Mechanism underlying synergic activation of Tyrosinase promoter by MITF and IRF4

Background: The transcription factor interferon regulatory factor 4 (IRF4) was identified to be involved in human pigmentation by genome-wide association studies (GWASs). The rs12203592-[T/C], which is located in intron 4 of IRF4, shows the strongest link to these pigmentation phenotypes including freckling, sun sensitivity, eye and hair color. Previous studies indicated a functional cooperation of IRF4 with Microphthalmia-associated transcription factor (MITF), a causing gene of Waardenburg syndrome (WS), to synergistically trans-activate Tyrosinase (TYR). However, the underlying mechanism is still unknown. Methods: To investigate the importance of DNA binding in the synergic effect of IRF4. Reporter plasmids with mutant TYR promoters was generated to locate the IRF4 DNA binding sites in the Tyrosinase minimal promoter. By building MITF and IRF4 truncated mutations plasmids, the necessary regions of the synergy functions of these two proteins were also located. Results: The cooperative effect between MITF and IRF4 was specific for TYR promoter. The DNA-binding of IRF4 was critical for the synergic function. IRF4 DNA binding sites in TYR promoter were identified. The Trans-activation domains in IRF4 (aa134-207, aa300-420) were both important for the synergic function, whereas the auto-mask domain (aa207-300) appeared to mask the synergic effect. Mutational analysis in MITF indicated that both DNA-binding and transcriptional activation domains were both required for this synergic effect. Conclusions: Here we showed that IRF4 potently synergized with MITF to activate the TYR promoter, which was dependent on DNA binding of IRF4. The synergic domains in both IRF4 and MITF were identified by mutational analysis. This identification of IRF4 as a partner for MITF in regulation of TYR may provide an important molecular function for IRF4 in the genesis of melanocytes and the pathogenic mechanism in WS

Genome-wide identification of regulatory elements in Sertoli cells

A current goal of molecular biology is to identify transcriptional networks that regulate cell differentiation. However, identifying functional gene regulatory elements has been challenging in the context of developing tissues where material is limited and cell types are mixed. To identify regulatory sites during sex determination, we subjected Sertoli cells from mouse fetal testes to DNaseI-seq and ChIP-seq for H3K27ac. DNaseI-seq identified putative regulatory sites around genes enriched in Sertoli and pregranulosa cells; however, active enhancers marked by H3K27ac were enriched proximal to only Sertoli-enriched genes. Sequence analysis identified putative binding sites of known and novel transcription factors likely controlling Sertoli cell differentiation. As a validation of this approach, we identified a novel Sertoli cell enhancer upstream of Wt1, and used it to drive expression of a transgenic reporter in Sertoli cells. This work furthers our understanding of the complex genetic network that underlies sex determination and identifies regions that potentially harbor non-coding mutations underlying disorders of sexual development

Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha

Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota

Exploring the safety, effectiveness, and cost-effectiveness of a Chinese patent medicine (Fufang E’jiao syrup) for alleviating cancer-related fatigue : a protocol for a randomized, double-blinded, placebo-controlled, multicenter trial

Objective: To provide higher level evidence on the benefits of a Chinese patent medicine (CPM) (Fufang E’jiao Syrup, FFEJS) for alleviating cancer-related fatigue (CRF), this article describes a protocol for a randomized controlled trial. Methods/design: We designed a double-blind, placebo-controlled stratified permuted block randomization clinical trial on CRF among 3 types of cancer in China. Participants will be equally allocated to FFEJS group or placebo group according to the randomization sequence and the hospitals they were enrolled at. Each patient will receive 20 ml of either the study formula FFEJS or a placebo formula, 3 times a day for 6 weeks. The follow-up period will be another 4 weeks for safety evaluation. The primary outcome is the difference in improvement of fatigue as measured with the Revised Piper Fatigue Scale-Chinese Version (RPFS-CV). Secondary outcomes include change in fatigue (measured by routine blood panel and hormones in peripheral blood) and QoL (measured by Edmonton symptom assessment scale and Functional Assessment of Cancer Therapy). Patient safety will be measured by liver, renal or cardiac damage, and the risk of FFEJS having a tumor promotion and progression effect will be monitored throughout this study. Cost-effectiveness will also be evaluated mainly by incremental cost per each quality-adjusted life year gained. Discussion: This article describes the study design of a CPM for CRF in patients with advanced cancer through exploring the effectiveness, safety, and cost-effectiveness of FFEJS. Trial registration: ClinicalTrials.gov, NCT04147312. Registered on 1 Sep 2019

Predicting pneumonia during hospitalization in flail chest patients using machine learning approaches

ObjectivePneumonia is a common pulmonary complication of flail chest, causing high morbidity and mortality rates in affected patients. The existing methods for identifying pneumonia have low accuracy, and their use may delay antimicrobial therapy. However, machine learning can be combined with electronic medical record systems to identify information and assist in quick clinical decision-making. Our study aimed to develop a novel machine-learning model to predict pneumonia risk in flail chest patients.MethodsFrom January 2011 to December 2021, the electronic medical records of 169 adult patients with flail chest at a tertiary teaching hospital in an urban level I Trauma Centre in Chongqing were retrospectively analysed. Then, the patients were randomly divided into training and test sets at a ratio of 7:3. Using the Fisher score, the best subset of variables was chosen. The performance of the seven models was evaluated by computing the area under the receiver operating characteristic curve (AUC). The output of the XGBoost model was shown using the Shapley Additive exPlanation (SHAP) method.ResultsOf 802 multiple rib fracture patients, 169 flail chest patients were eventually included, and 86 (50.80%) were diagnosed with pneumonia. The XGBoost model performed the best among all seven machine-learning models. The AUC of the XGBoost model was 0.895 (sensitivity: 84.3%; specificity: 80.0%).Pneumonia in flail chest patients was associated with several features: systolic blood pressure, pH value, blood transfusion, and ISS.ConclusionOur study demonstrated that the XGBoost model with 32 variables had high reliability in assessing risk indicators of pneumonia in flail chest patients. The SHAP method can identify vital pneumonia risk factors, making the XGBoost model's output clinically meaningful

Epigenomic Comparison Reveals Activation of “Seed” Enhancers during Transition from Naive to Primed Pluripotency

SummaryNaive mouse embryonic stem cells (mESCs) and primed epiblast stem cells (mEpiSCs) represent successive snapshots of pluripotency during embryogenesis. Using transcriptomic and epigenomic mapping we show that a small fraction of transcripts are differentially expressed between mESCs and mEpiSCs and that these genes show expected changes in chromatin at their promoters and enhancers. Unexpectedly, the cis-regulatory circuitry of genes that are expressed at identical levels between these cell states also differs dramatically. In mESCs, these genes are associated with dominant proximal enhancers and dormant distal enhancers, which we term seed enhancers. In mEpiSCs, the naive-dominant enhancers are lost, and the seed enhancers take up primary transcriptional control. Seed enhancers have increased sequence conservation and show preferential usage in downstream somatic tissues, often expanding into super enhancers. We propose that seed enhancers ensure proper enhancer utilization and transcriptional fidelity as mammalian cells transition from naive pluripotency to a somatic regulatory program

A dynamic H3K27ac signature identifies VEGFA-stimulated endothelial enhancers and requires EP300 activity

Histone modifications are now well-established mediators of transcriptional programs that distinguish cell states. However, the kinetics of histone modification and their role in mediating rapid, signal-responsive gene expression changes has been little studied on a genome-wide scale. Vascular endothelial growth factor A (VEGFA), a major regulator of angiogenesis, triggers changes in transcriptional activity of human umbilical vein endothelial cells (HUVECs). Here, we used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to measure genome-wide changes in histone H3 acetylation at lysine 27 (H3K27ac), a marker of active enhancers, in unstimulated HUVECs and HUVECs stimulated with VEGFA for 1, 4, and 12 h. We show that sites with the greatest H3K27ac change upon stimulation were associated tightly with EP300, a histone acetyltransferase. Using the variation of H3K27ac as a novel epigenetic signature, we identified transcriptional regulatory elements that are functionally linked to angiogenesis, participate in rapid VEGFA-stimulated changes in chromatin conformation, and mediate VEGFA-induced transcriptional responses. Dynamic H3K27ac deposition and associated changes in chromatin conformation required EP300 activity instead of altered nucleosome occupancy or changes in DNase I hypersensitivity. EP300 activity was also required for a subset of dynamic H3K27ac sites to loop into proximity of promoters. Our study identified thousands of endothelial, VEGFA-responsive enhancers, demonstrating that an epigenetic signature based on the variation of a chromatin feature is a productive approach to define signal-responsive genomic elements. Further, our study implicates global epigenetic modifications in rapid, signal-responsive transcriptional regulation

RANKL/RANK promotes the migration of gastric cancer cells by interacting with EGFR

BACKGROUND: The incidence and mortality rates of gastric cancer (GC) rank in top five among all malignant tumors. Chemokines and their receptor-signaling pathways reportedly play key roles in the metastasis of malignant tumor cells. Receptor activator of nuclear factor κB ligand (RANKL) is a member of the tumor necrosis factor family, with strong chemokine-like effects. Some studies have pointed out that the RANKL/RANK pathway is vital for the metastasis of cancer cells, but the specific mechanisms in GC remain poorly understood. RESULTS: This study reports original findings in cell culture models and in patients with GC. Flow cytometry and western blotting analyses showed that RANK was expressed in BGC-823 and SGC-7901 cells in particular. Chemotaxis experiments and wound healing assay suggested that RANKL spurred the migration of GC cells. This effect was offset by osteoprotegerin (OPG), a decoy receptor for RANKL. RANKL contributed to the activation of human epidermal growth factor receptor (HER) family pathways. The lipid raft core protein, caveolin 1 (Cav-1), interacted with both RANK and human epidermal growth factor receptor-1(EGFR). Knockdown of Cav-1 blocked the activation of EGFR and cell migration induced by RANKL. Moreover, RANK-positive GC patients who displayed higher levels of EGFR expression had poor overall survival. CONCLUSIONS: In summary, we confirmed that with the promotion of RANKL, RANK and EGFR can form complexes with the lipid raft core protein Cav-1, which together promote GC cell migration. The formation of the RANK-Cav-1-EGFR complex provides a novel mechanism for the metastasis of GC. These observations warrant confirmation in independent studies, in vitro and in vivo. They also inform future drug target discovery research and innovation in the treatment of GC progression