RESUMO
Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that govern gene expression; however, these networks are understudied due to a lack of methods for high-throughput profiling of DNA-bound TF-COF complexes. Here, we describe the Cofactor Recruitment (CoRec) method for rapid profiling of cell-specific TF-COF complexes. We define a lysine acetyltransferase (KAT)-TF network in resting and stimulated T cells. We find promiscuous recruitment of KATs for many TFs and that 35% of KAT-TF interactions are condition specific. KAT-TF interactions identify NF-κB as a primary regulator of acutely induced histone 3 lysine 27 acetylation (H3K27ac). Finally, we find that heterotypic clustering of CBP/P300-recruiting TFs is a strong predictor of total promoter H3K27ac. Our data support clustering of TF sites that broadly recruit KATs as a mechanism for widespread co-occurring histone acetylation marks. CoRec can be readily applied to different cell systems and provides a powerful approach to define TF-COF networks impacting chromatin state and gene regulation.
RESUMO
Embryonic development is arguably the most complex process an organism undergoes during its lifetime, and understanding this complexity is best approached with a systems-level perspective. The sea urchin has become a highly valuable model organism for understanding developmental specification, morphogenesis, and evolution. As a non-chordate deuterostome, the sea urchin occupies an important evolutionary niche between protostomes and vertebrates. Lytechinus variegatus (Lv) is an Atlantic species that has been well studied, and which has provided important insights into signal transduction, patterning, and morphogenetic changes during embryonic and larval development. The Pacific species, Strongylocentrotus purpuratus (Sp), is another well-studied sea urchin, particularly for gene regulatory networks (GRNs) and cis-regulatory analyses. A well-annotated genome and transcriptome for Sp are available, but similar resources have not been developed for Lv. Here, we provide an analysis of the Lv transcriptome at 11 timepoints during embryonic and larval development. Temporal analysis suggests that the gene regulatory networks that underlie specification are well-conserved among sea urchin species. We show that the major transitions in variation of embryonic transcription divide the developmental time series into four distinct, temporally sequential phases. Our work shows that sea urchin development occurs via sequential intervals of relatively stable gene expression states that are punctuated by abrupt transitions.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Lytechinus/embriologia , Transcriptoma/fisiologia , Animais , Strongylocentrotus purpuratus/embriologiaRESUMO
High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM (nuclear extract protein-binding microarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity.
Assuntos
Núcleo Celular/química , Redes Reguladoras de Genes , Análise Serial de Proteínas/métodos , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Extratos Celulares/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Células THP-1RESUMO
The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.
Assuntos
Padronização Corporal/genética , Proteoglicanas/metabolismo , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Análise de Sequência de RNA/métodos , Sulfatos/metabolismo , Animais , Padronização Corporal/efeitos dos fármacos , Proteínas de Transporte de Cátions/metabolismo , Diferenciação Celular/efeitos dos fármacos , Ectoderma/efeitos dos fármacos , Ectoderma/enzimologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Mesoderma/citologia , Modelos Biológicos , Níquel/toxicidade , Ouriços-do-Mar/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Non-coding DNA variants (NCVs) impact gene expression by altering binding sites for regulatory complexes. New high-throughput methods are needed to characterize the impact of NCVs on regulatory complexes. We developed CASCADE (Customizable Approach to Survey Complex Assembly at DNA Elements), an array-based high-throughput method to profile cofactor (COF) recruitment. CASCADE identifies DNA-bound transcription factor-cofactor (TF-COF) complexes in nuclear extracts and quantifies the impact of NCVs on their binding. We demonstrate CASCADE sensitivity in characterizing condition-specific recruitment of COFs p300 and RBBP5 (MLL subunit) to the CXCL10 promoter in lipopolysaccharide (LPS)-stimulated human macrophages and quantify the impact of all possible NCVs. To demonstrate applicability to NCV screens, we profile TF-COF binding to ~1,700 single-nucleotide polymorphism quantitative trait loci (SNP-QTLs) in human macrophages and identify perturbed ETS domain-containing complexes. CASCADE will facilitate high-throughput testing of molecular mechanisms of NCVs for diverse biological applications.
RESUMO
Nuclear factor-kappa B (NF-κB) transcription factors coordinate gene expression in response to a broad array of cellular signals. In vertebrates, there are five NF-κB proteins (c-Rel, RelA/p65, RelB, p50, and p52) that can form various dimeric combinations exhibiting both common and dimer-specific DNA-binding specificity. In this chapter, we describe the use of the nuclear extract protein-binding microarray (nextPBM), a high-throughput method to characterize the DNA binding of transcription factors present in cell nuclear extracts. NextPBMs allow for sensitive analysis of the DNA binding of NF-κB dimers and their interactions with cell-specific cofactors.
Assuntos
Análise Serial de Proteínas , Animais , DNA/genética , DNA/metabolismo , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Extratos Vegetais , Ligação Proteica , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The type II nuclear receptors (NRs) function as heterodimeric transcription factors with the retinoid X receptor (RXR) to regulate diverse biological processes in response to endogenous ligands and therapeutic drugs. DNA-binding specificity has been proposed as a primary mechanism for NR gene regulatory specificity. Here we use protein-binding microarrays (PBMs) to comprehensively analyze the DNA binding of 12 NR:RXRα dimers. We find more promiscuous NR-DNA binding than has been reported, challenging the view that NR binding specificity is defined by half-site spacing. We show that NRs bind DNA using two distinct modes, explaining widespread NR binding to half-sites in vivo. Finally, we show that the current models of NR specificity better reflect binding-site activity rather than binding-site affinity. Our rich dataset and revised NR binding models provide a framework for understanding NR regulatory specificity and will facilitate more accurate analyses of genomic datasets.