The Transcription Factor NFATp Plays a Key Role in Susceptibility to TB in Mice

In T cells, the transcription factor nuclear factor of activated T cells p (NFATp) is a key regulator of the cytokine genes tumor necrosis factor (TNF) and interferon-γ (IFN-γ). Here, we show that NFATp-deficient (NFATp^(−/−)) mice have a dramatic and highly significant increase in mortality after Mycobacterium tuberculosis (MTb) infection as compared to mortality of control animals after MTb infection. Animals deficient in NFATp have significantly impaired levels of TNF and IFN-γ transcription and protein expression in naïve or total CD4+ T cells, but display wild-type levels of TNF mRNA or protein from MTb-stimulated dendritic cells (DC). The rapid mortality and disease severity observed in MTb-infected NFATp^(−/−) mice is associated with dysregulated production of TNF and IFN-γ in the lungs, as well as with increased levels of TNF, in their serum. Furthermore, global blocking of TNF production by injection of a TNF neutralizaing agent at 6 weeks, but not 12 weeks, post-MTb-infection further decreased the survival rate of both wild-type and NFATp^(−/−) mice, indicating an early role for TNF derived from cells from the monocyte lineage in containment of infection. These results thus demonstrate that NFATp plays a critical role in immune containment of TB disease in vivo, through the NFATp-dependent expression of TNF and IFN-γ in T cells

NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, and activates TNF transcription during hypertonic stress alone

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays an important role in a variety of infectious and autoimmune disorders. Its transcription is regulated in a stimulus- and cell-type-specific manner via the recruitment of distinct DNA/activator complexes forming secondary structures or enhanceosomes. NFATp, a member of the nuclear factor of activated T cells (NFAT) family of transcription factors, plays a critical role in TNF gene regulation under a variety of conditions. In this study, we show that NFAT5, the most recently described NFAT family member, binds to the TNF promoter in a manner distinct from other NFAT proteins and is a key mediator in the activation of TNF gene transcription during hypertonic stress alone

NFAT5 Regulates HIV-1 in Primary Monocytes via a Highly Conserved Long Terminal Repeat Site

To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor–viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5–LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS

Chromatin profiling across the human tumour necrosis factor gene locus reveals a complex, cell type-specific landscape with novel regulatory elements

The TNF locus on chromosome 6p21 encodes a family of proteins with key roles in the immune response whose dysregulation leads to severe disease. Transcriptional regulation is important, with cell type and stimulus-specific enhancer complexes involving the proximal TNF promoter. We show how quantitative chromatin profiling across a 34 kb region spanning the TNF locus has allowed us to identify a number of novel DNase hypersensitive sites and characterize more distant regulatory elements. We demonstrate DNase hypersensitive sites corresponding to the lymphotoxin alpha (LTA) and tumour necrosis factor (TNF) promoter regions, a CpG island in exon 4 of lymphotoxin beta (LTB), the 3′ end of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1 (NFKBIL1) and 3.4 kb upstream of LTA. These sites co-localize to highly conserved DNA sequences and show evidence of cell type specificity when lymphoblastoid, Jurkat, U937, HeLa and HEK293T cell lines are analysed using Southern blotting. For Jurkat T cells, we define histone modifications across the locus. Peaks of acetylated histone H3 and H4, together with tri-methyl K4 of histone H3, correspond to hypersensitive sites, notably in exon 4 of LTB. We provide evidence of a functional role for an intergenic DNase I hypersensitive site distal to LTA in Jurkat cells based on reporter gene analysis, with evidence of recruitment of upstream stimulatory factors (USF) transcription factors

IL-10 inhibits transcription elongation of the human TNF gene in primary macrophages

IL-10 plays a central nonredundant role in limiting inflammation in vivo. However, the mechanisms involved remain to be resolved. Using primary human macrophages, we found that IL-10 inhibits selected inflammatory genes, primarily at a level of transcription. At the TNF gene, this occurs not through an inhibition of RNA polymerase II (Pol II) recruitment and transcription initiation but through a mechanism targeting the stimulation of transcription elongation by cyclin-dependent kinase (CDK) 9. We demonstrated an unanticipated requirement for a region downstream of the TNF 3′ untranslated region (UTR) that contains the nuclear factor κB (NF-κB) binding motif (κB4) both for induction of transcription by lipopolysaccharide (LPS) and its inhibition by IL-10. IL-10 not only inhibits the recruitment of RelA to regions containing κB sites at the TNF gene but also to those found at other LPS-induced genes. We show that although IL-10 elicits a general block in RelA recruitment to its genomic targets, the gene-specific nature of IL-10’s actions are defined through the differential recruitment of CDK9 and the control of transcription elongation. At TNF, but not NFKBIA, the consequence of RelA recruitment inhibition is a loss of CDK9 recruitment, preventing the stimulation of transcription elongation

Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment

Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling

Deregulated expression of CD40 ligand in HTLV-I infection: Distinct mechanisms of downregulation in HTLV-I-transformed cell lines and ATL patients

AbstractHTLV-I infection is associated with the development of adult T cell leukemia (ATL) and the neuroinflammatory disease HAM/TSP. There are quantitative and qualitative differences in the antiviral cytotoxic T cell (CTL) response in ATL and HAM/TSP although the underlying mechanisms are unclear. Here, we demonstrate that the HTLV-I Tax trans-activating protein is a transcriptional activator of CD40 ligand (CD40L), a critical regulator of dendritic cell maturation and adaptive immunity. Tax activates CD40L expression via a cyclosporin A insensitive pathway that is also independent of NF-κB. Although Tax upregulates CD40L gene expression, CD40L expression is absent in Tax-expressing HTLV-I-transformed cell lines via an epigenetic mechanism involving methylation. T lymphocytes cultured ex vivo from ATL patients, but not HAM/TSP or normal controls, exhibit a potent block in the induction of CD40L, but not CD69. However, the CD40L gene is not silenced by methylation in ATL patients, thus CD40L is downregulated by distinct mechanisms in HTLV-I-transformed cell lines and ATL patients

Primate TNF Promoters Reveal Markers of Phylogeny and Evolution of Innate Immunity

Background. Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry. Methodology/Principal findings. Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages. Conclusions/significance. Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF

Regulatory modules function in a non-autonomous manner to control transcription of the mbp gene

Multiple regulatory modules contribute to the complex expression programs realized by many loci. Although long thought of as isolated components, recent studies demonstrate that such regulatory sequences can physically associate with promoters and with each other and may localize to specific sub-nuclear transcription factories. These associations provide a substrate for putative interactions and have led to the suggested existence of a transcriptional interactome. Here, using a controlled strategy of transgenesis, we analyzed the functional consequences of regulatory sequence interaction within the myelin basic protein (mbp) locus. Interactions were revealed through comparisons of the qualitative and quantitative expression programs conferred by an allelic series of 11 different enhancer/inter-enhancer combinations ligated to a common promoter/reporter gene. In a developmentally contextual manner, the regulatory output of all modules changed markedly in the presence of other sequences. Predicted by transgene expression programs, deletion of one such module from the endogenous locus reduced oligodendrocyte expression levels but unexpectedly, also attenuated expression of the overlapping golli transcriptional unit. These observations support a regulatory architecture that extends beyond a combinatorial model to include frequent interactions capable of significantly modulating the functions conferred through regulatory modules in isolation