Divergent roles of RelA and c-Rel in establishing chromosomal loops upon activation of the Igkappa gene.
ABSTRACT: Precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening DNA, but how trans-acting regulatory proteins work to establish and maintain DNA loops during gene activation remains largely unexplored. LPS-induced transcription of the mouse Igkappa gene in B lymphocytes utilizes three distal enhancers and requires the transcription factor NF-kappaB, whose family members include RelA and c-Rel. Using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that LPS-induced Igkappa gene activation creates chromosomal loops by bridging together all three pairwise interactions between the distal enhancers and RNA polymerase II, the apparent molecular tie for the bases of these loops. RelA and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis, or c-Rel. We have thus identified both essential and nonessential events that establish higher order chromatin reorganization during Igkappa gene activation.
Project description:Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3'Igh regulatory region (3'IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-?B/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3'IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-?B/Rel binding site suggesting a potential reciprocal modulation of the 3'IghRR by AhR and NF-?B/Rel. The objective of the current study was to evaluate the role of NF-?B/Rel and the AhR on the 3'IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3'IghRR-regulated transgene reporter or an inducible I?B? (inhibitor kappa B-alpha protein) superrepressor (I?B?AA), we demonstrate an AhR- and NF-?B/Rel-dependent modulation of 3'IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-?B/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-?B/Rel protein binding profile within the 3'IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels.
Project description:Antibodies of the immunoglobulin G1 class are induced in mice by T-cell-dependent antigens but not by lipopolysaccharide (LPS). CD40 engagement contributes to this preferential isotype production by activating NF-kappaB/Rel to induce germ line gamma1 transcripts, which are essential for class switch recombination. Although LPS also activates NF-kappaB, it poorly induces germ line gamma1 transcripts. Western blot analyses show that CD40 ligand (CD40L) induces all NF-kappaB/Rel proteins, whereas LPS activates predominantly p50 and c-Rel. Electrophoretic mobility shift assays show that in CD40L-treated cells, p50-RelA and p50-RelB dimers are the major NF-kappaB complexes binding to the germ line gamma1 promoter, whereas in LPS-treated cells, p50-c-Rel and p50-p50 dimers are the major binding complexes. Transfection of expression plasmids for NF-kappaB/Rel fusion proteins (forced dimers) indicates that p50-RelA and p50-RelB dimers activate the germ line gamma1 promoter and that p50-c-Rel and p50-p50 dimers inhibit this activation by competitively binding to the promoter without activating the promoter. Therefore, germ line gamma1 transcription depends on the composition of NF-kappaB/Rel proteins.
Project description:The Rel/NF-kappaB transcription factors are constitutively activated in many human cancers. The Rel proteins in this family are implicated in leukemia/lymphomagenesis, but the mechanism is not completely understood. Previous studies showed that the transcription activation domains (TADs) of the viral oncoprotein v-Rel and its cellular Rel/NF-kappaB homologues c-Rel and RelA are key determinants of their different transforming activities in primary lymphocytes. Substitution of a Rel TAD for that of RelA conferred a strong transforming phenotype upon RelA, which otherwise failed to transform cells. To gain insights into protein interactions that influence cell transformation by the Rel TADs, we identified factors that interact with the TAD of v-Rel, the most oncogenic member of the Rel/NF-kappaB family. We report that the coactivator for transcription factors AP-1 and estrogen receptors, CAPERalpha, interacts with the v-Rel TAD and potently synergizes v-Rel-mediated transactivation. Importantly, coexpression of CAPERalpha markedly reduced and delayed v-Rel's transforming activity in primary lymphocytes, whereas a dominant-negative mutant enhanced the kinetics of v-Rel-mediated transformation. Furthermore, small interfering RNA-mediated knockdown of CAPERalpha in v-Rel-transformed lymphocytes significantly enhanced colony formation in soft agar. Since the potency of Rel-mediated transactivation is an important determinant of lymphocyte transformation, as is Rel's ability to induce transcriptional repression, these data suggest that CAPERalpha's interaction with the Rel TAD could modulate Rel/NF-kappaB's transforming activity by facilitating expression or dampening repression of specific gene subsets important for oncogenesis. Overall, this study identifies CAPERalpha as a new transcriptional coregulator for v-Rel and reveals an important role in modulating Rel's oncogenic activity.
Project description:Activation of the transcription factor NF-kappaB/Rel has been shown to be involved in inflammatory disease. Here we studied the role of RelA/p65, the main transactivating subunit, during acute pancreatitis using a Cre-loxP strategy. Selective truncation of the rela gene in pancreatic exocrine cells led to both severe injury of the acinar cells and systemic complications including lung and liver damage. Our data demonstrated that expression and induction of the protective pancreas-specific acute phase protein pancreatitis-associated protein 1 (PAP1) depended on RelA/p65. Lentiviral gene transfer of PAP1 cDNA reduced the extent of necrosis and infiltration in the pancreata of mice with selective truncation of RelA/p65. These results provide in vivo evidence for RelA/p65 protection of acinar cell death via upregulation of PAP1. Moreover, our data underscore the pancreas-specific role of NF-kappaB/Rel and suggest multidimensional roles of NF-kappaB/Rel in different cells and contexts during inflammation.
Project description:The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in low-grade adipose tissue inflammation and development of insulin resistance during obesity. In this context, nuclear factor ?-light-chain-enhancer of activated B cells (NF?B) is directly involved and required for the acute activation of the inflammatory gene program. Here, we show that the major transactivating subunit of NF?B, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), is also required for acute TNF-induced suppression of adipocyte genes. Notably, this repression does not involve RELA binding to the associated enhancers but rather loss of cofactors and enhancer RNA (eRNA) selectively from high-occupancy sites within super-enhancers. Based on these data, we have developed models that, with high accuracy, predict which enhancers and genes are repressed by TNF in adipocytes. We show that these models are applicable to other cell types where TNF represses genes associated with super-enhancers in a highly cell-type-specific manner. Our results propose a novel paradigm for NF?B-mediated repression, whereby NF?B selectively redistributes cofactors from high-occupancy enhancers, thereby specifically repressing super-enhancer-associated cell identity genes.
Project description:Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.
Project description:A fundamental goal in biology is to gain a quantitative understanding of how appropriate cell responses are achieved amid conflicting signals that work in parallel. Through live, single-cell imaging, we monitored both the dynamics of nuclear factor ?B (NF-?B) signaling and inflammatory cytokine transcription in macrophages exposed to the bacterial product lipopolysaccharide (LPS). Our analysis revealed a previously uncharacterized positive feedback loop involving induction of the expression of Rela, which encodes the RelA (p65) NF-?B subunit. This positive feedback loop rewired the regulatory network when cells were exposed to LPS above a distinct concentration. Paradoxically, this rewiring of NF-?B signaling in macrophages (a myeloid cell type) required the transcription factor Ikaros, which promotes the development of lymphoid cells. Mathematical modeling and experimental validation showed that the RelA positive feedback overcame existing negative feedback loops and enabled cells to discriminate between different concentrations of LPS to mount an effective innate immune response only at higher concentrations. We suggest that this switching in the relative dominance of feedback loops ("feedback dominance switching") may be a general mechanism in immune cells to integrate opposing feedback on a key transcriptional regulator and to set a response threshold for the host.
Project description:Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three trans-acting factors-NF-kappaB, STAT4, and T-bet-with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and corecruitment of STAT4. STAT4 and NF-kappaB therefore cooperate at multiple cis elements to enable NF-kappaB-dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in T helper 1 (Th1) and effector CD8(+) T (Tc1) cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals.
Project description:Determining the roles of Rel/NF-kappaB transcription factors in mouse skin development with loss-of-function mutants has been limited by redundancy among these proteins and by embryonic lethality associated with the absence of RelA. Using mice lacking RelA and c-rel, which survive throughout embryogenesis on a tumor necrosis factor alpha (TNF-alpha)-deficient background (rela(-/-) c-rel(-/-) tnfalpha(-/-)), we show that c-rel and RelA are required for normal epidermal development. Although mutant fetuses fail to form tylotrich hair and have a thinner epidermis, mutant keratinocyte progenitors undergo terminal differentiation to form an outer cornified layer. Mutant basal keratinocytes are abnormally small, exhibit a delay in G(1) progression, and fail to form keratinocyte colonies in culture. In contrast to the reduced proliferation of mutant keratinocytes during embryogenesis, skin grafting experiments revealed that the mutant epidermis develops a TNF-alpha-dependent hyperproliferative condition. Collectively, our findings indicate that RelA and c-rel control the development of the epidermis and associated appendages during embryogenesis and regulate epidermal homeostasis in a postnatal environment through the suppression of innate immune-mediated inflammation.
Project description:Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.