Modulation of hepatic gene expression by hepatocyte nuclear factor 1.

Hepatocyte nuclear factors 1 and 4 (HNF-1 and HNF-4) are liver-enriched transcription factors that function in the regulation of several liver-specific genes. HNF-1 activates genes containing promoters with HNF-1 binding sites. However, this factor negatively regulates its own expression and that of other HNF-4-dependent genes that lack HNF-1 binding sites in their promoter region. This repression is exerted by a direct interaction of HNF-1 with AF2, the main activation domain of HNF-4. The dual functions of gene activation and repression suggest that HNF-1 is a global regulator of the transcriptional network involved in the maintenance of hepatocyte-specific phenotype.

Chicken ovalbumin upstream promoter transcription factors act as auxiliary cofactors for hepatocyte nuclear factor 4 and enhance hepatic gene expression.

Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) strongly inhibit transcriptional activation mediated by nuclear hormone receptors, including hepatocyte nuclear factor 4 (HNF-4). COUP-TFs repress HNF-4-dependent gene expression by competition with HNF-4 for common binding sites found in several regulatory regions. Here we show that promoters, such as the HNF-1 promoter, which are recognized by HNF-4 but not by COUP-TFs are activated by COUP-TFI and COUP-TFII in conjunction with HNF-4 more than 100-fold above basal levels, as opposed to about 8-fold activation by HNF-4 alone. This enhancement was strictly dependent on an intact HNF-4 E domain. In vitro and in vivo evidence suggests that COUP-TFs enhance HNF-4 activity by a mechanism that involves their physical interaction with the amino acid 227 to 271 region of HNF-4. Our results indicate that in certain promoters, COUP-TFs act as auxiliary cofactors for HNF-4, orienting the HNF-4 activation domain in a more efficient configuration to achieve enhanced transcriptional activity. These findings provide new insights into the regulatory functions of COUP-TFs, suggesting their involvement in the initial activation and subsequent high-level expression of hepatic regulators, as well as in the positive and negative modulation of downstream target genes.

Regulatory mechanisms controlling human hepatocyte nuclear factor 4alpha gene expression.

Hepatocyte nuclear factor 4alpha (HNF-4alpha) (nuclear receptor 2A1) is an essential regulator of hepatocyte differentiation and function. Genetic and molecular evidence suggests that the tissue-restricted expression of HNF-4alpha is regulated mainly at the transcriptional level. As a step toward understanding the molecular mechanism involved in the transcriptional regulation of the human HNF-4alpha gene, we cloned and analyzed a 12.1-kb fragment of its upstream region. Major DNase I-hypersensitive sites were found at the proximal promoter, the first intron, and the more-upstream region comprising kb -6.5, -8.0, and -8.8. By the use of reporter constructs, we found that the proximal-promoter region was sufficient to drive high levels of hepatocyte-specific transcription in transient-transfection assays. DNase I footprint analysis and electrophoretic mobility shift experiments revealed binding sites for HNF-1alpha and -beta, Sp-1, GATA-6, and HNF-6. High levels of HNF-4alpha promoter activity were dependent on the synergism between either HNF-1alpha and HNF-6 or HNF-1beta and GATA-6, which implies that at least two alternative mechanisms may activate HNF-4alpha gene transcription. Chromatin immunoprecipitation experiments with human hepatoma cells showed stable association of HNF-1alpha, HNF-6, Sp-1, and COUP-TFII with the promoter. The last factor acts as a repressor via binding to a newly identified direct repeat 1 (DR-1) sequence of the human promoter, which is absent in the mouse homologue. We present evidence that this sequence is a bona fide retinoic acid response element and that HNF-4alpha expression is upregulated in vivo upon retinoic acid signaling.

Immunocytochemical detection of deoxycytidine kinase in haematological malignancies and solid tumours.

BACKGROUND: Deoxycytidine kinase (dCK) is responsible for the activation of several clinically important deoxynucleoside analogues used for the treatment of haematological and solid malignancies. AIM: To measure dCK expression in tumour cells from different origins. METHOD: A rabbit antihuman dCK antibody was used for the immunocytochemical detection of dCK expression in three leukaemic cell lines (HL60, U937, and CCRF-CEM) and 97 patient samples (paediatric acute myeloid leukaemia (AML) and lymphoid leukaemia (ALL), retinoblastoma, paediatric brain tumours, and adult non-small cell lung cancer (NSCLC)). RESULTS: CCRF-CEM, U937, and HL60 cells stained positively for dCK and the degree of expression correlated with dCK activity. dCK expression varied between tumour types and between individual patients within one tumour type. dCK was located predominantly in the cytoplasm. The staining intensity was scored as negative (0), low (1+), intermediate (2+), or high (3+). Expression of dCK was high in AML blasts. In contrast, brain tumour samples expressed low amounts of dCK. dCK staining ranged from low (1+) to high (3+) in ALL blasts, retinoblastoma, and NSCLC tissue samples. Staining was consistent (interobserver variability, 88%; kappa = 0.83) and specific. Western blotting detected the dCK protein appropriately at 30 kDa, without additional bands. CONCLUSIONS: Immunocytochemistry is an effective and reliable method for determining the expression of dCK in patient samples and requires little tumour material. This method enables large scale screening of dCK expression in tumour samples.

Isolation and characterization of a third isoform of human hepatocyte nuclear factor 4.

Hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of a large number of liver-specific genes. We report here the isolation of three HNF-4 isoforms from a human liver cDNA library. hHNF-4A and hHNF-4B, differing by the insertion of 10 amino acids in the C-terminal region, have been previously identified in mouse, rat and human liver. The novel isoform, hHNF-4C, is identical to hHNF-4A and B in the regions encompassing the DNA-binding and dimerization domains, but contains a different C-terminal domain. Similar to the other isoforms, hHNF-4C is produced in a limited number of tissues and represents 2.6-13% of the total hHNF-4 mRNA population, depending on the cell type. The chromosomal origin of all three isoforms has been localized to human chromosome 20. hHNF-4C can form heterodimers with hHNF-4A and B in vitro, and exhibits similar transactivation potential as hHNF-4A or B in transient transfection assays, suggesting that the divergent C-terminal region is not part of the transactivation domain.

Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes.

Deoxycytidine kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-beta-D-arabinofuranosylcytosine, difluorodeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl-Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

Immunocytochemical detection of deoxycytidine kinase in pediatric malignancies in relation to in vitro cytarabine sensitivity.

Deoxycytidine kinase (dCK) is essential for the phosphorylation of cytarabine (ara-C), a deoxycytidine analog active against acute leukemias. Resistance to ara-C has been linked to dCK deficiency. In this study we determined the expression of the dCK protein in pediatric malignancies, using immunocytochemistry and related the expression levels to in vitro ara-C sensitivity (measured with the MTT-assay). dCK expression was high in the AML and retinoblastoma samples, in the ALL samples dCK expression ranged from low to very high. The brain tumor samples expressed low levels of dCK. AML was significantly more sensitive in vitro to ara-C compared to ALL (p = 0.03). Retinoblastoma and brain tumor cells were extremely resistant in vitro, we were unable to detect more than 50% ara-C induced cell kill in the majority of samples. Samples were combined in groups according to dCK expression. Samples with low dCK expression were significantly more resistant to ara-C compared to samples with high dCK expression. In conclusion, dCK expression varies between individual samples and between different types of malignancies and may play a role in resistance to ara-C in particular tumor types.

Context-specific regulation of cancer epigenomes by histone and transcription factor methylation.

Altered expression or activity of histone lysine methylases and demethylases in cancer lead to aberrant chromatin modification patterns, which contribute to uncontrolled cell proliferation via cancer-specific deregulation of gene expression programs or the induction of genome instability. Several transcription factors that regulate growth-associated genes undergo lysine methylation, expanding the repertoire of regulatory targets modulated by histone-methylating enzymes during tumorigenesis. In certain specific tumor types or specific physiological conditions, these enzymes may trigger chromatin structure and/or transcription factor activity changes that result in opposite effects on cancer initiation or progression. The mechanisms of such context-specific dual functions and those involved in the crosstalk between factor and histone modifications are subject to extensive research, which is beginning to shed light into this novel level of complexity of cancer-related epigenetic pathways.

KMTase Set7/9 is a critical regulator of E2F1 activity upon genotoxic stress.

During the recent years lysine methyltransferase Set7/9 ((Su(var)-3-9, Enhancer-of-Zeste, Trithorax) domain containing protein 7/9) has emerged as an important regulator of different transcription factors. In this study, we report a novel function for Set7/9 as a critical co-activator of E2 promoter-binding factor 1 (E2F1)-dependent transcription in response to DNA damage. By means of various biochemical, cell biology, and bioinformatics approaches, we uncovered that cell-cycle progression through the G1/S checkpoint of tumour cells upon DNA damage is defined by the threshold of expression of both E2F1 and Set7/9. The latter affects the activity of E2F1 by indirectly modulating histone modifications in the promoters of E2F1-dependent genes. Moreover, Set7/9 differentially affects E2F1 transcription targets: it promotes cell proliferation via expression of the CCNE1 gene and represses apoptosis by inhibiting the TP73 gene. Our biochemical screening of the panel of lung tumour cell lines suggests that these two factors are critically important for transcriptional upregulation of the CCNE1 gene product and hence successful progression through cell cycle. These findings identify Set7/9 as a potential biomarker in tumour cells with overexpressed E2F1 activity.

Acetylation regulates transcription factor activity at multiple levels.

CREB-binding protein (CBP) possesses an intrinsic acetyltransferase activity capable of acetylating nucleosomal histones as well as several nonhistone proteins. Here, it is shown that CBP can acetylate hepatocyte nuclear factor-4 (HNF-4), a member of the nuclear hormone receptor family, at lysine residues within the nuclear localization sequence. CBP-mediated acetylation is crucial for the proper nuclear retention of HNF-4, which is otherwise transported out to the cytoplasm via the CRM1 pathway. Acetylation also increases HNF-4 DNA binding activity and its affinity of interaction with CBP itself and is required for target gene activation. The results show that acetylation is a key posttranslational modification that may affect several properties of a transcription factor critical for the execution of its biological functions.

Transcriptional activation by hepatocyte nuclear factor-1 requires synergism between multiple coactivator proteins.

Hepatocyte nuclear factor-1 (HNF-1) plays an important role in the regulation of a large number of genes expressed in the liver, kidney, and pancreatic beta-cells. In exploring the molecular mechanism involved in HNF-1-dependent gene activation in the in vivo chromatin context, we found that HNF-1 can physically interact with the histone acetyltransferases (HATs) CREB-binding protein (CBP), p300/CBP-associated factor (P/CAF), Src-1, and RAC3. The transcriptional activation potential of HNF-1 on a genome integrated promoter was strictly dependent on the synergistic action of CBP and P/CAF, which can independently interact with the N-terminal and C-terminal domain of HNF-1, respectively. Moreover, the HAT activity of both coactivators was important, as opposed to the selective requirement for the HAT activity of P/CAF in activation from a transiently transfected reporter. Interaction of CBP with the N-terminal domain of HNF-1 greatly increased the binding affinity for P/CAF with the C-terminal activation domain, which may represent the molecular basis for the observed functional synergism. The results support a model that involves the combined action of multiple coactivators recruited by HNF-1, which activate transcription by coupling nucleosome modification and recruitment of the general transcription machinery.

Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

Complex interactions between SP1 bound to multiple distal regulatory sites and HNF-4 bound to the proximal promoter lead to transcriptional activation of liver-specific human APOCIII gene.

Footprinting analysis of the human apoCIII promoter identified a set of four proximal (A-D) and six distal (E-J) regulatory elements between nucleotides -792 and -25 [Ogami, K., et al. (1990) J. Biol. Chem. 265, 9808-9815]. The distal regulatory elements of the apoCIII gene increase by 10-fold the strength of the homologous as well as of heterologous proximal promoters. Required for such transcriptional enhancement is the presence of an intact hormone response element (HRE) on the proximal promoter which binds a variety of nuclear hormone receptors. To understand the mechanism of this transcriptional activation, we identified the nature and the importance of the factors which bind to the upstream regulatory elements of the apoCIII promoter by DNA binding, competition, supershift, and transient transfection assays. These analyses showed that the upstream apoCIII promoter contains multiple binding sites for the ubiquitous transcription factor SP1, which recognizes the regulatory elements F, H, and I. The regulatory element G represents a specialized HRE which is recognized by the orphan receptors ARP-1 and EAR-3 but not by HNF-4. A single activity designated CIII J1 binds to the regulatory element J. The same or a similar activity binds as a minor component to the regulatory elements F and I where SP1 is the predominant binding activity. Finally, a minor activity designated CIII 15 binds to the regulatory element I.(ABSTRACT TRUNCATED AT 250 WORDS)

An indirect negative autoregulatory mechanism involved in hepatocyte nuclear factor-1 gene expression.

Recent studies have revealed that hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of another liver enriched transcription factor HNF-1, defining a transcriptional hierarchy between the two factors operating in hepatocytes. To assess the possible autoregulation of the HNF-1 gene we have examined the effect of HNF-1 on its own transcription. In transient transfection assays, HNF-1 strongly down-regulated transcription driven by its own promoter in HepG2 cells. In addition HNF-1 also repressed the activity of HNF-4 dependent ApoCIII and ApoAI promoters. The same effect was observed using vHNF-1, a distinct but highly related protein to HNF-1. Both HNF-1 and vHNF-1 downregulated HNF-4 activated transcription from intact and chimeric promoter constructs carrying various HNF-4 binding sites implying that they act by impeding HNF-4 binding or activity. DNA binding and cell free transcription experiments however failed to demonstrate any direct or indirect interaction of HNF-1 and vHNF-1 with the above regulatory regions. Both factors repressed HNF-4 induced transcription of the ApoCIII and HNF-1 genes in HeLa cells, arguing against the requirement of a hepatocyte specific function. These findings define an indirect negative autoregulatory mechanism involved in HNF-1 gene expression, which in turn may affect HNF-4 dependent transcription of other liver specific genes.

Transcriptional regulation of the apolipoprotein A-IV gene involves synergism between a proximal orphan receptor response element and a distant enhancer located in the upstream promoter region of the apolipoprotein C-III gene.

Apolipoprotein A-IV expression is limited to intestinal and hepatic cells, suggesting a tissue specific transcriptional regulation of its gene. To investigate the mechanism controlling apo A-IV transcription we have analysed its promoter region by in vitro DNA binding and transient transfection experiments. DNase I footprinting analysis of the proximal promoter with rat liver nuclear extracts revealed four protected regions: AIVA (-32 to -22), AIVB (-84 to -42), AIVC (-148 to -92) and AIVD (-274 to -250). Element AIVC which is necessary for maximal promoter activity, binds HNF-4, Arp-1 and Ear-3 with similar affinity in a mutually exclusive manner. HNF-4 transactivated chimeric constructs containing intact AIVC site in the context of either the apo A-IV promoter or the heterologous thymidine kinase minimal promoter, while Arp-1 and Ear-3 repressed this activation. Increasing amounts of HNF-4 alleviated Arp-1 or Ear-3 mediated repression, suggesting that the observed opposing effects is a result of direct competition of these factors for the same recognition site. In transient transfection assays the apo A-IV promoter region (-700 to +10) had a very low activity in cells of hepatic (HepG2) and intestinal (CaCo2) origin. This activity was increased 13 to 18-fold when the upstream elements of the distantly linked apo C-III gene were fused to the proximal promoter. Results obtained with different 5' and 3' deletion constructs indicated that the cis-acting elements F to J between the nucleotides -500 and -890 of the apo C-III promoter were absolutely necessary to drive maximal enhancement in HepG2 and CaCo2 cells. The apo C-III upstream elements enhanced the activity of the minimal AdML promoter or the apo A-IV site C mutant less efficiently than the intact apo A-IV or AdML promoter constructs containing single HNF-4 sites. The findings suggest that the enhancer effect is mediated by synergistic interactions between the trans-acting factors which recognize the apo C-III regulatory elements and HNF-4 which binds to the proximal apo A-IV promoter.

Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity.

CREB-binding protein (CBP) and CBP-associated factor (P/CAF) are coactivators possessing an intrinsic histone acetyltransferase (HAT) activity. They are positioned at promoter regions via association with sequence-specific DNA-binding factors and stimulate transcription in a gene-specific manner. The current view suggests that coactivator function depends mainly on the strength and specificity of transcription factor-coactivator interactions. Here we show that two dominant-negative mutants of hepatocyte nuclear factor-1alpha (HNF-1alpha), P447L and P519L, occurring in maturity onset diabetes of the young (MODY3) patients, exhibit paradoxically stronger interactions than the wild-type protein with either CBP or P/CAF. However, CBP and P/CAF recruited by these mutants lack HAT activity. In contrast, wild-type HNF-1alpha and other transcription factors, such as Sp1 or HNF-4, stimulated the HAT activity of CBP. The results suggest a more dynamic role for DNA-binding proteins in the transcription process than was considered previously. They are not only required for the recruitment of coactivators to the promoter but they may also modulate their enzymatic activity.

Distal apolipoprotein C-III regulatory elements F to J act as a general modular enhancer for proximal promoters that contain hormone response elements. Synergism between hepatic nuclear factor-4 molecules bound to the proximal promoter and distal enhancer sites.

Transient transfection assays have shown that the distal apoC-III promoter segments that contain the regulatory elements F to J enhance the strength of the tandemly linked proximal apoA-I promoter 5- to 13-fold in hepatic (HepG2) cells. Activation in intestinal (CaCo-2) cells to levels comparable to those obtained in HepG2 cells requires a larger apoA-I promoter sequence that extends to nucleotide -1500 as well as the presence of hepatic nuclear factor-4 (HNF-4). The distal apoC-III regulatory elements can also enhance 4- to 8-fold the strength of the heterologous apoB promoter in HepG2 and CaCo-2 cells. Finally, these elements in the presence of HNF-4 enhance 14.5- to 18.5-fold the strength of the minimal adenovirus major late promoter linked to two copies of the hormone response element (HRE) AID of apoA-I in both HepG2 and CaCo-2 cells. In vitro mutagenesis of the promoter/enhancer cluster established that the enhancer activity is lost by a mutation in the HRE present in the 3' end of the regulatory element I (-736 to -714) and is reduced significantly by point mutations or deletions in one or more of the regulatory elements F to J of the apoC-III enhancer. The enhancer activity also requires the HREs of the proximal apoA-I promoter. The apoC-III enhancer can also restore the activity of the proximal apoA-I and apoB promoters that have been inactivated by mutations in CCAAT/enhancers binding protein binding sites, indicating that C/EBP may not participate in the synergistic activation of the promoter/enhancer cluster. The findings suggest that the regulatory elements F to J of the apoC-III promoter act as a general modular enhancer that can potentiate the strength of proximal promoters that contain HREs. Such potentiation in the HepG2 cells can be accounted for by synergistic interactions between HNF-4 or other nuclear hormone receptors bound to the proximal and distal HREs and SP1 or other factors bound to the apoC-III enhancer. Additional factors may be required for optimal activity in CaCo-2 cells as well as for the function of this region as an intestinal enhancer.