HNF4A and HNF1A exhibit tissue specific target gene regulation in pancreatic beta cells and hepatocytes.

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A, we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3, HAAO, HNF1A, MAP3K11) and previously unidentified (ABCD3, CDKN2AIP, USH1C, VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs, the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1, GAD2 and HOPX gene expression, potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall, our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function, and set up a framework for gene discovery and functional validation.

Chromatin Immunoprecipitation in Human Pluripotent Stem Cell-Derived 3D Organoids to Analyze DNA-Protein Interactions.

Chromatin immunoprecipitation (ChIP) is a technique that has been widely used to interrogate DNA-protein interactions in cells. In recent years, human pluripotent stem cell (hPSC)-derived 3D organoids have emerged as a powerful model to understand human development and diseases. Performing ChIP in hPSC-derived 3D organoids is a useful approach to dissect the roles of transcription factors or co-factors and to understand the epigenetic landscape in human development and diseases. However, performing ChIP in 3D organoids is more challenging than monolayer cultures, and an optimized protocol is needed for interpretable data. Hence, in this chapter, we describe in detail a protocol for performing ChIP in hPSC-derived islet-like cells as an example, from organoid harvest to ChIP-qPCR data analysis. This chapter also highlights potential pitfalls and provides recommendations for troubleshooting.

Factors associated with false-positive mammography at first screen in an Asian population.

INTRODUCTION: False-positive recall is an issue in national screening programmes. The aim of this study is to investigate the recall rate at first screen and to identify potential predictors of false-positive recall in a multi-ethnic Asian population-based breast cancer screening programme. METHODS: Women aged 50-64 years attending screening mammography for the first time (n = 25,318) were included in this study. The associations between potential predictors (sociodemographic, lifestyle and reproductive) and false-positive recall were evaluated using multivariable logistic regression models. RESULTS: The recall rate was 7.6% (n = 1,923), of which with 93.8% were false-positive. Factors independently associated with higher false-positive recall included Indian ethnicity (odds ratio [95% confidence interval]: 1.52 [1.25 to 1.84]), premenopause (1.23 [1.04 to 1.44]), nulliparity (1.85 [1.57 to 2.17]), recent breast symptoms (1.72 [1.31 to 2.23]) and history of breast lump excision (1.87 [1.53 to 2.26]). Factors associated with lower risk of false-positive recall included older age at screen (0.84 [0.73 to 0.97]) and use of oral contraceptives (0.87 [0.78 to 0.97]). After further adjustment of percent mammographic density, associations with older age at screening (0.97 [0.84 to 1.11]) and menopausal status (1.12 [0.95 to 1.32]) were attenuated and no longer significant. CONCLUSION: For every breast cancer identified, 15 women without cancer were subjected to further testing. Efforts to educate Asian women on what it means to be recalled will be useful in reducing unnecessary stress and anxiety.

Intestinal microbiota regulate xenobiotic metabolism in the liver.

BACKGROUND: The liver is the central organ for xenobiotic metabolism (XM) and is regulated by nuclear receptors such as CAR and PXR, which control the metabolism of drugs. Here we report that gut microbiota influences liver gene expression and alters xenobiotic metabolism in animals exposed to barbiturates. PRINCIPAL FINDINGS: By comparing hepatic gene expression on microarrays from germfree (GF) and conventionally-raised mice (SPF), we identified a cluster of 112 differentially expressed target genes predominantly connected to xenobiotic metabolism and pathways inhibiting RXR function. These findings were functionally validated by exposing GF and SPF mice to pentobarbital which confirmed that xenobiotic metabolism in GF mice is significantly more efficient (shorter time of anesthesia) when compared to the SPF group. CONCLUSION: Our data demonstrate that gut microbiota modulates hepatic gene expression and function by altering its xenobiotic response to drugs without direct contact with the liver.

Gut flora, Toll-like receptors and nuclear receptors: a tripartite communication that tunes innate immunity in large intestine.

Separating the large intestine from gut flora is a robust layer of epithelial cells. This barrier is armed with an array of recognizing receptors that collectively set the host innate response. Here, we use nuclear receptors (NRs) and Toll-like receptors (TLRs), suggested to act as second messengers in the communication between microorganisms and epithelial cells, as probes to assess the impact of gut flora on innate immunity in germ-free (GF) mice. Using quantitative real-time polymerase chain reaction analyses, we show that 37/49 NRs are expressed in colonic cells of GF mice. Of these, 5 can be modulated by resident flora: LXRalpha, RORgamma and CAR show reduced expression and Nur77 and GCNF display elevated expression in conventionally raised mice compared with GF. Moreover, increased expression levels of TLR-2 and TLR-5 are observed in specific pathogen-free (SPF) mice compared with GF mice, and CAR expression is connected to the TLR-2 signalling pathway. Infections of GF or SPF mice with Yersinia pseudotuberculosis, show that GF intestinal epithelial cells fail to respond, except for CAR, which is downregulated. In contrast, SPF epithelial cells show a downregulation of all the NRs except CAR, which appears to be unaffected. Our findings indicate that gut flora contributes to the development of an intact barrier function.