Genome-Wide Location Analysis Reveals Distinct Transcriptional Circuitry by Paralogous Regulators Foxa1 and Foxa2

Gene duplication is a powerful driver of evolution. Newly duplicated genes acquire new roles that are relevant to fitness, or they will be lost over time. A potential path to functional relevance is mutation of the coding sequence leading to the acquisition of novel biochemical properties, as analyzed here for the highly homologous paralogs Foxa1 and Foxa2 transcriptional regulators. We determine by genome-wide location analysis (ChIP-Seq) that, although Foxa1 and Foxa2 share a large fraction of binding sites in the liver, each protein also occupies distinct regulatory elements in vivo. Foxa1-only sites are enriched for p53 binding sites and are frequently found near genes important to cell cycle regulation, while Foxa2-restricted sites show only a limited match to the forkhead consensus and are found in genes involved in steroid and lipid metabolism. Thus, Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the adult liver, ensuring the maintenance of both genes during evolution.Institute for Diabetes, Obesity and Metabolism. Diabetes Research Center (Functional Genomics Core P30-DK19525

Glucocorticoid Receptor-Dependent Gene Regulatory Networks

While the molecular mechanisms of glucocorticoid regulation of transcription have been studied in detail, the global networks regulated by the glucocorticoid receptor (GR) remain unknown. To address this question, we performed an orthogonal analysis to identify direct targets of the GR. First, we analyzed the expression profile of mouse livers in the presence or absence of exogenous glucocorticoid, resulting in over 1,300 differentially expressed genes. We then executed genome-wide location analysis on chromatin from the same livers, identifying more than 300 promoters that are bound by the GR. Intersecting the two lists yielded 53 genes whose expression is functionally dependent upon the ligand-bound GR. Further network and sequence analysis of the functional targets enabled us to suggest interactions between the GR and other transcription factors at specific target genes. Together, our results further our understanding of the GR and its targets, and provide the basis for more targeted glucocorticoid therapies

Hepatic metabolic regulatory networks of Foxa2

The winged helix transcription factor Foxa2 is an important regulator that plays a critical role in many tissues. Our group has found that Foxa2 facilitates maximal induction of hepatic genes in response to fasting, but the role of this transcription factor in the fed state is not yet clear. To determine the role of Foxa2 in the adult liver I used a functional genomics approach and a conditional knockout model, the Foxa2loxP/loxP Alfp.Cre mouse, where Foxa2 is deleted in the hepatocyte. Global location analysis ( ChIP-on-Chip ) of Foxa2 in the liver indicates that a cluster of categories with genes involved in lipid and steroid metabolism is over-represented in the set of targets bound by Foxa2 in vivo. Bile acids are oxidized derivatives of cholesterol produced by the liver to facilitate absorption of dietary lipids. Dysregulation of bile acid homeostasis can cause cholestatic liver disease. As suggested by the location analysis, deletion of Foxa2 in the hepatocyte leads to decreased transcription of genes encoding bile acid transporters on both the basolateral and canalicular membranes, resulting in intrahepatic cholestasis. Feeding Foxa2 mutants a cholic acid enriched diet results in harmful accretion of bile salts, ER stress, and liver injury. In addition to showing that Foxa2 is required for normal bile acid homeostasis in mice, I demonstrate that FOXA2 expression is severely reduced in livers of patients suffering from several cholestatic syndromes, suggesting that decreased FOXA2 levels could worsen the injury. To investigate why Foxa2-deficient mice exhibit a more dramatic phenotype on a cholic acid diet than on normal chow, in the second part of my dissertation I examine how Foxa2 regulates its targets in a cholic acid-dependent manner. I focus on direct targets, genes which were bound and whose expression was downregulated in Foxa2 mutants. Multiple cell-to-cell signaling pathways crucial for response of the liver to acute injury are suppressed in Foxa2 loxP/loxPAlfp.Cre mice, suggesting that the deletion of Foxa2 in the hepatocyte affects the liver on a global scale. I demonstrate that distinct feed-forward regulatory loops regulate Foxa2-dependent targets in a cholic acid dependent or independent manner and show that Foxa2 interacts with different partners to attain gene expression responses suitable for each physiological state

Hepatic metabolic regulatory networks of Foxa2

The winged helix transcription factor Foxa2 is an important regulator that plays a critical role in many tissues. Our group has found that Foxa2 facilitates maximal induction of hepatic genes in response to fasting, but the role of this transcription factor in the fed state is not yet clear. To determine the role of Foxa2 in the adult liver I used a functional genomics approach and a conditional knockout model, the Foxa2loxP/loxP Alfp.Cre mouse, where Foxa2 is deleted in the hepatocyte. Global location analysis ( ChIP-on-Chip ) of Foxa2 in the liver indicates that a cluster of categories with genes involved in lipid and steroid metabolism is over-represented in the set of targets bound by Foxa2 in vivo. Bile acids are oxidized derivatives of cholesterol produced by the liver to facilitate absorption of dietary lipids. Dysregulation of bile acid homeostasis can cause cholestatic liver disease. As suggested by the location analysis, deletion of Foxa2 in the hepatocyte leads to decreased transcription of genes encoding bile acid transporters on both the basolateral and canalicular membranes, resulting in intrahepatic cholestasis. Feeding Foxa2 mutants a cholic acid enriched diet results in harmful accretion of bile salts, ER stress, and liver injury. In addition to showing that Foxa2 is required for normal bile acid homeostasis in mice, I demonstrate that FOXA2 expression is severely reduced in livers of patients suffering from several cholestatic syndromes, suggesting that decreased FOXA2 levels could worsen the injury. To investigate why Foxa2-deficient mice exhibit a more dramatic phenotype on a cholic acid diet than on normal chow, in the second part of my dissertation I examine how Foxa2 regulates its targets in a cholic acid-dependent manner. I focus on direct targets, genes which were bound and whose expression was downregulated in Foxa2 mutants. Multiple cell-to-cell signaling pathways crucial for response of the liver to acute injury are suppressed in Foxa2 loxP/loxPAlfp.Cre mice, suggesting that the deletion of Foxa2 in the hepatocyte affects the liver on a global scale. I demonstrate that distinct feed-forward regulatory loops regulate Foxa2-dependent targets in a cholic acid dependent or independent manner and show that Foxa2 interacts with different partners to attain gene expression responses suitable for each physiological state

Changes in Nucleosome Occupancy Associated with Metabolic Alterations in Aged Mammalian Liver

Aging is accompanied by physiological impairments, which, in insulin-responsive tissues, including the liver, predispose individuals to metabolic disease. However, the molecular mechanisms underlying these changes remain largely unknown. Here, we analyze genome-wide profiles of RNA and chromatin organization in the liver of young (3 months) and old (21 months) mice. Transcriptional changes suggest that derepression of the nuclear receptors PPARα, PPARγ, and LXRα in aged mouse liver leads to activation of targets regulating lipid synthesis and storage, whereas age-dependent changes in nucleosome occupancy are associated with binding sites for both known regulators (forkhead factors and nuclear receptors) and candidates associated with nuclear lamina (Hdac3 and Srf) implicated to govern metabolic function of aging liver. Winged-helix transcription factor Foxa2 and nuclear receptor corepressor Hdac3 exhibit a reciprocal binding pattern at PPARα targets contributing to gene expression changes that lead to steatosis in aged liver

Impaired male fertility and atrophy of seminiferous tubules caused by haploinsufficiency for Foxa3

AbstractFoxa1, 2 and 3 (formerly HNF-3α, -β and -γ) constitute a sub-family of winged helix transcription factors with multiple roles in mammalian organ development. While all three Foxa mRNAs are present in endoderm derivatives including liver and pancreas, only Foxa3 is expressed in the testis. Here we demonstrate by genetic lineage tracing that Foxa3 is expressed in postmeiotic germ and interstitial Leydig cells. The germinal epithelium of Foxa3-deficient testes is characterized by a loss of germ cells secondary to an increase in germ cell apoptosis that ultimately leads to a Sertoli cell-only syndrome. Remarkably, not only the Foxa3−/− mice but also Foxa3+/− mice exhibited loss of germ cells. This cellular phenotype caused significantly reduced fertility and testis weight of both Foxa3−/− and Foxa3+/− mice. Using microarray analysis, we found a dramatic downregulation of the zinc finger protein 93 and the testicular tumor-associated paraneoplastic Ma antigen (PNMA) and increased expression of a number of genes including zinc finger protein 94 and several kallikrein 1-related peptidases which could account for at least part of the observed phenotype. In summary, we have identified Foxa3 as a transcriptional regulator with a dominant phenotype in germ cell maintenance and suggest FOXA3 as a potential candidate gene for subfertility in man

Foxa1-Specific Targets Are Enriched for p53 Binding Sites.

<p>(A) A set of Foxa1-only targets was defined as those sequences that had one or fewer sequence tags Foxa2 ChIP-Seq data set (yellow circle). Motif analysis of these sequences found in a weaker <i>forkhead</i> consensus. In addition to the <i>forkhead</i> consensus, several other motifs appeared in this set of sequences. The first (ACATG and ATG repeats with a spacer in the middle) comprises parts of the positional weight matrix (PWM) for p53. The second closely resembles the PWM of Klf12, also known as repressor of AP-2alpha (Tfap2a). Orthogonal analysis of sites bound only bound by Foxa1 (yellow circle) and gene expression changes in livers of Foxa1-deficient mice (light purple circle) shows that thirty-seven are direct targets of Foxa1. (B) Verification of Foxa1-only targets by qPCR. Filled bars, ChIP of liver chromatin with an anti-Foxa1 antibody, open bars, ChIP with an anti-Foxa2 antibody. Three negative control regions (Nc1, Nc2, and Nc3) with a low amount of reads are included Binding is expressed as enrichment of the PCR amplicon relative to input DNA in liver chromatin. (C) Binding of p53 to <i>cis</i>-regulatory elements of its previously identified targets (positive controls), the alpha-fetoprotein (AFP) and TNF receptor superfamily <i>Fas</i> genes and twelve additional Foxa1-only targets, including cell-cycle associated <i>Aatf</i> and <i>Zwint</i> genes in both wildtype (black bars) and Foxa1 mutant livers (white bars) (left panel) by ChIP of liver chromatin followed by qPCR. None of Foxa2-only sites tested were bound by p53 in wiltype livers (right panel) (D) Quantitative RT- PCR analysis for mRNA of Zwint, Zwint mRNA levels are significantly downregulated in livers of Foxa1 mutant mice by 30%, Values are represented as means plus standard error. P values were determined by Student's <i>t</i> test. * p-value<0.05 (E) Foxa1 targets are enriched in genes involved in cell differentiation, morphogenesis, movement, and cellular cycle. (F) A regulatory feed-forward loop involving Foxa1. In the set of Foxa1-specific targets, Foxa1 binds to regulatory elements of the nuclear receptor Esr2 (estrogen receptor), its co-activator Smyd3, and target gene Pgr (progesterone receptor). (G) Foxa1 only sites are more distributed more broadly surrounding TSS (+/−100 Kb) than dual Foxa1/Foxa2 targets (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002770#pgen-1002770-g004" target="_blank">Figure 4E</a>).</p

Dual Foxa1/Foxa2 Sites Contain a Perfect <i>Forkhead</i> Motif.

<p>(A) All sequences bound by both Foxa1 and Foxa2 in the liver contain at least one perfect match to the Foxa consensus (a 7-mer of 4 possible sequences, with variation in the second and fifth nucleotide, (T[A/G]TT[G/T]AC)), and often an additional Foxa-like motif, containing one or two degenerate nucleotides. Surprisingly, these sites show additional preferences for specific bases at positions flanking the <i>forkhead</i> motif, with a “C” occurring frequently one nucleotide upstream, and a “T” enriched immediately downstream of the core consensus sequence. (B) Confirmation of several common Foxa1/Foxa2 targets by quantitative RT-PCR and three negative control regions (with low amount of reads, Nc1, Nc2, and Nc3). Binding is expressed as enrichment of immunoprecipitated DNA relative to input DNA in liver chromatin. (C) Functional categories over-represented in the genes bound by both Foxa1 and Foxa2 include ‘embryonic development’, ‘regulation of transcription’, and ‘lipid metabolism’. Biological pathways enriched for Foxa1/Foxa2 targets are ‘metabolism of xenobiotics’, ‘vesicular transport’ and ‘receptor tyrosine kinase signaling’. (D) Foxa1 and Foxa2 bind to four distinct intronic regions of <i>Fto</i> (fat mass and obesity associated) gene, which is associated with the risk of diabetes. (E) Histogram of <i>cis</i>-regulatory elements bound by both Foxa paralogs shows a normal distribution near transcription start sites (TSS), with most sites within ten kilobases (Kb) from TSS.</p

Evolution of Vertebrate Foxa Paralogs and Functional Redundancy in the Fetal Liver.

<p>(A) Phylogenetic tree of the Foxa subfamily of transcriptional regulators. The putative Foxa4 gene was lost in vertebrates during evolution. (B) Sequence alignment of mouse Foxa1 and Foxa2 proteins by ClustalW2 algorithm. The winged-helix DNA binding domain is highlighted in blue. ‘*’, identical residues in all sequences, ‘:’ highly conserved amino acids; ‘.’ weakly conserved amino acids. (C) Venn diagram of the number of genes that are differentially expressed in fetal livers of Foxa1 and Foxa2 single mutants, as well as the double mutant (shaded in blue) on embryonic day 18.5. The number of genes dependent on each single factor is small compared to the number of genes that are differentially expressed in the double mutant.</p