RESUMEN
Adipocytes contribute to metabolic disorders such as obesity, diabetes, and atherosclerosis. Prior characterizations of the transcriptional network driving adipogenesis have overlooked transiently acting transcription factors (TFs), genes, and regulatory elements that are essential for proper differentiation. Moreover, traditional gene regulatory networks provide neither mechanistic details about individual regulatory element-gene relationships nor temporal information needed to define a regulatory hierarchy that prioritizes key regulatory factors. To address these shortcomings, we integrate kinetic chromatin accessibility (ATAC-seq) and nascent transcription (PRO-seq) data to generate temporally resolved networks that describe TF binding events and resultant effects on target gene expression. Our data indicate which TF families cooperate with and antagonize each other to regulate adipogenesis. Compartment modeling of RNA polymerase density quantifies how individual TFs mechanistically contribute to distinct steps in transcription. The glucocorticoid receptor activates transcription by inducing RNA polymerase pause release, whereas SP and AP-1 factors affect RNA polymerase initiation. We identify Twist2 as a previously unappreciated effector of adipocyte differentiation. We find that TWIST2 acts as a negative regulator of 3T3-L1 and primary preadipocyte differentiation. We confirm that Twist2 knockout mice have compromised lipid storage within subcutaneous and brown adipose tissue. Previous phenotyping of Twist2 knockout mice and Setleis syndrome Twist2 -/- patients noted deficiencies in subcutaneous adipose tissue. This network inference framework is a powerful and general approach for interpreting complex biological phenomena and can be applied to a wide range of cellular processes.
Asunto(s)
Adipocitos , Redes Reguladoras de Genes , Proteína 1 Relacionada con Twist , Animales , Ratones , Línea Celular , Adipocitos/citología , Adipocitos/metabolismo , Factores de Transcripción/metabolismo , Adipogénesis , Transcripción Genética , Elementos Reguladores de la Transcripción , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
Coupling molecular biology to high-throughput sequencing has revolutionized the study of biology. Molecular genomics techniques are continually refined to provide higher resolution mapping of nucleic acid interactions and structure. Sequence preferences of enzymes can interfere with the accurate interpretation of these data. We developed seqOutBias to characterize enzymatic sequence bias from experimental data and scale individual sequence reads to correct intrinsic enzymatic sequence biases. SeqOutBias efficiently corrects DNase-seq, TACh-seq, ATAC-seq, MNase-seq and PRO-seq data. We show that seqOutBias correction facilitates identification of true molecular signatures resulting from transcription factors and RNA polymerase interacting with DNA.
Asunto(s)
Algoritmos , Biología Computacional/métodos , ADN/metabolismo , Desoxirribonucleasas/metabolismo , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Sesgo , ADN/química , ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Desoxirribonucleasas/genética , Unión Proteica , Reproducibilidad de los Resultados , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Genome-wide association studies (GWAS) have discovered thousands loci associated with disease risk and quantitative traits, yet most of the variants responsible for risk remain uncharacterized. The majority of GWAS-identified loci are enriched for non-coding single-nucleotide polymorphisms (SNPs) and defining the molecular mechanism of risk is challenging. Many non-coding causal SNPs are hypothesized to alter transcription factor (TF) binding sites as the mechanism by which they affect organismal phenotypes. We employed an integrative genomics approach to identify candidate TF binding motifs that confer breast cancer-specific phenotypes identified by GWAS. We performed de novo motif analysis of regulatory elements, analyzed evolutionary conservation of identified motifs, and assayed TF footprinting data to identify sequence elements that recruit TFs and maintain chromatin landscape in breast cancer-relevant tissue and cell lines. We identified candidate causal SNPs that are predicted to alter TF binding within breast cancer-relevant regulatory regions that are in strong linkage disequilibrium with significantly associated GWAS SNPs. We confirm that the TFs bind with predicted allele-specific preferences using CTCF ChIP-seq data. We used The Cancer Genome Atlas breast cancer patient data to identify ANKLE1 and ZNF404 as the target genes of candidate TF binding site SNPs in the 19p13.11 and 19q13.31 GWAS-identified loci. These SNPs are associated with the expression of ZNF404 and ANKLE1 in breast tissue. This integrative analysis pipeline is a general framework to identify candidate causal variants within regulatory regions and TF binding sites that confer phenotypic variation and disease risk.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Estudio de Asociación del Genoma Completo , Alelos , Neoplasias de la Mama/patología , Cromatina/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Motivos de Nucleótidos/genética , Polimorfismo de Nucleótido Simple , Unión Proteica , Sitios de Carácter Cuantitativo/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genéticaRESUMEN
DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution.
Asunto(s)
Ensamble y Desensamble de Cromatina , Metilación de ADN , Poríferos/genética , Secuencia de Aminoácidos , Animales , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Fenotipo , Poríferos/metabolismo , Conformación ProteicaRESUMEN
The product of the meiosis-expressed gene 1 (MEIG1) is found in the cell bodies of spermatocytes and recruited to the manchette, a structure unique to elongating spermatids, by Parkin co-regulated gene (PACRG). This complex is essential for targeting cargo to the manchette during sperm flagellum assembly. Here we show that MEIG1 adopts a unique fold that provides a large surface for interacting with other proteins. We mutated 12 exposed and conserved amino acids and show that four of these mutations (W50A, K57E, F66A, Y68A) dramatically reduce binding to PACRG. These four amino acids form a contiguous hydrophobic patch on one end of the protein. Furthermore, each of these four mutations diminishes the ability of MEIG1 to stabilize PACRG when expressed in bacteria. Together these studies establish the unique structure and key interaction surface of MEIG1 and provide a framework to explore how MEIG1 recruits proteins to build the sperm tail.
Asunto(s)
Proteínas de Ciclo Celular/química , Modelos Moleculares , Proteínas Nucleares/química , Fosfoproteínas/química , Conformación Proteica , Proteínas/química , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ratones , Proteínas de Microfilamentos , Chaperonas Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Pliegue de Proteína , Estabilidad Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes , Solventes , Relación Estructura-ActividadRESUMEN
The MBD2-NuRD (Nucleosome Remodeling and Deacetylase) complex is an epigenetic reader of DNA methylation that regulates genes involved in normal development and neoplastic diseases. To delineate the architecture and functional interactions of the MBD2-NuRD complex, we previously solved the structures of MBD2 bound to methylated DNA and a coiled-coil interaction between MBD2 and p66α that recruits the CHD4 nucleosome remodeling protein to the complex. The work presented here identifies novel structural and functional features of a previously uncharacterized domain of MBD2 (MBD2IDR). Biophysical analyses show that the MBD2IDR is an intrinsically disordered region (IDR). However, despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical contact region requiring two contiguous amino acid residues, Arg(286) and Leu(287). Mutating these residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene PRSS8 in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Metilación de ADN , Proteínas de Unión al ADN/genética , Epigénesis Genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Cinética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting mCpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for mCpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference for mCpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to (m)CpG islands and promotes scanning by rapidly exchanging between successive mCpG sites which facilitates repair of nearby mCpG/TpG mismatches by the glycosylase domain.
Asunto(s)
Disparidad de Par Base , Islas de CpG , Metilación de ADN , ADN/química , Endodesoxirribonucleasas/química , Sitios de Unión , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Modelos Moleculares , Estructura Terciaria de Proteína , Cloruro de Sodio/químicaRESUMEN
Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.
Asunto(s)
Proteínas Aviares/química , Islas de CpG/fisiología , Proteínas de Unión al ADN/química , ADN/química , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Pollos , ADN/genética , ADN/metabolismo , Metilación de ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de ProteínaRESUMEN
The methyl-cytosine binding domain 2 (MBD2)-nucleosome remodeling and deacetylase (NuRD) complex recognizes methylated DNA and silences expression of associated genes through histone deacetylase and nucleosome remodeling functions. Our previous structural work demonstrated that a coiled-coil interaction between MBD2 and GATA zinc finger domain containing 2A (GATAD2A/p66α) proteins recruits the chromodomain helicase DNA-binding protein (CHD4/Mi2ß) to the NuRD complex and is necessary for MBD2-mediated DNA methylation-dependent gene silencing in vivo (Gnanapragasam, M. N., Scarsdale, J. N., Amaya, M. L., Webb, H. D., Desai, M. A., Walavalkar, N. M., Wang, S. Z., Zu Zhu, S., Ginder, G. D., and Williams, D. C., Jr. (2011) p66α-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex. Proc. Natl. Acad. Sci. U.S.A. 108, 7487-7492). The p66α-MBD2 interaction differs from most coiled-coils studied to date by forming an anti-parallel heterodimeric complex between two peptides that are largely monomeric in isolation. To further characterize unique features of this complex that drive heterodimeric specificity and high affinity binding, we carried out biophysical analyses of MBD2 and the related homologues MBD3, MBD3-like protein 1 (MBD3L1), and MBD3-like protein 2 (MBD3L2) as well as specific mutations that modify charge-charge interactions and helical propensity of the coiled-coil domains. Analytical ultracentrifugation analyses show that the individual peptides remain monomeric in isolation even at 300 µM in concentration for MBD2. Circular dichroism analyses demonstrate a direct correlation between helical content of the coiled-coil domains in isolation and binding affinity for p66α. Furthermore, complementary electrostatic surface potentials and inherent helical content of each peptide are necessary to maintain high-affinity association. These factors lead to a binding affinity hierarchy of p66α for the different MBD2 homologues (MBD2 ≈ MBD3 > MBD3L1 ≈ MBD3L2) and suggest a hierarchical regulatory model in tissue and life cycle stage-specific silencing by NuRD complexes.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Multimerización de Proteína , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Iones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/química , Homología de Secuencia de Aminoácido , Electricidad Estática , Temperatura , Dedos de ZincRESUMEN
Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal ß-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiled-coil interaction between MBD2 and p66α, a critical component of the MBD2-NuRD complex. We show that enforced expression of the isolated p66α coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2-NuRD complex that does not include native p66α or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.