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1.
Nat Metab ; 6(4): 697-707, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38413806

RESUMEN

Post-translational modifications (PTMs) on histones are a key source of regulation on chromatin through impacting cellular processes, including gene expression1. These PTMs often arise from metabolites and are thus impacted by metabolism and environmental cues2-7. One class of metabolically regulated PTMs are histone acylations, which include histone acetylation, butyrylation, crotonylation and propionylation3,8. As these PTMs can be derived from short-chain fatty acids, which are generated by the commensal microbiota in the intestinal lumen9-11, we aimed to define how microbes impact the host intestinal chromatin landscape, mainly in female mice. Here we show that in addition to acetylation, intestinal epithelial cells from the caecum and distal mouse intestine also harbour high levels of butyrylation and propionylation on lysines 9 and 27 of histone H3. We demonstrate that these acylations are regulated by the microbiota and that histone butyrylation is additionally regulated by the metabolite tributyrin. Tributyrin-regulated gene programmes are correlated with histone butyrylation, which is associated with active gene-regulatory elements and levels of gene expression. Together, our study uncovers a regulatory layer of how the microbiota and metabolites influence the intestinal epithelium through chromatin, demonstrating a physiological setting in which histone acylations are dynamically regulated and associated with gene regulation.


Asunto(s)
Microbioma Gastrointestinal , Regulación de la Expresión Génica , Histonas , Procesamiento Proteico-Postraduccional , Animales , Histonas/metabolismo , Ratones , Femenino , Mucosa Intestinal/metabolismo , Acetilación , Intestinos/microbiología , Triglicéridos/metabolismo , Ratones Endogámicos C57BL
2.
Cell Rep ; 41(11): 111809, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36516747

RESUMEN

The gut microbiota influences acetylation on host histones by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-coenzyme A (CoA), a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivo with 13C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis.


Asunto(s)
Colitis , Microbiota , Ratones , Animales , Acetilación , Histonas/metabolismo , Histona Acetiltransferasas/metabolismo , Isótopos/metabolismo , Carbono/metabolismo , Butiratos , Ácidos Grasos
3.
Nat Chem ; 12(6): 520-527, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32472103

RESUMEN

Elucidating the physiological binding partners of histone post-translational modifications (hPTMs) is key to understanding fundamental epigenetic regulatory pathways. Determining such interactomes will enable the study of how perturbations of these interactions affect disease. Here we use a synthetic biology approach to set a series of hPTM-controlled photo-affinity traps in native chromatin. Using quantitative proteomics, the local interactomes of these chemically customized chromatin landscapes are determined. We show that the approach captures transiently interacting factors such as methyltransferases and demethylases, as well as previously reported and novel hPTM reader proteins. We also apply this in situ proteomics approach to a recently disclosed cancer-associated histone mutation, H3K4M, revealing a number of perturbed interactions with the mutated tail. Collectively our studies demonstrate that modifying and interrogating native chromatin with chemical precision is a powerful tool for exploring epigenetic regulation and dysregulation at the molecular level.


Asunto(s)
Cromatina/química , Epigénesis Genética , Histonas/química , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Cromatina/genética , Cromatina/metabolismo , Código de Histonas , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Metiltransferasas/metabolismo , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Etiquetas de Fotoafinidad
4.
Cell Metab ; 31(4): 852-861.e6, 2020 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-32268116

RESUMEN

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress. The mechanisms by which ATF4 couples metabolic stresses to specific transcriptional outputs remain unknown. Using functional genomics, we identified transcription factors that regulate the responses to distinct amino acid deprivation conditions. While ATF4 is universally required under amino acid starvation, our screens yielded a transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as uniquely essential under asparagine deprivation. ZBTB1 knockout cells are unable to synthesize asparagine due to reduced expression of asparagine synthetase (ASNS), the enzyme responsible for asparagine synthesis. Mechanistically, ZBTB1 binds to the ASNS promoter and promotes ASNS transcription. Finally, loss of ZBTB1 sensitizes therapy-resistant T cell leukemia cells to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Our work reveals a critical regulator of the nutrient stress response that may be of therapeutic value.


Asunto(s)
Asparagina/biosíntesis , Aspartatoamoníaco Ligasa/metabolismo , Leucemia , Proteínas Represoras/fisiología , Animales , Asparagina/deficiencia , Línea Celular Tumoral , Proliferación Celular , Regulación de la Expresión Génica , Humanos , Leucemia/metabolismo , Leucemia/patología , Ratones Endogámicos NOD , Ratones SCID , Transcripción Genética
5.
Oncogene ; 37(33): 4581-4598, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29748621

RESUMEN

Approximately 75% of breast cancers are estrogen receptor alpha (ERα)-positive and are treatable with endocrine therapies, but often patients develop lethal resistant disease. Frequent mutations (10-40%) in the ligand-binding domain (LBD) codons in the gene encoding ERα (ESR1) have been identified, resulting in ligand-independent, constitutively active receptors. In addition, ESR1 chromosomal translocations can occur, resulting in fusion proteins that lack the LBD and are entirely unresponsive to all endocrine treatments. Thus, identifying coactivators that bind to these mutant ERα proteins may offer new therapeutic targets for endocrine-resistant cancer. To define coactivator candidate targets, a proteomics approach was performed profiling proteins recruited to the two most common ERα LBD mutants, Y537S and D538G, and an ESR1-YAP1 fusion protein. These mutants displayed enhanced coactivator interactions as compared to unliganded wild-type ERα. Inhibition of these coactivators decreased the ability of ESR1 mutants to activate transcription and promote breast cancer growth in vitro and in vivo. Thus, we have identified specific coactivators that may be useful as targets for endocrine-resistant breast cancers.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/genética , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Humanos , Células MCF-7 , Unión Proteica/genética , Proteómica , Transcripción Genética/genética , Activación Transcripcional/genética , Translocación Genética/genética
6.
Trends Biochem Sci ; 42(12): 977-989, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29122461

RESUMEN

Particular chromatin modifications are associated with different states of gene transcription, yet our understanding of which modifications are causal 'drivers' in promoting transcription is incomplete. Here, we discuss new developments describing the ordered, mechanistic role of select histone marks occurring during distinct steps in the RNA polymerase II (Pol II) transcription cycle. In particular, we highlight the interplay between histone marks in specifying the 'next step' of transcription. While many studies have described correlative relationships between histone marks and their occupancy at distinct gene regions, we focus on studies that elucidate clear functional consequences of specific histone marks during different stages of transcription. These recent discoveries have refined our current mechanistic understanding of how histone marks promote Pol II transcriptional progression.


Asunto(s)
Código de Histonas/genética , ARN Polimerasa II/metabolismo , Transcripción Genética/genética , Humanos
7.
J Biol Chem ; 292(35): 14456-14472, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28717009

RESUMEN

The transition from transcription initiation to elongation is a key regulatory step in gene expression, which requires RNA polymerase II (pol II) to escape promoter proximal pausing on chromatin. Although elongation factors promote pause release leading to transcription elongation, the role of epigenetic modifications during this critical transition step is poorly understood. Two histone marks on histone H3, lysine 4 trimethylation (H3K4me3) and lysine 9 acetylation (H3K9ac), co-localize on active gene promoters and are associated with active transcription. H3K4me3 can promote transcription initiation, yet the functional role of H3K9ac is much less understood. We hypothesized that H3K9ac may function downstream of transcription initiation by recruiting proteins important for the next step of transcription. Here, we describe a functional role for H3K9ac in promoting pol II pause release by directly recruiting the super elongation complex (SEC) to chromatin. H3K9ac serves as a substrate for direct binding of the SEC, as does acetylation of histone H4 lysine 5 to a lesser extent. Furthermore, lysine 9 on histone H3 is necessary for maximal pol II pause release through SEC action, and loss of H3K9ac increases the pol II pausing index on a subset of genes in HeLa cells. At select gene promoters, H3K9ac loss or SEC depletion reduces gene expression and increases paused pol II occupancy. We therefore propose that an ordered histone code can promote progression through the transcription cycle, providing new mechanistic insight indicating that SEC recruitment to certain acetylated histones on a subset of genes stimulates the subsequent release of paused pol II needed for transcription elongation.


Asunto(s)
Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Lisina/metabolismo , Modelos Biológicos , Procesamiento Proteico-Postraduccional , Elongación de la Transcripción Genética , Iniciación de la Transcripción Genética , Acetilación , Sustitución de Aminoácidos , Animales , Drosophila , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epigénesis Genética , Células HeLa , Histonas/antagonistas & inhibidores , Histonas/química , Histonas/genética , Humanos , Mutación , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
8.
Nucleic Acids Res ; 45(16): 9348-9360, 2017 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-28637181

RESUMEN

In eukaryotic cells, the gene expression status is strictly controlled by epigenetic modifications on chromatin. The repressive status of chromatin largely contributes to HIV latency. Studies have shown that modification of histone H3K27 acts as a key molecular switch for activation or suppression of many cellular genes. In this study, we found that K27-acetylated histone H3 specifically recruited Super Elongation Complex (SEC), the transcriptional elongation complex essential for HIV-1 long terminal repeat (LTR)-mediated and general cellular transcription. Interestingly, H3K27 acetylation further stimulates H3R26 methylation, which subsequently abrogates the recruitment of SEC, forming a negative feedback regulatory loop. Importantly, by inhibiting methyltransferase activity of CARM1, the enzyme responsible for H3R26 methylation, HIV-1 transcription is reactivated in several HIV latency cell models, including a primary resting CD4+ T cell model. When combined with other latency disrupting compounds such as JQ1 or vorinostat/SAHA, the CARM1 inhibitor achieved synergistic effects on HIV-1 activation. This study suggests that coordinated and dynamic modifications at histone H3K27 and H3R26 orchestrate HIV-1 LTR-mediated transcription, and potentially opens a new avenue to disrupt latent HIV-1 infection by targeting specific epigenetic enzymes.


Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/genética , Código de Histonas , Histonas/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Latencia del Virus/genética , Acetilación , Compuestos de Bencilideno/química , Compuestos de Bencilideno/farmacología , Línea Celular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Duplicado del Terminal Largo de VIH , VIH-1/efectos de los fármacos , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Metilación , Piperidonas/química , Piperidonas/farmacología , Elongación de la Transcripción Genética , Factores de Transcripción/metabolismo , Latencia del Virus/efectos de los fármacos
9.
Mol Cell ; 60(5): 769-783, 2015 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-26611104

RESUMEN

A central mechanism for controlling circadian gene amplitude remains elusive. We present evidence for a "facilitated repression (FR)" model that functions as an amplitude rheostat for circadian gene oscillation. We demonstrate that ROR and/or BMAL1 promote global chromatin decondensation during the activation phase of the circadian cycle to actively facilitate REV-ERB loading for repression of circadian gene expression. Mechanistically, we found that SRC-2 dictates global circadian chromatin remodeling through spatial and temporal recruitment of PBAF members of the SWI/SNF complex to facilitate loading of REV-ERB in the hepatic genome. Mathematical modeling highlights how the FR model sustains proper circadian rhythm despite fluctuations of REV-ERB levels. Our study not only reveals a mechanism for active communication between the positive and negative limbs of the circadian transcriptional loop but also establishes the concept that clock transcription factor binding dynamics is perhaps a central tenet for fine-tuning circadian rhythm.


Asunto(s)
Cromatina/metabolismo , Ritmo Circadiano , Hígado/metabolismo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Factores de Transcripción ARNTL/metabolismo , Animales , Regulación de la Expresión Génica , Ratones , Modelos Biológicos , Coactivador 2 del Receptor Nuclear/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo
10.
Chem Res Toxicol ; 28(7): 1508-16, 2015 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-26114498

RESUMEN

Furan, a possible human carcinogen, is found in heat treated foods and tobacco smoke. Previous studies have shown that humans are capable of converting furan to its reactive metabolite, cis-2-butene-1,4-dial (BDA), and therefore may be susceptible to furan toxicity. Human risk assessment of furan exposure has been stymied because of the lack of mechanism-based exposure biomarkers. Therefore, a sensitive LC-MS/MS assay for six furan metabolites was applied to measure their levels in urine from furan-exposed rodents as well as in human urine from smokers and nonsmokers. The metabolites that result from direct reaction of BDA with lysine (BDA-N(α)-acetyllysine) and from cysteine-BDA-lysine cross-links (N-acetylcysteine-BDA-lysine, N-acetylcysteine-BDA-N(α)-acetyllysine, and their sulfoxides) were targeted in this study. Five of the six metabolites were identified in urine from rodents treated with furan by gavage. BDA-N(α)-acetyllysine, N-acetylcysteine-BDA-lysine, and its sulfoxide were detected in most human urine samples from three different groups. The levels of N-acetylcysteine-BDA-lysine sulfoxide were more than 10 times higher than that of the corresponding sulfide in many samples. The amount of this metabolite was higher in smokers relative to that in nonsmokers and was significantly reduced following smoking cessation. Our results indicate a strong relationship between BDA-derived metabolites and smoking. Future studies will determine if levels of these biomarkers are associated with adverse health effects in humans.


Asunto(s)
Furanos/orina , Nicotiana/química , Fumar , Aldehídos/química , Animales , Biomarcadores/orina , Cromatografía Líquida de Alta Presión , Femenino , Furanos/química , Furanos/metabolismo , Humanos , Masculino , Ratones , Ratas , Ratas Endogámicas F344 , Espectrometría de Masas en Tándem , Nicotiana/metabolismo
11.
Drug Metab Dispos ; 42(7): 1132-6, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24751574

RESUMEN

Furan is a liver toxicant and carcinogen in rodents. Although humans are most likely exposed to furan through a variety of sources, the effect of furan exposure on human health is still unknown. In rodents, furan requires metabolism to exert its toxic effects. The initial product of the cytochrome P450 2E1-catalyzed oxidation is a reactive α,ß-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). BDA is toxic and mutagenic and consequently is considered responsible for the toxic effects of furan. The urinary metabolites of furan in rats are derived from the reaction of BDA with cellular nucleophiles, and precursors to these metabolites are detected in furan-exposed hepatocytes. Many of these precursors are 2-(S-glutathionyl)butanedial-amine cross-links in which the amines are amino acids and polyamines. Because these metabolites are derived from the reaction of BDA with cellular nucleophiles, their levels are a measure of the internal dose of this reactive metabolite. To compare the ability of human hepatocytes to convert furan to the same metabolites as rodent hepatocytes, furan was incubated with cryopreserved human and rodent hepatocytes. A semiquantitative liquid chromatography with tandem mass spectrometry assay was developed for a number of the previously characterized furan metabolites. Qualitative and semiquantitative analysis of the metabolites demonstrated that furan is metabolized in a similar manner in all three species. These results indicate that humans may be susceptible to the toxic effects of furan.


Asunto(s)
Criopreservación , Furanos/metabolismo , Hepatocitos/metabolismo , Animales , Cromatografía Liquida , Glutatión/metabolismo , Humanos , Ratones , Ratas , Espectrometría de Masas en Tándem
12.
Mol Cell ; 51(2): 185-99, 2013 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-23850489

RESUMEN

Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pull-down assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17ß-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an "activated" state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a "natural" ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Receptor alfa de Estrógeno/metabolismo , Regulación Neoplásica de la Expresión Génica , Nucleosomas/metabolismo , Proteómica , Elementos de Respuesta/genética , Neoplasias de la Mama/genética , Inmunoprecipitación de Cromatina , ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Receptor alfa de Estrógeno/genética , Estrógenos/farmacología , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HeLa , Humanos , Células MCF-7 , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coactivador 3 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/metabolismo , Nucleosomas/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transactivadores , Transcripción Genética , Activación Transcripcional
13.
Drug Metab Dispos ; 40(3): 596-601, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22187484

RESUMEN

Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,ß-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.


Asunto(s)
Aldehídos/metabolismo , Carcinógenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Furanos/metabolismo , Microsomas Hepáticos/metabolismo , Acetilcisteína/metabolismo , Animales , Cromatografía Líquida de Alta Presión/métodos , Citocromo P-450 CYP2E1/metabolismo , Glutatión/metabolismo , Humanos , Lisina/metabolismo , Ratones , Microsomas Hepáticos/enzimología , Oxidación-Reducción , Oxidorreductasas/metabolismo , Ratas , Espectrometría de Masas en Tándem/métodos
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