Significance We identified a function for a member of the extracellular matrix in the regulation of autophagy. Decorin, a member of the small leucine-rich proteoglycan family and an established pan-receptor tyrosine kinase inhibitor, evokes endothelial cell autophagy and inhibits angiogenesis. This process is mediated by a high-affinity interaction with VEGFR2 which leads to increased levels of Peg3, a tumor-suppressor gene. We provide mechanistic evidence that Peg3 is required to maintain basal levels of Beclin 1, a major autophagic marker. These data provide a paradigmatic shift for other soluble matrix constituents to regulate autophagy.
Background: Decorin functions as a soluble tumor repressor via binding receptor-tyrosine kinases, such as Met, to curb rampant tumor neovascularization. Results: Decorin evokes tumor cell mitophagy through dynamic co-regulation of PGC-1␣ and mitostatin via physical interactions between PGC-1␣ and mitostatin Conclusion: Decorin requires mitostatin to evoke mitophagy as the underlying basis for angiogenic attenuation. Significance: We have identified mitostatin as a novel mitophagic effector.
Systemic lupus erythematosus (SLE) is mediated by autoreactive antibodies that damage multiple tissues. Genome-wide association studies (GWAS) link >60 loci with SLE risk, but the causal variants and effector genes are largely unknown. We generated high-resolution spatial maps of SLE variant accessibility and gene connectivity in human follicular helper T cells (TFH), a cell type required for anti-nuclear antibodies characteristic of SLE. Of thẽ 400 potential regulatory variants identified, 90% exhibit spatial proximity to genes distant in the 1D genome sequence, including variants that loop to regulate the canonical TFH genes BCL6 and CXCR5 as confirmed by genome editing. SLE 'variant-to-gene' maps also implicate genes with no known role in TFH/SLE disease biology, including the kinases HIPK1 and MINK1. Targeting these kinases in TFH inhibits production of IL-21, a cytokine crucial for class-switched B cell antibodies. These studies offer mechanistic insight into the SLEassociated regulatory architecture of the human genome.
We previously discovered that systemic delivery of decorin for treatment of breast carcinoma xenografts induces paternally expressed gene 3 (Peg3), an imprinted gene encoding a zinc finger transcription factor postulated to function as a tumor suppressor. Here we found that de novo expression of Peg3 increased Beclin 1 promoter activity and protein expression. This process required the full-length Peg3 as truncated mutants lacking either the N-terminal SCAN domain or the zinc fingers failed to translocate to the nucleus and promote Beclin 1 transcription. Importantly, overexpression of Peg3 in endothelial cells stimulated autophagy and concurrently inhibited endothelial cell migration and evasion from a 3D matrix. Mechanistically, we found that Peg3 induced the secretion of the powerful angiostatic glycoprotein Thrombospondin 1 independently of Beclin 1 transcriptional induction. Thus, we provide a new mechanism whereby Peg3 can simultaneously evoke autophagy in endothelial cells and attenuate angiogenesis.
The IL1A and IL1B genes lie in close proximity on chromosome 2 near the gene for their natural inhibitor, IL1RN. Despite diverse functions, they are all three inducible through TLR4 signaling but with distinct kinetics. This study analyzed transcriptional induction kinetics, chromosome looping, and enhancer RNA production to understand the distinct regulation of these three genes in human cells. IL1A, IL1B, and IL1RN were rapidly induced after stimulation with LPS; however, IL1B mRNA production was less inhibitable by iBET151, suggesting it does not use pause-release regulation. Surprisingly, chromatin looping contacts between IL1A and IL1B were highly intermingled, although those of IL1RN were distinct, and we focused on comparing IL1A and IL1B transcriptional pathways. Our studies demonstrated that enhancer RNAs were produced from a subset of the regulatory regions, that they were critical for production of the mRNAs, and that they bound a diverse array of RNA binding proteins, including p300 but not CBP. We, furthermore, demonstrated that recruitment of p300 was dependent on MAPKs. Integrator is another RNA binding protein recruited to the promoters and enhancers, and its recruitment was more dependent on NF-kB than MAPKs. We found that integrator and NELF, an RNA polymerase II pausing protein, were associated with RNA in a manner that facilitated interaction. We conclude that IL1A and IL1B share many regulatory contacts, signaling pathways, and interactions with enhancer RNAs. A complex of protein interactions with enhancer RNAs emphasize the role of enhancer RNAs and the overall structural aspects of transcriptional regulation.
22Systemic lupus erythematosus (SLE) is a complex inflammatory disease mediated by 23 autoreactive antibodies that damages multiple tissues in children and adults. wide association studies (GWAS) have statistically implicated hundreds of loci in the 25 susceptibility to human disease, including SLE, but the majority have failed to identify the 26 causal variants or the effector genes. As a physicochemical approach to detecting 27 functional variants and connecting them to target genes, we generated comprehensive, 28 high-resolution maps of SLE variant accessibility and gene connectivity in the context of 29 the three-dimensional chromosomal architecture of human tonsillar follicular helper T 30 cells (TFH), a cell type required for the production of anti-nuclear antibodies characteristic 31 of SLE. These spatial epigenomic maps identified a shortlist of over 400 potentially 32 functional variants across 48 GWAS-implicated SLE loci. Twenty percent of these 33 variants were located in open promoters of highly-expressed TFH genes, while 80% 34 reside in non-promoter genomic regions that are connected in 3D to genes that likewise 35 tend to be highly expressed in TFH. Importantly, we find that 90% of SLE-associated 36 variants exhibit spatial proximity to genes that are not nearby in the 1D sequence of the 37 genome, and over 60% of variants 'skip' the nearest gene to physically interact only with 38 the promoters of distant genes. Gene ontology confirmed that genes in spatial proximity 39 to SLE variants reside in highly SLE-relevant networks, including accessible variants that 40 loop 200-1000 kb to interact with the promoters of the canonical TFH genes BCL6 and 41 CXCR5. CRISPR-Cas9 genome editing confirmed that these variants reside in novel, 42 distal regulatory elements required for normal BCL6 and CXCR5 expression by T cells. 43Furthermore, SLE-associated SNP-promoter interactomes implicated a set of novel 44 51 52 GWAS has been an important tool in understanding the genetic basis of complex, 53 heritable diseases and traits. However, GWAS is typically powered to identify relatively 54 large blocks of the genome containing dozens to hundreds of single nucleotide 55 polymorphisms (SNP) in linkage disequilibrium (LD), any one of which could be 56 responsible for the association of the entire locus with disease susceptibility. Moreover, 57 ~90% of GWAS-implicated SNP are intergenic or intronic, and do not affect the coding 58 sequence of proteins. Therefore, the location of the GWAS signal per se does not identify 59 the culprit gene(s). Examples of this are the FTO GWAS signal in obesity 1,2 , and the 60 TCF7L2 GWAS signal in type 2 diabetes 3 , in which each suspected causal variant resides 61 in an intron of the local gene, but were shown instead to regulate expression of the distant 62 genes IRX3/5 and ACSL5, respectively. 63Systemic lupus erythematosus (SLE) is a complex inflammatory disease mediated by 64 autoreactive antibodies that damage skin, joints, kidneys, brain and other tissues in 65 children and ...
Costimulation of T cells through the antigen receptor and the CD28 receptor engages an ‘effector’ gene expression program and a burst of proliferation. Conversely, antigen receptor engagement in the absence of costimulation induces a hypo-responsive state, referred to as anergy, that is associated with active silencing of effector genes. Our ATAC-seq analysis of CD28-dependent chromatin remodeling in anergic and effector CD4+ T cells has identified p21-activated kinase 6 (PAK6) as a gene that becomes accessible in anergic but not effector T cells. PAK6 is a member of a family of kinases comprised of two groups, group I (PAK1-3) and group II (PAK4-6). PAK6 and other group II members lack a regulatory domain that is present in group I, causing it to function differently. Previous studies by others have shown that PAK2, a group I PAK, was a positive regulator of CD3/CD28 T cell activation. Here, we demonstrate that PAK6 has the opposite role in T cell activation as PAK2, where it inhibits IL2 secretion, a hallmark characteristic of T cell anergy. Knockdown of PAK6 in anergic and effector T cells, using a CRISPR/CAS9 approach, resulted in a 50% increase in IL2 secretion, and 5-fold more cell expansion in anergic cultures upon restimulation with CD3/CD28. This suggests that PAK6 may be involved in a signaling pathway which inhibits cellular proliferation or promotes apoptosis. The highest level of PAK6 expression was observed upon re-stimulation of anergic cells, suggesting that PAK6 may be involved in the maintenance of T cell anergy. This study identifies a novel role for PAK6 in T cell anergy and a potential new target for regulating T cell function in autoimmune diseases.
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