Ördög, Tamás

ABSTRACT The Ca 2+ ‐activated Cl − channel, anoctamin 1 (Ano1, also known as transmembrane protein 16A) contributes to intestinal pacemaking, fluid secretion, cellular excitability, and tissue development. The human ANO1 promoter contains binding sites for the glioma‐associated oncogene (Gli) proteins. We investigated regulation of ANO1 transcription by Gli. ANO1 promoter activity was determined using a luciferase reporter system. Binding and functional effects of Glis on ANO1 transcription and expression were demonstrated by chromatin immunoprecipitation, small interfering RNA knockdown, PCR, immunolabeling, and recordings of Ca 2+ ‐activated Cl − currents in human embryonic kidney 293 (HEK293) cells. Results from previous genome‐wide association studies were used to test ANO1 promoter polymorphisms for association with disease. Gli1 and Gli2 bound to the promoter and repressed ANO1 transcription. Repression depended on Gli binding to a site close to the ANO1 transcriptional start site. Mutation of this site prevented Gli binding and transcriptional repression. Knockdown of Gli expression and inhibition of Gli activity increased expression of ANO1 RNA and Ca 2+ ‐activated Cl − currents in HEK293 cells. A single‐nucleotide polymorphism prevented Gli binding and showed association with irritable bowel syndrome. We conclude that Gli1 and Gli2 repress ANO1 by a novel mechanism that is independent of Gli cleavage and that has a role in gastrointestinal function.—Mazzone, A., Gibbons, S. J., Eisenman, S. T., Strege, P. R., Zheng, T., D'Amato, M., Ordog, T., Fernandez‐Zapico, M. E., Farrugia, G. Direct repression of anoctamin 1 (ANO1) gene transcription by Gli proteins. FASEB J. 33, 6632–6642 (2019). www.fasebj.org

CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.

e13073 Background: We observed several patients with a personal history of both RCC and myeloid malignancy, and aimed to explore a possible association, especially given the similarity in their epigenetic landscape, with both being characterized by widespread aberrant hypermethylation (Hu C et al, CCR 2014; Jiang Y et al, Blood 2009). Methods: Mayo Clinic’s ‘Advanced Cohort Explorer’ database was used to identify patients with a history of both RCC and a myeloid malignancy - Acute Myelogenous Leukemia (AML), Myeloproliferative Neoplasms (MPN), Myelodysplastic syndromes (MDS) - and to determine the clinical characteristics. The incidence of MDS in patients ≥65 years with a history of RCC was compared to that in the general population ≥65 years (SEER- Medicare database) as well as the general patient population at Mayo Clinic, using one sample test of proportions. Results: A total of 59 patients were identified, with both biopsy proven RCC and a myeloid malignancy during their life time (12 AML, 9 MPN, 9 low risk MDS and 29 intermediate- high risk MDS). The cohort was characterized by marked male predominance (4.4: 1). Median age at RCC diagnosis was 64 years (range 37-87) and myeloid malignancy was 75 years (range 44-90). 46/59 patients had the RCC antecedent, 10/59 concurrent and 3/59 subsequent to the myeloid malignancy with a median time of myeloid malignancy diagnosis after RCC diagnosis of 7.7 years. For patients ≥65 years, the risk of developing MDS with a history of stage I/II RCC and nephrectomy was 5.26 times that of the general population based on the SEER-Medicare database (Cogle, Blood 2011)(395/100,000 vs 75/100,000; p value < 0.001), and 3.07 times that of the general population at Mayo Clinic (395/100,000 vs 128.4/100,000; p value < 0. 001). Conclusions: We observed an association between RCC and myeloid malignancies, particularly MDS; with a history of RCC conferring a substantially increased risk of developing MDS. We hypothesize that the perturbation of epigenetic landscape in the form of widespread hypermethylation may explain, in part, the association between the two malignancies; and aim to explore the potential aberrancy of epigenetic regulators in our patient cohort with a genomic, epigenomic and transcriptomic analysis.

A microfluidic device was designed to efficiently fragment chromatin for downstream epigenetic assays.

Significance Focal adhesion kinase (FAK) is an intensely studied protein involved in many medically relevant biological processes, including cancer. Despite the large interest in FAK, a promising strategy to target FAK therapeutically is elusive. Here, we show that a region within the FAK protein that contains autophosphorylation site tyrosine (Y) 397 is essential for FAK activity in vivo. Myosin-1E (MYO1E), an actin-dependent molecular motor protein, directly interacts with FAK to induce Y397 autophosphorylation, which, in turn, causes changes in gene expression commonly observed in aggressive cancer. Our findings are significant because they further delineate FAK function in vivo and identify the MYO1E–FAK interaction as a possible Achilles’ heel for cancer.

464 Background: Although the von Hippel-Lindau (VHL) tumor suppressor gene is mutated in 60% of ccRCC, deletion of VHL in mice is insufficient for tumorigenesis. Sequencing of ccRCC tumors identified mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. We hypothesize that loss of SETD2 methyltransferase activity alters the genome wide pattern of H3K36 trimethylation (H3K36me3) in ccRCC, and contributes to the cancer phenotype. Methods: To generate a genome-wide profile of H3K36me3 in frozen nephrectomy samples and RCC cell lines, we optimized a chromatin immunoprecipitation (ChIP) protocol for the isolation of DNA associated with H3K36me3. H3K36me3 is associated with open chromatin and an H3K36me3-specific antibody was used for immunoprecipitation of endogenous H3K36me3-bound DNA. ChIP PCR primers were optimized for active genes, such as actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a “gene desert” on chromosome 12 (negative control). ChIP libraries were then generated from 3 paired uninvolved kidney and RCC and 2 RCC cell lines. In order to identify H3K36Me3 upregulated regions in uninvolved kidney and RCC, reads from the ChIP sequencing were mapped to the human genome using Burrows-Wheeler Aligner and SICER algorithms. Results: Using ChIP PCR, we found that active genomic regions were enriched 15-30 fold over the negative controls indicating that the quality and yield of immunoprecipitated DNA/chromatin complexes from frozen tissue was sufficient for ChIP sequencing. A preliminary ChIP sequencing analysis of RCC cell lines and frozen ccRCC tissue indicates that H3K36me3 enriched DNA sequences were mapped to exons (31.3%) compared to introns (13.5%, p<0.001), consistent with the role of H3K36me3 in transcription. Conclusions: Genomic regions enriched for H3K36Me3 binding were identified from patient-derived tissue and RCC cell lines. Current efforts are focused on comparing the H3K36me3 profiles between matched tumor and uninvolved kidney ChIP libraries to generate a genome wide map of dysregulated H3K36me3 modifications.

Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl–channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Flmice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+transients’ synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+transients.NEW & NOTEWORTHY The Ca2+-activated Cl−channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+transients in myenteric ICC but Ca2+transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.View this article's corresponding video summary at https://youtu.be/cyPtDP0KLY4 .