RESUMO
An understanding of the mechanisms and logic by which transcription factors coordinate gene regulation requires delineation of their genomic interactions at a genome-wide scale. Chromatin immunoprecipitation-sequencing (ChIP-seq) and more recent techniques, including CUT&Tag, typically reveal thousands of genomic interactions by transcription factors, but without insight into their functional roles. Due to cost and time considerations, optimization of ChIP experimental conditions is typically carried out only with representative interaction sites rather than through genome-wide analyses. Here, we describe insights gained from the titration of two chemical crosslinking reagents in genome-wide ChIP-seq experiments examining two members of the NF-κB family of transcription factors: RelA and c-Rel. We also describe a comparison of ChIP-seq and CUT&Tag. Our results highlight the large impact of ChIP-seq experimental conditions on the number of interactions detected, on the enrichment of consensus and non-consensus DNA motifs for the factor, and on the frequency with which the genomic interactions detected are located near potential target genes. We also found considerable consistency between ChIP-seq and CUT&Tag results, but with a substantial fraction of genomic interactions detected with only one of the two techniques. Together, the results demonstrate the dramatic impact of experimental conditions on the results obtained in a genome-wide analysis of transcription factor binding, highlighting the need for further scrutiny of the functional significance of these condition-dependent differences.
RESUMO
Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1-/- tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.
Assuntos
Fator Regulador 1 de Interferon , Animais , Humanos , Camundongos , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Imunidade , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/genética , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/genética , Fator de Transcrição STAT1/metabolismo , Masculino , FemininoRESUMO
The five NF-κB family members and three nuclear IκB proteins play important biological roles, but the mechanisms by which distinct members of these protein families contribute to selective gene transcription remain poorly understood, especially at a genome-wide scale. Using nascent transcript RNA-seq, we observed considerable overlap between p50-dependent and IκBζ-dependent genes in Toll-like receptor 4 (TLR4)-activated macrophages. Key immunoregulatory genes, including Il6, Il1b, Nos2, Lcn2, and Batf, are among the p50-IκBζ-codependent genes. IκBζ-bound genomic sites are occupied at earlier time points by NF-κB dimers. However, p50-IκBζ codependence does not coincide with preferential binding of either p50 or IκBζ, as RelA co-occupies hundreds of genomic sites with the two proteins. A common feature of p50-IκBζ-codependent genes is a nearby p50/RelA/IκBζ-cobound site exhibiting p50-dependent binding of both RelA and IκBζ. This and other results suggest that IκBζ acts in concert with RelA:p50 heterodimers. Notably, p50-IκBζ-codependent genes comprise a high percentage of genes exhibiting the greatest differential expression between TLR4-stimulated and tumor necrosis factor receptor (TNFR)-stimulated macrophages. Thus, our genome-centric analysis reveals a defined p50-IκBζ pathway that selectively activates a set of key immunoregulatory genes and serves as an important contributor to differential TNFR and TLR4 responses.
Assuntos
Regulação da Expressão Gênica , Macrófagos , Subunidade p50 de NF-kappa B , Transdução de Sinais , Receptor 4 Toll-Like , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inflamação/genética , Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Ligação Proteica , Transdução de Sinais/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , MasculinoRESUMO
Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.
Assuntos
Processamento Alternativo , Diferenciação Celular , Cromatina , Ribonucleoproteínas Nucleares Heterogêneas , Neurônios , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Fatores de Transcrição , Processamento Alternativo/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Animais , Diferenciação Celular/genética , Cromatina/metabolismo , Camundongos , Neurônios/metabolismo , Neurônios/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Transcrição Gênica , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Éxons/genética , Humanos , Autorrenovação Celular/genéticaRESUMO
Inducible nucleosome remodeling at hundreds of latent enhancers and several promoters shapes the transcriptional response to Toll-like receptor 4 (TLR4) signaling in macrophages. We aimed to define the identities of the transcription factors that promote TLR-induced remodeling. An analysis strategy based on ATAC-seq and single-cell ATAC-seq that enriched for genomic regions most likely to undergo remodeling revealed that the transcription factor nuclear factor κB (NF-κB) bound to all high-confidence peaks marking remodeling during the primary response to the TLR4 ligand, lipid A. Deletion of NF-κB subunits RelA and c-Rel resulted in the loss of remodeling at high-confidence ATAC-seq peaks, and CRISPR-Cas9 mutagenesis of NF-κB-binding motifs impaired remodeling. Remodeling selectivity at defined regions was conferred by collaboration with other inducible factors, including IRF3- and MAP-kinase-induced factors. Thus, NF-κB is unique among TLR4-activated transcription factors in its broad contribution to inducible nucleosome remodeling, alongside its ability to activate poised enhancers and promoters assembled into open chromatin.
Assuntos
NF-kappa B , Receptor 4 Toll-Like , NF-kappa B/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Nucleossomos , Transdução de Sinais , Regulação da Expressão Gênica , Fator de Transcrição RelA/metabolismoRESUMO
Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.