High glucose promotes IL-17A-induced gene expression through histone acetylation in retinal pigment epithelium cells.

Diabetic retinopathy (DR) is a complication of diabetes mellitus (DM) that can cause visual impairment and blindness. Inflammation plays a critical role in its development and progression. Retinal pigment epithelium (RPE) cells secrete inflammatory factors that modulate ocular immune response. However, it is unclear how diabetes regulates the expression of inflammatory factors in RPE cells. In this study, streptozocin (STZ) was applied to induce diabetic alterations in the retinas of mice, and RPE cells were further purified to profile gene expressions. The IL-17 signaling pathway was the most significantly enriched and the only enriched inflammation pathway in the profile via KEGG analysis. IL-17A induced the expression of targeted genes, which was enhanced by high glucose levels, suggesting a synergistic effect of IL-17A and high glucose. High glucose did not affect the mRNA stability of IL-17A-targeted genes or the activity of IL-17A signaling transduction, but it boosted the histone acetylation on IL-17A-targeted genes. Curcumin, an inhibitor of histone acetyltransferase, abolished high glucose-enhanced histone acetylation of IL-17A-targeted genes and blocked the promotion of high glucose levels on gene expression induced by IL-17A. In conclusion, high glucose levels promote IL-17A-induced gene expression via histone acetylation in RPE cells.

Single cell RNA sequencing (scRNA-Seq) deciphering pathological alterations in streptozotocin-induced diabetic retinas.

Diabetic retinopathy (DR) is an irreversible and progressive diabetic complication leading to visual impairment, even blindness. Due to the delicate and complicated structure of the retina, the pathology of DR has not been completely elucidated yet. We constructed a transcriptome atlas of >14,000 single cells from healthy and streptozotocin (STZ)-induced diabetic murine retinas to decipher pathological alterations of DR. We found four stress-inducible genes Cirbp, Rmb3, Mt1 and Mt2 commonly induced in most types of retinal cells. Bipolar cells were little affected on both number and gene expression. Diabetes increased expression of inflammatory factor genes in retinal microglia, and stimulated expression of immediate early genes (IEGs) in retinal astrocytes. A large number of genes were deregulated in diabetic vascular endothelial cells (ECs), and the differentially expressed genes were paired to the pathways functioning in metabolism, shear stress and vascular permeability. These pathways were mapped by more deregulated genes in a subpopulation of ECs specifically presented in diabetic retinas (diabetic retinal ECs, DRECs). Moreover, several inflammation pathways were activated in DRECs, and the most significant one is the IL-17 signaling pathway. According to the EC markers, DRECs were mainly capillary ECs, confirmed by immunofluorescent staining of S100a9, a target gene of the IL-17 signaling pathway. This study deciphered pathological alterations of DR, and provided clues for potential targets for DR therapy.

DNA Sensing in Mismatch Repair-Deficient Tumor Cells Is Essential for Anti-tumor Immunity.

Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-ß in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.

MLH1 Deficiency-Triggered DNA Hyperexcision by Exonuclease 1 Activates the cGAS-STING Pathway.

Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased single-strand DNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.

High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods.

BACKGROUND: Next generation sequencing (NGS) is being increasingly applied for assisting cancer molecular diagnosis. However, it is still needed to validate NGS accuracy on detection of DNA alternations based on a large number of clinical samples, especially for DNA rearrangements and copy number variations (CNVs). This study is to set up basic parameters of targeted NGS for clinical diagnosis and to understand advantage of targeted NGS in comparison with the conventional methods of molecular diagnosis. METHODS: Genomic DNA from 1000 Genomes Project and DNA from cancer cell lines have been used to establish the basic parameters for targeted NGS. The following confirmation was conducted by clinical samples. The multiple variants tested by amplification-refractory mutation system (ARMS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were evaluated by targeted NGS to determine the sensitivity. Furthermore, the multiple variants detected by targeted NGS were confirmed by current conventional methods to elucidate the specificity. RESULTS: At sequencing depth of 500×, the maximal sensitivities on detecting single nucletic variances (SNVs) and small insertions/deletions (Indels) can reach 99% and 98.7% respectively, and in 20% of cancer cells, CNV detection can reach to the maximal level. The following confirmation of the sensitivity and specificity was conducted by a large cohort of clinical samples. For SNV and indel detection in clinical samples, targeted NGS can identify all hotspot mutations with 100% sensitivity and specificity. On ALK fusion detection, about 86% IHC-identified cases could be identified by targeted NGS and all ALK fusion detected by targeted NGS were confirmed by IHC. For HER2-amplification, 14 HER2-amplification cases identified by target NGS were all confirmed by FISH and about 93.3% of Her-2 IHC (3+) cases were identified by targeted NGS. Finally, the targeted NGS platform developed here has accurately detected EGFR hotspot mutations in 215 NSCLC patients. CONCLUSIONS: DNA from cancer cell lines is better than standard DNA as a reference to establish basic parameters for targeted NGS. Comparison of the conventional methods using a large cohort of patient samples confirmed the high preformance of targeted NGS on detecting DNA alterations.

AF9 YEATS domain links histone acetylation to DOT1L-mediated H3K79 methylation.

The recognition of modified histones by "reader" proteins constitutes a key mechanism regulating gene expression in the chromatin context. Compared with the great variety of readers for histone methylation, few protein modules that recognize histone acetylation are known. Here, we show that the AF9 YEATS domain binds strongly to histone H3K9 acetylation and, to a lesser extent, H3K27 and H3K18 acetylation. Crystal structural studies revealed that AF9 YEATS adopts an eight-stranded immunoglobin fold and utilizes a serine-lined aromatic "sandwiching" cage for acetyllysine readout, representing a novel recognition mechanism that is distinct from that of known acetyllysine readers. ChIP-seq experiments revealed a strong colocalization of AF9 and H3K9 acetylation genome-wide, which is important for the chromatin recruitment of the H3K79 methyltransferase DOT1L. Together, our studies identified the evolutionarily conserved YEATS domain as a novel acetyllysine-binding module and established a direct link between histone acetylation and DOT1L-mediated H3K79 methylation in transcription control.

Gcn5 and PCAF negatively regulate interferon-ß production through HAT-independent inhibition of TBK1.

Viral infection triggers innate immune signaling, which in turn induces interferon-ß (IFN-ß) production to establish innate antiviral immunity. Previous studies showed that Gcn5 (Kat2a), a histone acetyltransferase (HAT) with partial functional redundancy with PCAF (Kat2b), and Gcn5/PCAF-mediated histone H3K9 acetylation (H3K9ac) are enriched on the active IFNB gene promoter. However, whether Gcn5/PCAF and H3K9ac regulate IFN-ß production is unknown. Here, we show that Gcn5/PCAF-mediated H3K9ac correlates well with, but is surprisingly dispensable for, the expression of endogenous IFNB and the vast majority of active genes in fibroblasts. Instead, Gcn5/PCAF repress IFN-ß production and innate antiviral immunity in several cell types in a HAT-independent and non-transcriptional manner: by inhibiting the innate immune signaling kinase TBK1 in the cytoplasm. Our results thus identify Gcn5 and PCAF as negative regulators of IFN-ß production and innate immune signaling.

Gcn5 and PCAF regulate PPARγ and Prdm16 expression to facilitate brown adipogenesis.

The acetyltransferase Gcn5 is critical for embryogenesis and shows partial functional redundancy with its homolog PCAF. However, the tissue- and cell lineage-specific functions of Gcn5 and PCAF are still not well defined. Here we probe the functions of Gcn5 and PCAF in adipogenesis. We found that the double knockout (DKO) of Gcn5/PCAF inhibits expression of the master adipogenic transcription factor gene PPARγ, thereby preventing adipocyte differentiation. The adipogenesis defects in Gcn5/PCAF DKO cells are rescued by ectopic expression of peroxisome proliferator-activated receptor γ (PPARγ), suggesting Gcn5/PCAF act upstream of PPARγ to facilitate adipogenesis. The requirement of Gcn5/PCAF for PPARγ expression was unexpectedly bypassed by prolonged treatment with an adipogenic inducer, 3-isobutyl-1-methylxanthine (IBMX). However, neither PPARγ ectopic expression nor prolonged IBMX treatment rescued defects in Prdm16 expression in DKO cells, indicating that Gcn5/PCAF are essential for normal Prdm16 expression. Gcn5/PCAF regulate PPARγ and Prdm16 expression at different steps in the transcription process, facilitating RNA polymerase II recruitment to Prdm16 and elongation of PPARγ transcripts. Overall, our study reveals that Gcn5/PCAF facilitate adipogenesis through regulation of PPARγ expression and regulate brown adipogenesis by influencing Prdm16 expression.

Promoter-bound p300 complexes facilitate post-mitotic transmission of transcriptional memory.

A central hallmark of epigenetic inheritance is the parental transmission of changes in patterns of gene expression to progeny without modification of DNA sequence. Although, the trans-generational conveyance of this molecular memory has been traditionally linked to covalent modification of histone and/or DNA, recent studies suggest a role for proteins that persist or remain bound within chromatin to "bookmark" specific loci for enhanced or potentiated responses in daughter cells immediately following cell division. In this report we describe a role for p300 in enabling gene bookmarking by pre-initiation complexes (PICs) containing RNA polymerase II (pol II), Mediator and TBP. Once formed these complexes require p300 to enable reacquisition of protein complex assemblies, chromatin modifications and long range chromatin interactions that facilitate post-mitotic transmission of transcriptional memory of prior environmental stimuli.

UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/ß-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

EZH2 Mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3, and NGFR.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb protein histone methyltransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibiton with 3-deazaneplanocin A increased CASZ1 expression, inhibited NB cell growth, and induced neurite extension. Similarly, EZH2(-/-) mouse embryonic fibroblasts (MEF) displayed 3-fold higher levels of CASZ1 mRNA compared with EZH2(+/+) MEFs. In cells with increased expression of CASZ1, treatment with histone deacetylase (HDAC) inhibitor decreased expression of EZH2 and the Polycomb Repressor complex component SUZ12. Under steady-state conditions, H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR, and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors.

Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation.

Histone acetyltransferases (HATs) GCN5 and PCAF (GCN5/PCAF) and CBP and p300 (CBP/p300) are transcription co-activators. However, how these two distinct families of HATs regulate gene activation remains unclear. Here, we show deletion of GCN5/PCAF in cells specifically and dramatically reduces acetylation on histone H3K9 (H3K9ac) while deletion of CBP/p300 specifically and dramatically reduces acetylations on H3K18 and H3K27 (H3K18/27ac). A ligand for nuclear receptor (NR) PPARδ induces sequential enrichment of H3K18/27ac, RNA polymerase II (Pol II) and H3K9ac on PPARδ target gene Angptl4 promoter, which correlates with a robust Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9ac, but not H3K18/27ac, on the Angptl4 promoter. Finally, we show GCN5/PCAF and GCN5/PCAF-mediated H3K9ac correlate with, but are surprisingly dispensable for, NR target gene activation. In contrast, CBP/p300 and their HAT activities are essential for ligand-induced Pol II recruitment on, and activation of, NR target genes. These results highlight the substrate and site specificities of HATs in cells, demonstrate the distinct roles of GCN5/PCAF- and CBP/p300-mediated histone acetylations in gene activation, and suggest an important role of CBP/p300-mediated H3K18/27ac in NR-dependent transcription.

Histone H3K27 methyltransferase Ezh2 represses Wnt genes to facilitate adipogenesis.

Wnt/beta-catenin signaling inhibits adipogenesis. Genome-wide profiling studies have revealed the enrichment of histone H3K27 methyltransferase Ezh2 on Wnt genes. However, the functional significance of such a direct link between the two types of developmental regulators in mammalian cells, and the role of Ezh2 in adipogenesis, remain unclear. Here we show Ezh2 and its H3K27 methyltransferase activity are required for adipogenesis. Ezh2 directly represses Wnt1, -6, -10a, and -10b genes in preadipocytes and during adipogenesis. Deletion of Ezh2 eliminates H3K27me3 on Wnt promoters and derepresses Wnt expression, which leads to activation of Wnt/beta-catenin signaling and inhibition of adipogenesis. Ectopic expression of the wild-type (WT) Ezh2, but not the enzymatically inactive F667I mutant, prevents the loss of H3K27me3 and the defects in adipogenesis in Ezh2(-/-) preadipocytes. The adipogenesis defects in Ezh2(-/-) cells can be rescued by expression of adipogenic transcription factors PPARgamma, C/EBPalpha, or inhibitors of Wnt/beta-catenin signaling. Interestingly, Ezh2(-/-) cells show marked increase of H3K27 acetylation globally as well as on Wnt promoters. These results indicate that H3K27 methyltransferase Ezh2 directly represses Wnt genes to facilitate adipogenesis and suggest that acetylation and trimethylation on H3K27 play opposing roles in regulating Wnt expression.

C/EBPalpha regulates SIRT1 expression during adipogenesis.

SIRT1 plays an important role in adipogenesis, but how SIRT1 is regulated in adipogenesis is largely unknown. In this study, we show that both SIRT1 protein and mRNA levels were increased along with CCAAT/enhancer-binding protein alpha (C/EBPalpha) during adipocyte differentiation. C/EBPalpha, but not C/EBPalphap30, activated SIRT1 promoter in both HeLa cells and 3T3-L1 preadipocytes. Furthermore, C/EBPalpha upregulated SIRT1 mRNA and protein levels in HeLa cells and increased SIRT1 expression in a p53-independent manner in Soas2 cells. In preadipocytes, ectopic expression of C/EBPalpha upregulated SIRT1 protein level and knockdown of C/EBPalpha led to the decrease of SIRT1 protein level. Moreover, by promoter deletion analysis, gel shift assay and chromatin immunoprecipitation, we found that C/EBPalpha bound to the SIRT1 promoter at a consensus C/EBPalpha binding site. These data demonstrate that C/EBPalpha regulates SIRT1 expression during adipogenesis by directly binding to the SIRT1 promoter.

Patt1, a novel protein acetyltransferase that is highly expressed in liver and downregulated in hepatocellular carcinoma, enhances apoptosis of hepatoma cells.

Protein acetylation is increasingly recognized as an important post-translational modification. Although a lot of protein acetyltransferases have been identified, a few putative acetyltransferases are yet to be studied. In this study, we identified a novel protein acetyltransferase, Patt1, which belongs to GNAT family. Patt1 exhibited histone acetyltransferase activity and auto-acetylation activity. Deletion and mutation analysis of the predicted acetyltransferase domain in Patt1 showed that the conserved Glu139 was an important residue for its protein acetyltransferase activity. Furthermore, we found that Patt1 was highly expressed in liver and significantly downregulated in hepatocellular carcinoma tissues. In addition, we showed that overexpression of Patt1 enhanced the apoptosis of hepatoma cells dependent on its acetyltransferase activity, whereas knockdown of Patt1 significantly protected Chang liver cells from apoptosis. These data suggest that Patt1 might be involved in the development of hepatocellular carcinoma, and could be served as a potential therapy target for hepatocellular carcinoma.

Histone methylation regulator PTIP is required for PPARgamma and C/EBPalpha expression and adipogenesis.

PPARgamma and C/EBPalpha cooperate to control preadipocyte differentiation (adipogenesis). However, the factors that regulate PPARgamma and C/EBPalpha expression during adipogenesis remain largely unclear. Here, we show PTIP, a protein that associates with histone H3K4 methyltransferases, regulates PPARgamma and C/EBPalpha expression in mouse embryonic fibroblasts (MEFs) and during preadipocyte differentiation. PTIP deletion in MEFs leads to marked decreases of PPARgamma expression and PPARgamma-stimulated C/EBPalpha expression. Further, PTIP is essential for induction of PPARgamma and C/EBPalpha expression during preadipocyte differentiation. Deletion of PTIP impairs the enrichment of H3K4 trimethylation and RNA polymerase II on PPARgamma and C/EBPalpha promoters. Accordingly, PTIP(-/-) MEFs and preadipocytes all show striking defects in adipogenesis. Rescue of the adipogenesis defect in PTIP(-/-) MEFs requires coexpression of PPARgamma and C/EBPalpha. Finally, deletion of PTIP in brown adipose tissue significantly reduces tissue weight. Thus, by regulating PPARgamma and C/EBPalpha expression, PTIP plays a critical role in adipogenesis.

PCAF acetylates {beta}-catenin and improves its stability.

beta-Catenin plays an important role in development and tumorigenesis. However, the effect of a key acetyltransferase p300/CBP-associated factor (PCAF) on beta-catenin signaling is largely unknown. In this study, we found PCAF could increase the beta-catenin transcriptional activity, induce its nuclear translocation, and up-regulate its protein level by inhibiting its ubiquitination and improving its stability. Further studies showed that PCAF directly binds to and acetylates beta-catenin. The key ubiquitination sites Lys-19 and Lys-49 of beta-catenin were shown as the critical residues for PCAF-induced acetylation and stabilization. Knockdown of PCAF in colon cancer cells markedly reduced the protein level, transcriptional activity, and acetylation level of beta-catenin; promoted cell differentiation; inhibited cell migration; and repressed xenografted tumorigenesis and tumor growth in nude mice. All these data demonstrate that PCAF acetylates beta-catenin and regulates its stability, and they raise the prospect that therapies targeting PCAF may be of clinical use in beta-catenin-driven diseases, such as colon cancer.

GSK3beta mediates the induced expression of synaptic acetylcholinesterase during apoptosis.

Besides its role in terminating acetylcholine-mediated neurotransmission, acetylcholinesterase (AChE) is found to be expressed and participate in the process of apoptosis in various cell types. However, the mechanisms underlying AChE up-regulation in neuronal cells remain elusive. Herein we demonstrated that glycogen synthase kinase-3beta (GSK3beta) mediates induced AChE-S expression during apoptosis. In this study, A23187 and thapsigargin (TG) were employed to induce apoptosis in neuroendocrine PC12 cells. The results showed that exposure of PC12 cells to A23187 and TG up-regulated AChE activity significantly. The same treatment also led to activation of GSK3beta. Two different inhibitors of GSK3beta (lithium and GSK3beta-specific inhibitor VIII) could block A23187- or TG-induced up-regulation of AChE activity, AChE-S mRNA level and protein expression. However, lithium could not inhibit the induction of AChE-R mRNA and protein under similar conditions. Taken together, our results show that GSK3beta is specifically involved in the induction of AChE-S expression in PC12 cells during apoptosis.

Cytoplasm-localized SIRT1 enhances apoptosis.

In general, SIRT1 is localized in nuclei. Here, we showed that endogenous and exogenous SIRT1 were both able to partially localize in cytoplasm in certain cell lines, and cytoplasm-localized SIRT1 was associated with apoptosis and led to increased sensitivity to apoptosis. Furthermore, we demonstrated that translocation of nucleus-localized SIRT1 from nuclei to cytoplasm was the main pathway leading to localization of SIRT1 in cytoplasm. In HeLa cells, wild type SIRT1 was completely localized in nuclei. By truncation of two predicted nuclear localization signals or fusion with an exogenous nuclear export signal, SIRT1 was partially localized in cytoplasm of HeLa cells and resulted in increased sensitivity to apoptosis. The apoptosis enhanced by cytoplasm-localized SIRT1 was independent of its deacetylase activity, but dependent on caspases. SIRT1 was distributed in cytoplasm at metaphase during mitosis, and overexpression of SIRT1 significantly augmented apoptosis for cells at metaphase. In summary, we found SIRT1 is able to localize in cytoplasm, and cytoplasm-localized SIRT1 enhances apoptosis.