RNF20 and RNF40 regulate vitamin D receptor-dependent signaling in inflammatory bowel disease

Despite the identification of several genetic factors linked to increased susceptibility to inflammatory bowel disease (IBD), underlying molecular mechanisms remain to be elucidated in detail. The ubiquitin ligases RNF20 and RNF40 mediate the monoubiquitination of histone H2B at lysine 120 (H2Bub1) and were shown to play context-dependent roles in the development of inflammation. Here, we aimed to examine the function of the RNF20/RNF40/H2Bub1 axis in intestinal inflammation in IBD patients and mouse models. For this purpose, intestinal sections from IBD patients were immunohistochemically stained for H2Bub1. Rnf20 or Rnf40 were conditionally deleted in the mouse intestine and mice were monitored for inflammation-associated symptoms. Using mRNA-seq and chromatin immunoprecipitation (ChIP)-seq, we analyzed underlying molecular pathways in primary intestinal epithelial cells (IECs) isolated from these animals and confirmed these findings in IBD resection specimens using ChIP-seq.The majority (80%) of IBD patients displayed a loss of H2Bub1 levels in inflamed areas and the intestine-specific deletion of Rnf20 or Rnf40 resulted in spontaneous colorectal inflammation in mice. Consistently, deletion of Rnf20 or Rnf40 promoted IBD-associated gene expression programs, including deregulation of various IBD risk genes in these animals. Further analysis of murine IECs revealed that H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and VDR target genes is RNF20/40-dependent. Finally, these effects were confirmed in a subgroup of Crohn\u27s disease patients which displayed epigenetic and expression changes in RNF20/40-dependent gene signatures. Our findings reveal that loss of H2B monoubiquitination promotes intestinal inflammation via decreased VDR activity thereby identifying RNF20 and RNF40 as critical regulators of IBD

RNF40 regulates gene expression in an epigenetic context-dependent manner

Background Monoubiquitination of H2B (H2Bub1) is a largely enigmatic histone modification that has been linked to transcriptional elongation. Because of this association, it has been commonly assumed that H2Bub1 is an exclusively positively acting histone modification and that increased H2Bub1 occupancy correlates with increased gene expression. In contrast, depletion of the H2B ubiquitin ligases RNF20 or RNF40 alters the expression of only a subset of genes. Results Using conditional Rnf40 knockout mouse embryo fibroblasts, we show that genes occupied by low to moderate amounts of H2Bub1 are selectively regulated in response to Rnf40 deletion, whereas genes marked by high levels of H2Bub1 are mostly unaffected by Rnf40 loss. Furthermore, we find that decreased expression of RNF40-dependent genes is highly associated with widespread narrowing of H3K4me3 peaks. H2Bub1 promotes the broadening of H3K4me3 to increase transcriptional elongation, which together lead to increased tissue-specific gene transcription. Notably, genes upregulated following Rnf40 deletion, including Foxl2, are enriched for H3K27me3, which is decreased following Rnf40 deletion due to decreased expression of the Ezh2 gene. As a consequence, increased expression of some RNF40-“suppressed” genes is associated with enhancer activation via FOXL2. Conclusion Together these findings reveal the complexity and context-dependency whereby one histone modification can have divergent effects on gene transcription. Furthermore, we show that these effects are dependent upon the activity of other epigenetic regulatory proteins and histone modifications

Cell Type-Specific Induction of Inflammation-Associated Genes in Crohn’s Disease and Colorectal Cancer

Based on the rapid increase in incidence of inflammatory bowel disease (IBD), the identification of susceptibility genes and cell populations contributing to this condition is essential. Previous studies suggested multiple genes associated with the susceptibility of IBD; however, due to the analysis of whole-tissue samples, the contribution of individual cell populations remains widely unresolved. Single-cell RNA sequencing (scRNA-seq) provides the opportunity to identify underlying cellular populations. We determined the enrichment of Crohn’s disease (CD)-induced genes in a publicly available Crohn’s disease scRNA-seq dataset and detected the strongest induction of these genes in innate lymphoid cells (ILC1), highly activated T cells and dendritic cells, pericytes and activated fibroblasts, as well as epithelial cells. Notably, these genes were highly enriched in IBD-associated neoplasia, as well as sporadic colorectal cancer (CRC). Indeed, the same six cell populations displayed an upregulation of CD-induced genes in a CRC scRNA-seq dataset. Finally, after integrating and harmonizing the CD and CRC scRNA-seq data, we demonstrated that these six cell types display a gradual increase in gene expression levels from a healthy state to an inflammatory and tumorous state. Together, we identified cell populations that specifically upregulate CD-induced genes in CD and CRC patients and could, therefore, contribute to inflammation-associated tumor development

Effect of the lipoxygenase inhibitor baicalein on bone tissue and bone healing in ovariectomized rats

Abstract Background Osteoporosis is one of the world’s major medical burdens in the twenty-first century. Pharmaceutical intervention currently focusses on decelerating bone loss, but phytochemicals such as baicalein, which is a lipoxygenase inhibitor, may rescue bone loss. Studies evaluating the effect of baicalein in vivo are rare. Methods We administered baicalein to sixty-one three-month-old female Sprague-Dawley rats. They were divided into five groups, four of which were ovariectomized (OVX) and one non-ovariectomized (NON-OVX). Eight weeks after ovariectomy, bilateral tibial osteotomy with plate osteosynthesis was performed and bone formation quantified. Baicalein was administered subcutaneously using three doses (C1: 1 mg/kg BW; C2: 10 mg/kg BW; and C3: 100 mg/kg BW) eight weeks after ovariectomy for four weeks. Finally, femora and tibiae were collected. Biomechanical tests, micro-CT, ashing, histological and gene expression analyses were performed. Results Biomechanical properties were unchanged in tibiae and reduced in femora. In tibiae, C1 treatment enhanced callus density and cortical width and decreased callus area. In the C3 group, callus formation was reduced during the first 3 weeks after osteotomy, correlating to a higher mRNA expression of Osteocalcin, Tartrate-resistant acid phosphatase and Rankl. In femora, baicalein treatments did not alter bone parameters. Conclusions Baicalein enhanced callus density and cortical width but impaired early callus formation in tibiae. In femora, it diminished the biomechanical properties and calcium-to-phosphate ratio. Thus, it is not advisable to apply baicalein to treat early bone fractures. To determine the exact effects on bone healing, further studies in which baicalein treatments are started at different stages of healing are needed

The histone methyltransferase DOT1L is required for proper DNA damage response, DNA repair, and modulates chemotherapy responsiveness

Abstract Background Disruptor of telomeric silencing 1-like (DOT1L) is a non-SET domain containing methyltransferase known to catalyze mono-, di-, and tri-methylation of histone 3 on lysine 79 (H3K79me). DOT1L-mediated H3K79me has been implicated in chromatin-associated functions including gene transcription, heterochromatin formation, and DNA repair. Recent studies have uncovered a role for DOT1L in the initiation and progression of leukemia and other solid tumors. The development and availability of small molecule inhibitors of DOT1L may provide new and unique therapeutic options for certain types or subgroups of cancer. Methods In this study, we examined the role of DOT1L in DNA double-strand break (DSB) response and repair by depleting DOT1L using siRNA or inhibiting its methyltransferase activity using small molecule inhibitors in colorectal cancer cells. Cells were treated with different agents to induce DNA damage in DOT1L-depleted or -inhibited cells and analyzed for DNA repair efficiency and survival. Further, rectal cancer patient samples were analyzed for H3K79me3 levels in order to determine whether it may serve as a potential marker for personalized therapy. Results Our results indicate that DOT1L is required for a proper DNA damage response following DNA double-strand breaks by regulating the phosphorylation of the variant histone H2AX (γH2AX) and repair via homologous recombination (HR). Importantly, we show that small molecule inhibitors of DOT1L combined with chemotherapeutic agents that are used to treat colorectal cancers show additive effects. Furthermore, examination of H3K79me3 levels in rectal cancer patients demonstrates that lower levels correlate with a poorer prognosis. Conclusions In this study, we conclude that DOT1L plays an important role in an early DNA damage response and repair of DNA double-strand breaks via the HR pathway. Moreover, DOT1L inhibition leads to increased sensitivity to chemotherapeutic agents and PARP inhibition, which further highlights its potential clinical utility. Our results further suggest that H3K79me3 can be useful as a predictive and or prognostic marker for rectal cancer patients

Usp22 is an intracellular regulator of systemic emergency hematopoiesis

International audienceEmergency hematopoiesis is a concerted response aimed toward enhanced protection from infection, involving multiple cell types and developmental stages across the immune system. Despite its importance, the underlying molecular regulation remains poorly understood. The deubiquitinase USP22 regulates the levels of monoubiquitinated histone H2B (H2Bub1), which is associated with activation of interferon responses upon viral infection. Here, we show that in the absence of infection or inflammation, mice lacking Usp22 in all hematopoietic cells display profound systemic emergency hematopoiesis, evident by increased hematopoietic stem cell proliferation, myeloid bias, and extramedullary hematopoiesis. Functionally, loss of Usp22 results in elevated phagocytosis by neutrophilic granulocytes and enhanced innate protection against Listeria monocytogenes infection. At the molecular level, we found this state of emergency hematopoiesis associated with transcriptional signatures of myeloid priming, enhanced mitochondrial respiration, and innate and adaptive immunity and inflammation. Augmented expression of many inflammatory genes was linked to elevated locus-specific H2Bub1 levels. Collectively, these results demonstrate the existence of a tunable epigenetic state that promotes systemic emergency hematopoiesis in a cell-autonomous manner to enhance innate protection, identifying potential paths toward immune enhancement