Transcription Factor HNF1 Research Articles

Multiple roles and regulatory mechanisms of the transcription factor HNF4 in the intestine.

Hepatocyte nuclear factor 4-alpha (HNF4α) drives a complex array of transcriptional programs across multiple organs. Beyond its previously documented function in the liver, HNF4α has crucial roles in the kidney, intestine, and pancreas. In the intestine, a multitude of functions have been attributed to HNF4 and its accessory transcription factors, including but not limited to, intestinal maturation, differentiation, regeneration, and stem cell renewal. Functional redundancy between HNF4α and its intestine-restricted paralog HNF4γ, and co-regulation with other transcription factors drive these functions. Dysregulated expression of HNF4 results in a wide range of disease manifestations, including the development of a chronic inflammatory state in the intestine. In this review, we focus on the multiple molecular mechanisms of HNF4 in the intestine and explore translational opportunities. We aim to introduce new perspectives in understanding intestinal genetics and the complexity of gastrointestinal disorders through the lens of HNF4 transcription factors.

Comparative analysis of nutritional and transcriptional regulation of hacd1 in large yellow croaker (Larimichthys crocea) and rainbow trout (Oncorhynchus mykiss).

3-hydroxyacyl-CoA dehydratases 1 (Hacd1) is a critical enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. The difference in expression of hacd1 might account for the stronger capacity of LC-PUFA biosynthesis in freshwater fish than in marine fish, but little is known about fish hacd1. Therefore, this study compared the responses of large yellow croaker and rainbow trout hacd1 to different oil sources or fatty acids, and also examined transcriptional regulation of this gene. In this study, hacd1 was highly expressed in the liver of large yellow croaker and rainbow trout, which is the main organ for LC-PUFA biosynthesis. Therefore, we cloned the hacd1 coding sequence, with a phylogenetic analysis showing that this gene is evolutionarily conserved. Its localization to the endoplasmic reticulum (ER), likely also indicates a conserved structure and function. The expression of hacd1 in the liver was significantly decreased after the substitution of soybean oil (SO) for fish oil but was not significantly affected after palm oil (PO) substitution. Linoleic acid (LA) incubation significantly promoted hacd1 expression in primary hepatocytes of large yellow croaker and eicosapentaenoic acid (EPA) incubation significantly promoted hacd1 expression in primary hepatocytes of rainbow trout. Transcription factors STAT4, C/EBPα, C/EBPβ, HNF1, HSF3 and FOXP3 were identified in both large yellow croaker and rainbow trout. HNF1 had a stronger activation effect in rainbow trout than in large yellow croaker. FOXP3 inhibited hacd1 promoter activity in large yellow croaker but had no effect in rainbow trout. Therefore, the differences between HNF1 and FOXP3 affected the expression of hacd1 in the liver thus being responsible for the high capacity of LC-PUFA biosynthesis in rainbow trout.

Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia

Enterotoxigenic E. coli (ETEC) produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and commonly cause diarrhea in resource-poor regions. ETEC have been linked repeatedly to sequelae in children including enteropathy, malnutrition, and growth impairment. Although cellular actions of ETEC enterotoxins leading to diarrhea are well-established, their contributions to sequelae remain unclear. LT increases cellular cAMP to activate protein kinase A (PKA) that phosphorylates ion channels driving intestinal export of salt and water resulting in diarrhea. As PKA also modulates transcription of many genes, we interrogated transcriptional profiles of LT-treated intestinal epithelia. Here we show that LT significantly alters intestinal epithelial gene expression directing biogenesis of the brush border, the major site for nutrient absorption, suppresses transcription factors HNF4 and SMAD4 critical to enterocyte differentiation, and profoundly disrupts microvillus architecture and essential nutrient transport. In addition, ETEC-challenged neonatal mice exhibit substantial brush border derangement that is prevented by maternal vaccination with LT. Finally, mice repeatedly challenged with toxigenic ETEC exhibit impaired growth recapitulating the multiplicative impact of recurring ETEC infections in children. These findings highlight impacts of ETEC enterotoxins beyond acute diarrheal illness and may inform approaches to prevent major sequelae of these common infections including malnutrition that impact millions of children.

Invivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient invivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression invivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

GATA binding protein 4 promotes the expression and transcription of hepatitis B virus by facilitating hepatocyte nuclear factor 4 alpha in vitro

BackgroundGATA binding protein 4 (GATA4) has been reported as a potential target of gene therapy for hepatocellular carcinoma (HCC). It is well known that the main cause of HCC is the chronic infection of hepatitis B virus (HBV). However, whether the effect of GATA4 on HBV has not yet been reported.MethodsIn this study, the regulation of GATA4 on HBV was analyzed in vitro. In turn, the effect of HBV on GATA4 was also observed in vitro, in vivo, and clinical HCC patients. Subsequently, we analyzed whether the effect of GATA4 on HBV was related to hepatocyte nuclear factor 4 alpha (HNF4α) in vitro.ResultsThe results showed that GATA4 significantly promoted the secretion of HBV surface antigen (HBsAg) and HBV e antigen in the cell culture medium, improved the replication of HBV genomic DNA, and increased the level of HBV 3.5 kb pre-genomic RNA and HBV total RNA (P < 0.05). Moreover, it was showed that HBV had no significant effect on GATA4 in vitro and in vivo (P > 0.05). At the same time, GATA4 expression was decreased in 78.9% (15/19) of HCC patients regardless of the HBV and HBsAg status. Among them, there were 76.9% (10/13) in HBV-associated patients with HCC (HBV-HCC), and 83.3% (5/6) in non-HBV-HCC patients. In addition, the expression of HNF4α was also up-regulated or down-regulated accordingly when stimulating or interfering with the expression of GATA4. Furthermore, stimulating the expression of HNF4α could only alleviate the HBsAg level and HBV transcription levels, but had no significant effect on GATA4.ConclusionsIn summary, this study found that GATA4 has a positive effect on HBV, and the potential pathway may be related to another transcription factor HNF4α that regulates HBV.

CBP/p300 HAT maintains the gene network critical for \u03b2 cell identity and functional maturity

Loss of β cell identity and functional immaturity are thought to be involved in β cell failure in type 2 diabetes. CREB-binding protein (CBP) and its paralogue p300 act as multifunctional transcriptional co-activators and histone acetyltransferases (HAT) with extensive biological functions. However, whether the regulatory role of CBP/p300 in islet β cell function depends on the HAT activity remains uncertain. In this current study, A-485, a selective inhibitor of CBP/p300 HAT activity, greatly impaired glucose-stimulated insulin secretion from rat islets in vitro and in vivo. RNA-sequencing analysis showed a comprehensive downregulation of β cell and α cell identity genes in A-485-treated islets, without upregulation of dedifferentiation markers and derepression of disallowed genes. A-485 treatment decreased the expressions of genes involved in glucose sensing, not in glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation. In the islets of prediabetic db/db mice, CBP/p300 displayed a significant decrease with key genes for β cell function. The deacetylation of histone H3K27 as well as the transcription factors Hnf1α and Foxo1 was involved in CBP/p300 HAT inactivation-repressed expressions of β cell identity and functional genes. These findings highlight the dominant role of CBP/p300 HAT in the maintenance of β cell identity by governing transcription network.

Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23-32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114-E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263-273 (2017)]. We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.

GSK‑3&amp;beta; mediates the effects of HNF‑1&amp;beta; overexpression in ovarian clear cell carcinoma

Deubiquitinase USP28 is a target gene of the transcription factor HNF1 homeobox β (HNF-1β), which promotes the survival of ovarian clear cell carcinoma (OCCC) cell lines. However, the pharmacological inhibition of HNF-1β can cause several adverse effects as it is abundantly expressed in numerous organ systems, including the kidney, liver, pancreas and digestive tract. Therefore, small interfering RNA (siRNA) screening was performed in the current study to identify other potential downstream targets of the HNF-1β-mediated pathway. The results revealed that glycogen synthase kinase-3β (GSK-3β) may be a potential downstream target affecting cell viability. To further clarify the effects of GSK-3β, two human OCCC cell lines, TOV-21G (HNF-1β overexpressing line) and ES2 (HNF-1β negative) were transfected with siRNA targeting GSK-3β or control vectors. Loss-of-function studies using RNAi-mediated gene silencing indicated that HNF-1β facilitated GSK-3β expression, resulting in the loss of phosphorylated nuclear factor-κB (p-NFκB) and the reduction of TOV-21G cell proliferation. The cell proliferation assay also revealed that GSK-3β inhibitors rescued the effects of HNF-1β silencing on cell viability in a dose-dependent manner. Furthermore, the GSK-3β inhibitor, AR-A014418, effectively inhibited tumor cell proliferation in a xenograft mouse model. In conclusion and to the best of our knowledge, the current study was the first to determine that GSK-3β is a target gene of HNF-1β. In addition, the results of the present study revealed the novel HNF-1β-GSK-3β-p-NFκB pathway, occurring in response to DNA damage. Targeting this pathway may therefore represent a putative, novel, anticancer strategy in patients with OCCC.

Transcriptional Regulation of the Natural Cytotoxicity Receptor NKp44 Gene in Human NK Cell Leukemia

The natural cytotoxicity receptor NKp44 is an important regulator of NK cell function. We have previously reported that NKp44 has binding affinity to heparin and the transferrin containing multimeric sialyl Lewis X produced by human hepatoma HepG2 cells (HepTF) is more highly expressed than the other natural cytotoxicity receptors. To further understand the differences between natural cytotoxicity receptors, we sought to understand the gene transcriptional regulation of NKp44 in KHYG-1 human NK leukemia cells. 5′-rapid amplification of cDNA ends revealed the location of the NKp44 transcription initiation sites allowing identification of the 5′ untranscribed region. The result of the dual luciferase assay suggested that regulation of NKp44 gene expression is affected by the -1963 to -1599 nt region and the -352 to -231 nt region relative to the transcription initiation sites. The -410 to +1 nt region contained potential binding sites for the transcription factors AP-1, Oct-1, HNF-4, and Pax-4. These results suggest that defined regions in the 5′-flanking region of NKp44 gene is important for NKp44 transcription and regulation.

SUN-106 Loss of Coordination in the Expression of Hepatic Lipase and Endoplasmic Reticulum Stress Genes in Steatosis

The retention of cellular homeostasis is essential for the efficient execution of all biological processes which is reflected to the coordinated expression of genes that belong to same transcriptional networks. During pathology, homeostasis is abolished and cells read just their transcriptional program in a manner according to which several transcripts are being up- or down-regulated. Conventionally, genes exhibiting the highest overexpression or downregulation are considered as the most relevant for disease development. Despite this aberrant expression, coordination of individual genes and certain transcriptional networks is likely retained unless this disturbance of concerted expression is linked to disease development. To test this hypothesis we used induced hepatic steatosis as a model that was inflicted in outbred deer mice (Peromyscus) by high fat/sucrose diet. Our transcriptional analysis focused on genes associated with the unfolded protein response (UPR), lipophagy (ATG5, ATG14 and LAMP1) and lipid metabolism (lipoprotein lipase (LPL), hepatic lipase (HL) or trafficking (low density lipoprotein receptor, LDLR). qPCR studies showed that long-term administration of high-fat diet caused overexpression of all targets tested regardless of the onset of steatosis, suggesting that activation of gene expression was induced by the diet and not by the disease. However, when the coordination with the UPR was analyzed, it was discovered that HL was the only target gene exhibiting coordination with the UPR genes in all liver specimens except those that had evidence of hepatic steatosis. Subsequent studies involving cultured rat hepatocytes showed that during ER stress the expression of HL is suppressed by a manner that involves the PERK arm of the UPR and specifically by CHOP. This effect is likely mediated by the transcription factor HNF4 that is an established activator of HL. These findings illustrate the power of coordination analysis by using genetically diverse disease models. Furthermore, they challenge conventional views on the impact of specific genes in disease and suggest that besides abundance, the maintenance of coordination between transcriptional networks may be equally or even more important for disease development.

The Role of CREG1 as a Master Regular of Liver Function

Mammalian development involves a complex system of regulatory signals and reactions resulting in highly differentiated cell types with specific structure and function. The liver is a major organ that has been studied extensively to understand underlying genetic processes responsible for specification, establishment and maintenance of tissue identity. Hepatoma and hepatoma variant cell lines have been used as a model to understand genetic networks responsible for liver function. Whole genome microarray analysis of hepatocyte cell lines has revealed candidate genes that may serve as regulators or master regulators of liver specificity. In two previous studies, the role of candidate gene Cellular Repressor of E1A Stimulated Gene (CREG1) on regulation of liver‐specific gene expression was determined using transfection studies combined with utilization of quantitative real‐time polymerase chain reaction (qRTPCR). Both studies found strong activation (10–100‐fold) of transcription factor Hnf6 and the downstream gene Serpina1 (a marker gene used to identify liver function). These findings suggested that CREG1 might act through Hnf6 to regulate Serpina1 via a Locus Control Region (LCR). However, while one study showed that of CREG1 overexpression in a hepatoma variant cell line resulted in modest activation of liver‐specific transcription factors Hnf1, Hnf4 and Hnf3, the second study suggested robust activation of these genes as measured by qRT‐PCR. In the current study, a CREG1 expression vector was reintroduced into two variant cell lines and gene activation profiles monitored to establish validity of the previous studies. However, we postulated that CREG‐1 overexpression, validated by qRT‐PCR, can activate expression of hepatocyte transcription factor Hnf6 and the downstream gene Serpina1.However, analysis of gene silencing of liver and non‐liver cell types using whole genome microarray screening will help to identify, compare and elucidate the effects of various candidate tissue specific genes. Moreover, we are currently interested to see the reintroduction of liver‐enriched factors in hepatoma variants fully reactivates liver gene expression. Thus, understanding the underlying mechanism and role of each gene helps to develop therapeutic treatment targets for treating liver diseases.Support or Funding InformationEastern Illinois University, Department of Biological ScienceThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential effects, on oncogenic pathway signalling, by derivatives of the HNF4 \u03b1 inhibitor BI6015

BackgroundGastric cancer (GC) is a highly heterogeneous disease with few “targeted” therapeutic options. Previously, we demonstrated involvement of the transcription factor HNF4α in human GC tumours, and the developmental signal mediator, WNT5A, as a prognostic GC biomarker. One previously developed HNF4α antagonist, BI6015, while not advancing beyond preclinical stages, remains useful for studying GC.MethodsHere, we characterised the antineoplastic signalling activity of derivatives of BI6015, including transfer of the nitro group from the para position, relative to a methyl group on its benzene ring, to the ortho- and meta positions. We assessed binding efficacy, through surface plasmon resonance and docking studies, while biologic activity was assessed by antimitogenic efficacy against a panel of GC cell lines, and dysregulated transcriptomes, followed by pathway and subpathway analysis.ResultsThe para derivative of BI6105 was found substantially more growth inhibitory, and effective, in downregulating numerous oncogenic signal pathways, including the embryonic cascade WNT. The ortho and meta derivatives, however, failed to downregulate WNT or other embryonic signalling pathways, unable to suppress GC growth.ConclusionStraightforward strategies, employing bioinformatics analyses, to facilitate the effective design and development of “druggable” transcription factor inhibitors, are useful for targeting specific oncogenic signalling pathways, in GC and other cancers.

Abstract P377: Effect of Salt Diet on Haplotype Specific Expression of Human Angiotensinogen Gene

Human Angiotensinogen gene (hAGT) is an important component of the Renin- Angiotensin System. The 2.5 Kb promoter of the hAGT gene has ten polymorphisms that form two haplotype (Hap) blocks. Hap-I containing -6A, -20A, -217A, -532T, -793A, -1074T, -1178G, -1561T, -1562C, and -1670A is associated with increased blood pressure whereas Hap-II containing -6G, -20A, -217G, -532C, -793G, -1074G, -1178A, -1561G, -1562G, and -1670G is associated with normal blood pressure in human subjects. In order to understand the role of these SNPs in the transcriptional regulation, we have generated novel transgenic mice with the hAGT gene, targeted to the mHPRT locus with either Hap I or II variants. In the present study, we have examined the effect of high salt diet on the hAGT gene expression and transcriptional regulation in the 8 weeks old male transgenic mice containing these two haplotypes. Quantitative real time PCR analysis revealed an increase in hAGT expression in the liver of Hap-I as compared to Hap-II in basal condition (4.09 fold), Hap-I high salt vs Hap-I basal (2.15 fold), Hap-II high salt vs Hap-II basal (1.9 fold) and Hap-I high salt vs Hap-II high salt (2.47 fold) with p&lt;0.05 in these transgenic mice. Immunoblot analysis also indicated the upregulation of the hAGT in plasma in all the study groups, viz; Hap-I compared to that of Hap-II (2.5 fold), Hap-I high salt vs Hap-I basal (1.9 fold) and Hap-II high salt vs Hap-II basal (1.7 fold) with p&lt;0.05. Our studies show that the transgenic mice fed high salt diet have increased mRNA levels in the liver for the transcription factors HNF1, FOXO1, FOXA1 and MR (4, 3.13, 3.93 and 5.4 fold increase respectively; p&lt;0.05). CHIP assay also complements the above observations. Our results strongly suggest that SNPs in Hap-I promote increased transcription and expression of the hAGT gene in multiple tissues, with resultant elevation of plasma hAGT levels. Our studies would provide new insights into the susceptibility of individuals to salt induced hypertension and explain the novel paradigm in salt induced transcriptional regulation of human angiotensinogen gene.

Bioinformatic analysis of gene expression profiling of intracranial aneurysm.

Intracranial aneurysm (IA) is a severe clinical condition of primary concern and currently, there is no effective therapeutic reagent. The present study aimed to investigate the molecular mechanism of IA via bioinformatic analysis. Various gene expression profiles (GSE26969) were downloaded from the Gene Expression Omnibus database, including 3 IA and 3 normal superficial temporal artery samples. Firstly, the limma package in R language was used to identify differentially expressed genes (DEGs; P-value <0.01 and |log2 FC|≥1). Secondly, the database for annotation, visualization and integrated discovery software was utilized to perform pathway and functional enrichment analyses (false discovery rate ≤0.05). Finally, protein-protein interaction (PPI) network and sub-network clustering analyses were performed using the biomolecular interaction network database and ClusterONE software, respectively. Following this, a transcription factor regulatory network was identified from the PPI network. A total of 1,124 DEGs were identified, of which 989 were upregulated and 135 downregulated. Pathway and functional enrichment analyses revealed that the DEGs primarily participated in RNA splicing, functioning of the spliceosome, RNA processing and the mRNA metabolic process. Following PPI network analysis, 1 hepatocyte nuclear factor (HNF) 4A (transcription factor)-centered regulatory network and 5 DEG-centered sub-networks were identified. On analysis of the transcription factor regulatory network, 6 transcription factors (HNF6, HNF4A, E2F4, YY1, H4 and H31T) and a regulatory pathway (HNF6-HNF4-E2F4) were identified. The results of the present study suggest that activating transcription factor-5, Jun proto-oncogene, activator protein-1 transcription factor subunit, HNF6, HNF4 and E2F4 may participate in IA progression via vascular smooth muscle cell apoptosis, inflammation, vessel wall remodeling and damage and the tumor necrosis factor-β signaling pathway. However, further experimental studies are required to validate these predictions.

Valproic Acid Increases the Hepatic Differentiation Potential of Salivary Gland Cells

The studies of cell plasticity and differentiation abilities are important problems in modern cellular biology. The use of histone deacetylase inhibitor - valproic acid is a promising approach to increasing the differentiation efficiency of various cell types. In this paper we investigate the ability of mouse submandibular salivary gland cells to differentiate into the hepatic direction and the effect of valproic acid on the efficiency of this differentiation. It was shown that the gene expression levels of hepatocyte markers (Aat, Afp, G6p, Pepck, Tat, Cyp3a13) and liver-enriched transcription factors (Hnf-3α, Hnf-3β, Hnf-4α, Hnf-6) were increased after differentiation in salivary gland cells. Valproic acid increases the specificity of hepatic differentiation, reducing the expression levels of the ductal (Krt19, Hhex1, Cyp7a1) and acinar (Ptf1a) markers. After valproic acid exposure, the efficiency of hepatic differentiation also increases, as evidenced by the increase in the gene expression level of Alb and Tdo, and increase in urea production by differentiated cells. No change was found in DNA methylation of the promoter regions of the genes; however, valproic acid treatment and subsequent hepatic differentiation largely affected the histone H3 methylation of liver-enriched genes. Thus, mouse submandibular salivary gland cells are capable of effective differentiation in the hepatic direction. Valproic acid increases the specificity and efficiency of the hepatic differentiation of these cells.

Cross Talk Between GH-Regulated Transcription Factors HNF6 and CUX2 in Adult Mouse Liver.

Hepatocyte-enriched nuclear factor (HNF)6 and CUX2 are GH and STAT5-regulated homeobox transcription factors. CUX2 shows female-specific expression and contributes to liver sex differences by repressing many male-biased genes and inducing many female-biased genes, whereas HNF6 is expressed at similar levels in male and female liver. In cell-based transfection studies, CUX2 inhibited HNF6 transcriptional regulation of the sex-specific gene promoters CYP2C11 and CYP2C12, blocking HNF6 repression of CYP2C11 and HNF6 activation of CYP2C12. These inhibitory actions of CUX2 can be explained by competition for HNF6 DNA binding, as demonstrated by in vitro EMSA analysis and validated in vivo by global analysis of the HNF6 cistrome. Approximately 40 000 HNF6-binding sites were identified in mouse liver chromatin, including several thousand sites showing significant sex differences in HNF6 binding. These sex-biased HNF6-binding sites showed strong enrichment for correspondingly sex-biased DNase hypersensitive sites and for proximity to genes showing local sex-biased chromatin marks and a corresponding sex-biased expression. Further, approximately 90% of the genome-wide binding sites for CUX2 were also bound by HNF6. These HNF6/CUX2 common binding sites were enriched for genomic regions more accessible in male than in female mouse liver chromatin and showed strongest enrichment for male-biased genes, suggesting CUX2 displacement of HNF6 as a mechanism to explain the observed CUX2 repression of male-biased genes in female liver. HNF6 binding was sex independent at a majority of its binding sites, and HNF6 peaks were frequently associated with cobinding by multiple other liver transcription factors, consistent with HNF6 playing a global regulatory role in both male and female liver.

Cis-regulatory elements involved in species-specific transcriptional regulation of the SVCT1 gene in rat and human hepatoma cells

Ascorbic acid is transported into cells by the sodium-coupled vitamin C transporters (SVCTs). Recently, we obtained evidence of differential regulation of SVCT expression in response to acute oxidative stress in cells from species that differ in their capacity to synthesize vitamin C, with a marked decrease in SVCT1 mRNA and protein levels in rat hepatoma cells that was not observed in human hepatoma cells. To better understand the regulatory aspects involved, we performed a structural and functional analysis of the proximal promoter of the SVCT1 rat gene. We cloned a 1476-bp segment containing the proximal promoter of the rat SVCT1 gene and generated deletion-derived truncated promoters of decreasing sizes and mutant promoters by modification of consensus binding sites for transcription factors by site-directed mutagenesis. We next analyzed their capacity to direct the transcription of a reporter gene after transfection into rat H4IIE and human HepG2 hepatoma cells, in experiments involving the coexpression of transcription factors whose consensus binding sequences are present in the SVCT1 promoter. This analysis revealed the presence of two critical cis-regulatory elements of the transcriptional activity of the rat SVCT1 gene promoter, sites containing consensus sequences for the binding of the transcription factors Bach1 and HNF4 that are not present in equivalent locations in the human SVCT1 gene promoter. Moreover, a consensus site for HNF1 that is crucial for the regulation of the human SVCT1 promoter is present in the SVCT1 rat promoter but has no effect on its transcriptional activity. These findings imply that regulation of vitamin C metabolism in the rat, a species with the capacity to synthesize large amounts of ascorbic acid, may differ from that of humans, a species that must obtain ascorbic acid from the diet through a transport mechanism that depends on proper SVCT1 expression.

Interactions with the Bifunctional Interface of the Transcriptional Coactivator DCoH1 Are Kinetically Regulated

Pterin-4a-carbinolamine dehydratase (PCD) is a highly conserved enzyme that evolved a second, unrelated function in mammals, as a transcriptional coactivator. As a coactivator, PCD is known as DCoH or dimerization cofactor of the transcription factor HNF-1. These two activities are associated with a change in oligomeric state: from two dimers interacting as an enzyme in the cytoplasm to a dimer interacting with a dimer of HNF-1 in the nucleus. The same interface of DCoH forms both complexes. To determine how DCoH partitions between its two functions, we studied the folding and stability of the DCoH homotetramer. We show that the DCoH1 homotetramer is kinetically trapped, meaning once it forms it will not dissociate to interact with HNF-1. In contrast, DCoH2, a paralog of DCoH1, unfolds within hours. A simple mutation in the interface of DCoH2 from Ser-51 to Thr, as found in DCoH1, increases the kinetic stability by 9 orders of magnitude, to τ(½) ∼ 2 million years. This suggests that the DCoH1·HNF-1 complex must co-fold to interact. We conclude that simple mutations can dramatically affect the dissociation kinetics of a complex. Residue 51 represents a "kinetic hot spot" instead of a "thermodynamic hot spot." Kinetic regulation allows PCD to adopt two distinct functions. Mutations in DCoH1 associated with diabetes affect both functions of DCoH1, perhaps by disrupting the balance between the two DCoH complexes.

Dissecting reprogramming, differentiation and conversion with network biology.

Dissecting reprogramming, differentiation and conversion with network biology.

Up regulation of liver-enriched transcription factors HNF4a and HNF6 and liver-specific microRNA (miR-122) by inhibition of let-7b in mesenchymal stem cells.

MicroRNAs are small non-coding RNAs that regulate key processes of the stem cells. Although, microRNAs have emerged as powerful regulators of differentiation, few studies have been focused on the post-transcriptional regulation of hepatic differentiation in mesenchymal stem cells (MSCs) by microRNAs. The aim of this study was to evaluate the specific effect of let-7 microRNAs in particular let-7b in hepatic commitment of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs). The dynamic expression profile of let-7a, b, c microRNAs and two liver-enriched transcription factors (LETFs) HNF4a and HNF6 was studied during in vitro hepatic differentiation of hAT-MSCs. Let-7b was used for transient overexpression and knockdown investigations. It was shown that the expression of LETFs is inversely correlated with those of let-7 miRNAs during differentiation progress (p < 0.05). Inhibition of let-7b caused upregulation of LETFs, an increase in the expression of miR-122 (p < 0.01) emulating the features of functional hepatocytes, and accumulation of hAT-MSCs in the G0 /G1 phase of cell cycle, triggering initiation of hepatic commitment. In conclusion, transient inhibition of let-7b activates hepatic differentiation of hAT-MSCs. The findings of this work might help optimization of in vitro hepatogenic differentiation utilizing microRNAs and hAT-MSCs that could be used for therapeutic purposes.