, known as proteolysis targeting chimeras (PROTACs). Idasanutlin inhibits the MDM2-P53 protein-protein interaction and is in clinical trials. Although two MDM2-recruiting heterobifunctional molecules reduced HDAC1 and HDAC2 abundance with complete selectivity over HDAC3 and reduced HDAC1/2 corepressor components LSD1 and SIN3A, we were surprised to observe that idasanutlin alone was also capable of this effect. This finding suggests an association between the MDM2 E3 ligase and HDAC1/2 corepressor complexes, which could be important for designing future dual/bifunctional HDAC- and MDM2-targeting therapeutics, such as PROTACs.
RESUMEN
HDACs (histone deacetylases) 1 and 2 are ubiquitous long-lived proteins, which are often found together in three major multiprotein co-repressor complexes: Sin3, NuRD (nucleosome remodelling and deacetylation) and CoREST (co-repressor for element-1-silencing transcription factor). Although there is a burgeoning number of non-histone proteins within the acetylome, these complexes contain multiple DNA/chromatin-recognition motifs, which, in combination with transcription factors, target HDAC1/2 to chromatin. Their physiological roles should therefore be viewed within the framework of chromatin manipulation. Classically, HDACs were thought to be recruited predominantly by transcriptional repressors to facilitate local histone deacetylation and transcriptional repression. More recently, genome-wide assays have mapped HDAC1/2 and their associated proteins to transcriptionally active loci and have provided alternative context-specific functions, whereby their repressive functions are subtly exerted to balance transcriptional activation and repression. With a few significant exceptions (early embryogenesis, brain development), HDAC1 and HDAC2 are functionally redundant. In most mouse knockout studies, deletion of both enzymes is required in order to produce a substantial phenotype. HDAC1/2 activity has been implicated in the development of numerous tissue and cell types, including heart, skin, brain, B-cells and T-cells. A common feature in all HDAC1/2-knockout, -knockdown and small-molecule inhibitor studies is a reduction in cell proliferation. A generic role in cell cycle progression could be exploited in cancer cells, by blocking HDAC1/2 activity with small-molecule inhibitors, making them potentially useful drug targets.
Asunto(s)
Histona Desacetilasa 1/fisiología , Histona Desacetilasa 2/fisiología , Acetilación , Animales , Proteínas Co-Represoras/metabolismo , Proteínas Co-Represoras/fisiología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Histonas/metabolismo , Histonas/fisiología , Humanos , Ratones , Ratones Noqueados , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
Histone deacetylases (HDAC) 1 and 2 are highly similar enzymes that help regulate chromatin structure as the core catalytic components of corepressor complexes. Although tissue-specific deletion of HDAC1 and HDAC2 has demonstrated functional redundancy, germ-line deletion of HDAC1 in the mouse causes early embryonic lethality, whereas HDAC2 does not. To address the unique requirement for HDAC1 in early embryogenesis we have generated conditional knockout embryonic stem (ES) cells in which HDAC1 or HDAC2 genes can be inactivated. Deletion of HDAC1, but not HDAC2, causes a significant reduction in the HDAC activity of Sin3A, NuRD, and CoREST corepressor complexes. This reduced corepressor activity results in a specific 1.6-fold increase in histone H3 K56 acetylation (H3K56Ac), thus providing genetic evidence that H3K56Ac is a substrate of HDAC1. In culture, ES cell proliferation was unaffected by loss of either HDAC1 or HDAC2. Rather, we find that loss of HDAC1 affects ES cell differentiation. ES cells lacking either HDAC1 or HDAC2 were capable of forming embryoid bodies (EBs), which stimulates differentiation into the three primary germ layers. However, HDAC1-deficient EBs were significantly smaller, showed spontaneous rhythmic contraction, and increased expression of both cardiomyocyte and neuronal markers. In summary, our genetic study of HDAC1 and HDAC2 in ES cells, which mimic the embryonic epiblast, has identified a unique requirement for HDAC1 in the optimal activity of HDAC1/2 corepressor complexes and cell fate determination during differentiation.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Animales , Biomarcadores , Línea Celular , Proliferación Celular , Regulación Enzimológica de la Expresión Génica , Histona Desacetilasa 1/deficiencia , Histona Desacetilasa 2/deficiencia , Ratones , Unión ProteicaRESUMEN
Lysine specific demethylase 1 (LSD1) regulates gene expression as part of the CoREST complex, along with co-repressor of REST (CoREST) and histone deacetylase 1 (HDAC1). CoREST is recruited to specific genomic loci by core components and numerous transient interactions with chromatin-associated factors and transcription factors. We hypothesise that many of these weaker and transient associations may be difficult to identify using traditional co-immunoprecipitation methods. We have therefore employed proximity-dependent biotin-identification (BioID) with four different members of the CoREST complex, in three different cell types, to identify a comprehensive network of LSD1/CoREST associated proteins. In HEK293T cells, we identified 302 CoREST-associated proteins. Among this group were 16 of 18 known CoREST components and numerous novel associations, including readers (CHD3, 4, 6, 7 and 8), writers (KMT2B and KMT2D) and erasers (KDM2B) of histone methylation. However, components of other HDAC1 containing complexes (e.g. Sin3) were largely absent. To examine the dynamic nature of the CoREST interactome in a primary cell type, we replaced endogenous LSD1 with BirA*-LSD1 in embryonic stem (ES) cells and performed BioID in pluripotent, early- and late-differentiating environments. We identified 156 LSD1-associated proteins of which 67 were constitutively associated across all three time-points (43%), including novel associations with the MMB and ChAHP complexes, implying that the majority of interactors are both dynamic and cell type dependent. In total, we have performed 16 independent BioID experiments for LSD1 in three different cell types, producing a definitive network of LSD1-assoicated proteins that should provide a major resource for the field.
Asunto(s)
Biotina , Histona Demetilasas , Diferenciación Celular , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Células HEK293 , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Proteínas del Tejido Nervioso/genéticaRESUMEN
Click chemistry was utilised to prepare a library of PROTACs based on entinostat a class I histone deacetylase (HDAC) inhibitor in clinical trials. A novel PROTAC JMC-137 was identified as a HDAC1/2 and HDAC3 degrader in HCT116 cells. However, potency was compromised compared to previously identified class I HDAC PROTACs highlighting the importance in the choice of HDAC ligand, functional group for linker attachment and positioning in PROTAC design.
RESUMEN
Class I histone deacetylase (HDAC) enzymes 1, 2, and 3 organize chromatin as the catalytic subunits within seven distinct multiprotein corepressor complexes and are established drug targets. We report optimization studies of benzamide-based Von Hippel-Lindau (VHL) E3-ligase proteolysis targeting chimeras (PROTACs) and for the first time describe transcriptome perturbations resulting from these degraders. By modifying the linker and VHL ligand, we identified PROTACs 7, 9, and 22 with submicromolar DC50 values for HDAC1 and/or HDAC3 in HCT116 cells. A hook effect was observed for HDAC3 that could be negated by modifying the position of attachment of the VHL ligand to the linker. The more potent HDAC1/2 degraders correlated with greater total differentially expressed genes and enhanced apoptosis in HCT116 cells. We demonstrate that HDAC1/2 degradation by PROTACs correlates with enhanced global gene expression and apoptosis, important for the development of more efficacious HDAC therapeutics with reduced side effects.
Asunto(s)
Histona Desacetilasas , Neoplasias , Apoptosis , Quimera/metabolismo , Histona Desacetilasa 1/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ligandos , Neoplasias/tratamiento farmacológico , Proteolisis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.
Asunto(s)
Desarrollo Embrionario , Histona Desacetilasas/metabolismo , Complejos Multiproteicos/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatina/metabolismo , Cromosomas de los Mamíferos/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Redes Reguladoras de Genes , Heterocigoto , Homocigoto , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Mitosis , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Proteínas Nucleares/metabolismo , Dominios Proteicos , Multimerización de ProteínaRESUMEN
Recruitment of the histone deacetylase (HDAC)-associated Sin3 corepressor is an obligatory step in many eukaryotic gene silencing pathways. Here we show that HBP1, a cell cycle inhibitor and regulator of differentiation, represses transcription in a HDAC/Sin3-dependent manner by targeting the mammalian Sin3A (mSin3A) PAH2 domain. HBP1 is unrelated to the Mad1 repressor for which high-resolution structures in complex with PAH2 have been described. We show that like Mad1, the HBP1 transrepression domain binds through a helical structure to the hydrophobic cleft of mSin3A PAH2. Notably, the HBP1 helix binds PAH2 in a reversed orientation relative to Mad1 and, equally unexpectedly, this is correlated with a chain reversal of the minimal Sin3 interaction motifs. These results not only provide insights into how multiple, unrelated transcription factors recruit the same coregulator, but also have implications for how sequence similarity searches are conducted.
Asunto(s)
Proteínas del Grupo de Alta Movilidad/metabolismo , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Calorimetría , Proteínas de Ciclo Celular , Diferenciación Celular , Línea Celular , Glutatión Transferasa/metabolismo , Histona Desacetilasas , Humanos , Espectroscopía de Resonancia Magnética , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Estereoisomerismo , Transcripción Genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Packaging the long and fragile genomes of eukaryotic species into nucleosomes is all well and good, but how do cells gain access to the DNA again after it has been bundled away? The solution, in every species from yeast to man, is to post-translationally modify histones, altering their chemical properties to either relax the chromatin, label it for remodelling or make it more compact still. Histones are subject to a myriad of modifications: acetylation, methylation, phosphorylation, ubiquitination etc. This review focuses on histone acylations, a diverse group of modifications which occur on the ε-amino group of Lysine residues and includes the well-characterised Lysine acetylation. Over the last 50 years, histone acetylation has been extensively characterised, with the discovery of histone acetyltransferases (HATs) and histone deacetylases (HDACs), and global mapping experiments, revealing an association of hyperacetylated histones with accessible, transcriptionally active chromatin. More recently, there has been an explosion in the number of unique short chain 'acylations' identified by MS, including: propionylation, butyrylation, crotonylation, succinylation, malonylation and 2-hydroxyisobutyrylation. These novel modifications add a range of chemical environments to histones, and similar to acetylation, appear to accumulate at transcriptional start sites and correlate with gene activity.
Asunto(s)
Histonas/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Animales , ADN/genética , ADN/metabolismo , Regulación de la Expresión Génica , Histonas/química , Histonas/genética , Humanos , Lisina/química , Nucleosomas/química , Nucleosomas/genéticaRESUMEN
The corepressor mSin3A is the core component of a chromatin-modifying complex that is recruited by multiple gene-specific transcriptional repressors. In order to understand the role of mSin3A during development, we generated constitutive germ line as well as conditional msin3A deletions. msin3A deletion in the developing mouse embryo results in lethality at the postimplantation stage, demonstrating that it is an essential gene. Blastocysts derived from preimplantation msin3A null embryos and mouse embryo fibroblasts (MEFs) lacking msin3A display a significant reduction in cell division. msin3A null MEFs also show mislocalization of the heterochromatin protein, HP1alpha, without alterations in global histone acetylation. Heterozygous msin3A(+/-) mice with a systemic twofold decrease in mSin3A protein develop splenomegaly as well as kidney disease indicative of a disruption of lymphocyte homeostasis. Conditional deletion of msin3A from developing T cells results in reduced thymic cellularity and a fivefold decrease in the number of cytotoxic (CD8) T cells, while helper (CD4) T cells are unaffected. We show that CD8 development is dependent on mSin3A at a step downstream of T-cell receptor signaling and that loss of mSin3A specifically decreases survival of double-positive and CD8 T cells. Thus, msin3A is a pleiotropic gene which, in addition to its role in cell cycle progression, is required for the development and homeostasis of cells in the lymphoid lineage.