Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and posttranscriptional mechanisms ...contributing to the regulatory network. We examined proinflammatory gene regulation in activated macrophages by performing RNA-seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the subcellular locations of coding and noncoding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of coexpressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.
Display omitted
► Coding and noncoding transcripts exhibit characteristic subcellular distributions ► The most potently induced genes favor promoters with low CpG content ► Full-length, incompletely spliced transcripts accumulate on the chromatin ► Delayed transcript release may reflect a requirement for the completion of splicing
Sequencing analysis of subcellular fractions allows the separation of synthesis of nascent transcripts from RNA processing and the accumulation of mature mRNAs, revealing a high-resolution view of transcript dynamics of proinflammatory genes. Surprisingly, full-length yet incompletely spliced transcripts accumulate on chromatin, suggesting that splicing often follows the completion of transcription.
We describe a broad mechanistic framework for the transcriptional induction of mammalian primary response genes by Toll-like receptors and other stimuli. One major class of primary response genes is ...characterized by CpG-island promoters, which facilitate promiscuous induction from constitutively active chromatin without a requirement for SWI/SNF nucleosome remodeling complexes. The low nucleosome occupancy at promoters in this class can be attributed to the assembly of CpG islands into unstable nucleosomes, which may lead to SWI/SNF independence. Another major class consists of non-CpG-island promoters that assemble into stable nucleosomes, resulting in SWI/SNF dependence and a requirement for transcription factors that promote selective nucleosome remodeling. Some stimuli, including serum and tumor necrosis factor-α, exhibit a strong bias toward activation of SWI/SNF-independent CpG-island genes. In contrast, interferon-β is strongly biased toward SWI/SNF-dependent non-CpG-island genes. By activating a diverse set of transcription factors, Toll-like receptors induce both classes and others for an optimal response to microbial pathogens.
Regulatory T cells (Tregs) play a pivotal role in the inhibition of anti-tumor immune responses. Understanding the mechanisms governing Treg homeostasis may therefore be important for development of ...effective tumor immunotherapy. We have recently demonstrated a key role for the canonical nuclear factor κB (NF-κB) subunits, p65 and c-Rel, in Treg identity and function. In this report, we show that NF-κB c-Rel ablation specifically impairs the generation and maintenance of the activated Treg (aTreg) subset, which is known to be enriched at sites of tumors. Using mouse models, we demonstrate that melanoma growth is drastically reduced in mice lacking c-Rel, but not p65, in Tregs. Moreover, chemical inhibition of c-Rel function delayed melanoma growth by impairing aTreg-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. Our studies therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.
Display omitted
•NF-κB c-Rel regulates the transcriptional landscape of activated Tregs•c-Rel activity in Tregs restricts anti-tumor responses•Chemical c-Rel inhibition reduces melanoma growth and potentiates anti-PD-1 therapy
NF-κB c-Rel is critical for the function of activated Tregs and serves as a target to reduce Treg function in the tumor microenvironment without compromising systemic tolerance or causing autoimmunity.
Both conventional T (Tconv) cells and regulatory T (Treg) cells are activated through ligation of the T cell receptor (TCR) complex, leading to the induction of the transcription factor NF-κB. In ...Tconv cells, NF-κB regulates expression of genes essential for T cell activation, proliferation, and function. However the role of NF-κB in Treg function remains unclear. We conditionally deleted canonical NF-κB members p65 and c-Rel in developing and mature Treg cells and found they have unique but partially redundant roles. c-Rel was critical for thymic Treg development while p65 was essential for mature Treg identity and maintenance of immune tolerance. Transcriptome and NF-κB p65 binding analyses demonstrated a lineage specific, NF-κB-dependent transcriptional program, enabled by enhanced chromatin accessibility. These dual roles of canonical NF-κB in Tconv and Treg cells highlight the functional plasticity of the NF-κB signaling pathway and underscores the need for more selective strategies to therapeutically target NF-κB.
Display omitted
•Canonical NF-κB signaling promotes regulatory T (Treg) cell development•Conditional deletions reveal that c-Rel and p65 play unique but partly redundant roles•NF-κB maintains Treg cell homeostasis and immune tolerance•NF-κB establishes Treg cell transcriptional identity
NF-κB regulates genes essential for conventional T cell activation and function, but its role in Treg cell function remains unclear. Oh et al. use Treg cell-specific conditional deletions of canonical NF-κB subunits to demonstrate central roles for p65 and c-Rel in orchestrating Treg cell development, suppressive function, and molecular identity.
The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the ...relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3' end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3'-processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.
Virtually all living organisms have evolved mechanisms to adapt to their environment by sensing environmental stresses and inducing the transcription of appropriate sets of response genes in a ...coordinated fashion. In the vertebrate immune system, the highly selective response to an environmental stimulus, often an invading microorganism, plays an especially important role in regulating the activities of, and interactions among, the many cell types involved in innate and adaptive immunity. It is now widely appreciated that the selective response to a stimulus requires the concerted action of signal transduction pathways, transcription factors, and chromatin structure. Many proteins and pathways that help to regulate a response have been characterized. However, our understanding of the gene-specific and global logic through which a highly selective response is elicited has only recently begun to emerge.
Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and post-transcriptional mechanisms ...contributing to the regulatory network. We examined pro-inflammatory gene regulation in activated macrophages by performing RNA-Seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the sub-cellular locations of coding and non-coding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of co-expressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.
We describe a broad mechanistic framework for the transcriptional induction of mammalian primary response genes by Toll-like receptors and other stimuli. One major class of primary response genes is ...characterized by CpG-island promoters, which facilitate promiscuous induction from constitutively active chromatin without a requirement for SWI/SNF nucleosome remodeling complexes. The low nucleosome occupancy at promoters in this class can be attributed to the assembly of CpG islands into unstable nucleosomes, which may lead to SWI/SNF independence. Another major class consists of non-CpG-island promoters that assemble into stable nucleosomes, resulting in SWI/SNF dependence and a requirement for transcription factors that promote selective nucleosome remodeling. Some stimuli, including serum and tumor necrosis factor- alpha , exhibit a strong bias toward activation of SWI/SNF-independent CpG-island genes. In contrast, interferon- beta is strongly biased toward SWI/SNF-dependent non-CpG-island genes. By activating a diverse set of transcription factors, Toll-like receptors induce both classes and others for an optimal response to microbial pathogens.
In response to pathogenic stimulation, cells of the innate immune system initiate an inflammatory response and upregulate the expression of numerous pro-inflammatory genes. It is critical that the ...expression of these genes be restricted to a pathogenic situation. In an effort to understand the mechanisms of selectivity, previous studies have shown that chromatin remodeling of promoter elements is a critical regulatory mechanism to restrict inflammatory gene expression. This dissertation describes two studies that seek to understand the regulatory mechanisms that govern differentially expressed inflammatory genes. The first study expands the analyses of chromatin remodeling during the inflammatory response. We explore the significance of the observation that rapidly-induced, remodeling-independent genes are distinguished by the prevalence of CpG island elements at their promoters, while slowly induced remodeling-dependent genes are devoid of these elements. Chromatin immunoprecipitation and in vitro nucleosome assembly experiments show that these CpG island genes have a poised chromatin structure at promoters due to the intrinsic instability of these sequences to form nucleosomes. From this we establish a regulatory framework to understand the inducible expression of inflammatory genes. The second study attempts to explore the genomewide significance of this framework. We utilize high-throughput sequencing technologies to explore chromatin-associated nascent transcripts alongside other subcellular RNA populations. We find that induced genes display diverse expression profiles, and that rapidly induced genes are largely associated with CpG island promoters. Furthermore, this novel approach gives us a unique view of transcription, allowing visualization of interesting phenomena such as partially elongating nascent transcripts and the detection of co-transcriptional splicing events at a genome-wide level.
PDF
