Epistasis of Transcriptomes Reveals Synergism between Transcriptional Activators Hnf1α and Hnf4α

The transcription of individual genes is determined by combinatorial interactions between DNA–binding transcription factors. The current challenge is to understand how such combinatorial interactions regulate broad genetic programs that underlie cellular functions and disease. The transcription factors Hnf1α and Hnf4α control pancreatic islet β-cell function and growth, and mutations in their genes cause closely related forms of diabetes. We have now exploited genetic epistasis to examine how Hnf1α and Hnf4α functionally interact in pancreatic islets. Expression profiling in islets from either Hnf1a+/− or pancreas-specific Hnf4a mutant mice showed that the two transcription factors regulate a strikingly similar set of genes. We integrated expression and genomic binding studies and show that the shared transcriptional phenotype of these two mutant models is linked to common direct targets, rather than to known effects of Hnf1α on Hnf4a gene transcription. Epistasis analysis with transcriptomes of single- and double-mutant islets revealed that Hnf1α and Hnf4α regulate common targets synergistically. Hnf1α binding in Hnf4a-deficient islets was decreased in selected targets, but remained unaltered in others, thus suggesting that the mechanisms for synergistic regulation are gene-specific. These findings provide an in vivo strategy to study combinatorial gene regulation and reveal how Hnf1α and Hnf4α control a common islet-cell regulatory program that is defective in human monogenic diabetes

Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.

Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.LB is supported by an EMBO Postdoctoral fellowship (EMBO ALTF 794-2014). CH is supported by a Cambridge Stem Cell Institute Seed Fund award and the Herchel Smith Fund. BK is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society. MH is a Wellcome Trust Sir Henry Dale Fellow and is jointly funded by the Wellcome Trust and the Royal Society (104151/Z/14/Z).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nprot.2016.097

Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites

<p>Abstract</p> <p>Background</p> <p>The transcription factors peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key transcriptional regulators of adipocyte differentiation and function. We and others have previously shown that binding sites of these two transcription factors show a high degree of overlap and are associated with the majority of genes upregulated during differentiation of murine 3T3-L1 adipocytes.</p> <p>Results</p> <p>Here we have mapped all binding sites of C/EBPα and PPARγ in human SGBS adipocytes and compared these with the genome-wide profiles from mouse adipocytes to systematically investigate what biological features correlate with retention of sites in orthologous regions between mouse and human. Despite a limited interspecies retention of binding sites, several biological features make sites more likely to be retained. First, co-binding of PPARγ and C/EBPα in mouse is the most powerful predictor of retention of the corresponding binding sites in human. Second, vicinity to genes highly upregulated during adipogenesis significantly increases retention. Third, the presence of C/EBPα consensus sites correlate with retention of both factors, indicating that C/EBPα facilitates recruitment of PPARγ. Fourth, retention correlates with overall sequence conservation within the binding regions independent of C/EBPα and PPARγ sequence patterns, indicating that other transcription factors work cooperatively with these two key transcription factors.</p> <p>Conclusions</p> <p>This study provides a comprehensive and systematic analysis of what biological features impact on retention of binding sites between human and mouse. Specifically, we show that the binding of C/EBPα and PPARγ in adipocytes have evolved in a highly interdependent manner, indicating a significant cooperativity between these two transcription factors.</p

ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation

The work was supported by a grant A12008 from CR-UK (L. Dumartin, N.R. Lemoine and T. Crnogorac-Jurcevic)

Targeted Deficiency of the Transcriptional Activator Hnf1α Alters Subnuclear Positioning of Its Genomic Targets

DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary β-cells and hepatocytes freshly isolated from mice lacking Hnf1α, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3). We show that in Hnf1a−/− cells inactive endogenous Hnf1α-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1α-targets in Hnf1a−/− cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease

Genomics Meets Glycomics—The First GWAS Study of Human N-Glycome Identifies HNF1α as a Master Regulator of Plasma Protein Fucosylation

Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders

Epigenetic remodelling licences adult cholangiocytes for organoid formation and liver regeneration.

Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.RCUK Cancer Research UK ERC H2020 Wellcome Trus