HNF4 Binding Site Research Articles

Differential Immediate Early Gene Expression in Conditional Hepatitis B Virus pX-transforming VersusNontransforming Hepatocyte Cell Lines

We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1alpha and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1beta and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation. Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. The pX-transforming 3pX lineage displays early, pX-dependent induction of ATF3 and prolonged induction of c-fos. The nontransforming 4pX cells display an absence of pX-dependent ATF3 induction and transient induction of c-fos. Our results support the direct link of pX expression to oncogenic transformation in 3pX lineage clones and underscore the advantage of this conditional cellular model system for studying mechanisms of pX-mediated oncogenesis.

DNA Curvature and Phosphate Neutralization: An Important Aspect of Specific Protein Binding

A theoretical study is presented of the influence of salt bridges between protein cationic side chains and DNA phosphates on DNA conformation and flexibility. Two DNA sequences are studied containing respectively the HNF3 and CAP binding sites. The effect of salt bridges is modelled by the neutralisation of net phosphate charges for the groups involved in such interactions in the complex. Energy optimised conformations are obtained by molecular mechanics calculations using the JUMNA program. Base sequence dependence is studied by moving the phosphate neutralisation pattern along the sequence, while normal mode analysis is used to evaluate DNA flexibility. The results show that phosphate neutralisation has a strong influence on DNA conformation. For the HNF3 binding sequence, the free oligomer is bent in direction very different from that observed in the protein complex. Phosphate neutralisation changes this direction by 45° to within only 4° of the direction in the complex, without changing the bending angle. For the CAP binding sequence, the free oligomer is already intrinsically curved in the direction favoured by the protein, but phosphate neutralisation increases the bending angle. For both oligomers studied these effects are strongly sequence dependent. It is also shown that oligomer flexibility cannot be explained by a simple superposition of the properties of successive dinucleotide steps. Important long range coupling effects are observed. However, for both sequence studied, phosphate neutralisation however leads to a reduction in oligomer flexibility.

SRC-1 and GRIP1 Coactivate Transcription with Hepatocyte Nuclear Factor 4

Hepatocyte nuclear factor-4 (HNF4), a member of the nuclear receptor superfamily, plays an important role in tissue-specific gene expression, including genes involved in hepatic glucose metabolism. In this study, we show that SRC-1 and GRIP1, which act as coactivators for various nuclear receptors, associate with HNF4 in vivo and enhance its transactivation potential. The AF-2 domain of HNF4 is required for this interaction and for the potentiation of transcriptional activity by these coactivators. p300 can also serve as a coactivator with HNF4, and it synergizes with SRC-1 to further augment the activity of HNF4. HNF4 is also a key regulator of the expression of hepatocyte nuclear factor-1 (HNF1). The overexpression of SRC-1 or GRIP1 enhances expression from a HNF1 gene promoter-reporter in HepG2 hepatoma cells, and this requires an intact HNF4-binding site in the HNF1 gene promoter. Type 1 maturity onset diabetes of young (MODY), which is characterized by abnormal glucose-mediated insulin secretion, is caused by mutations of the HNF4 gene. A mutation of the HNF4-binding site in the HNF1 gene promoter has also been associated with MODY. Thus, HNF4 is involved in the regulation of glucose homeostasis at several levels and along with the SRC-1, GRIP1, and p300 may play an important role in the pathophysiology of non-insulin-dependent diabetes mellitus.

Vitamin D-binding Protein Gene Transcription Is Regulated by the Relative Abundance of Hepatocyte Nuclear Factors 1α and 1β

Vitamin D-binding protein (DBP)/Gc-globulin, the major carrier of vitamin D and its metabolites in blood, is synthesized predominantly in the liver in a developmentally regulated fashion. By transient transfection analysis, we identified three regions in the 5'-flanking region of the rat DBP gene, segments F-2, B, and A, that contain tissue-specific transcriptional determinants. Gel mobility shift and DNase I footprinting analyses showed that all three regions contained binding sites for the hepatocyte nuclear factor 1 (HNF1), a transcriptional regulator composed of HNF1alpha and HNF1beta hetero- and homodimers. The activity of the most proximal segment A (coordinates -141 to -43) was DBP promoter-specific, position-dependent, and positively controlled by HNF1alpha. In contrast, the two more distal determinants (segments F-2 and B; coordinates -1844 to -1621 and -254 to -140, respectively) acted as classical enhancers in transfected hepatocyte-derived HepG2 cells; their activities were promoter- and orientation-independent, and disruption of their respective HNF1-binding sites resulted in marked loss of DBP gene expression. Remarkably, the activities of these two distal elements depended upon the relative levels of HNF1alpha and HNF1beta; HNF1alpha had a major stimulatory effect, whereas HNF1beta acted as a trans-dominant inhibitor of HNF1alpha-mediated enhancer activity. These results suggested that the net expression of the DBP gene reflected a balance between the two major HNF1 species; the relative abundance of HNF1alpha and HNF1beta proteins in a cell may thus play a critical role in determining the pattern of gene expression.

Transactivation of the ApoCIII promoter by ATF-2 and repression by members of the Jun family.

It was shown previously that cytokines such as tumor necrosis factor-alpha that stimulate signal transduction pathways involving transcription factors ATF-2 and Jun repress apoCIII promoter activity in HepG2 cells. In the present study, DNase I footprinting analysis established that ATF-2 protected three regions in the apoCIII promoter. One region (-747/-726) present in the apoCIII enhancer is within the previously identified footprint I and has overlapping boundaries with the binding sites of Sp1 (-764/-742) and HNF-4 (-736/-714). The other two regions represent new footprints and have been designated D/E (-219/-199) and B/C (-102/-75). The B/C region overlaps with the previously identified footprint B which contains an HNF-4 binding site (-87/-63). Cotransfection experiments in HepG2 cells showed that ATF-2 transactivated the -890/+24 apoCIII promoter 1.6-fold. In addition, mutations in the proximal D/E (-219/-199) and distal I (-747/-726) ATF-2-binding sites reduced the apoCIII promoter strength to 33 and 9% of control, respectively, indicating that ATF-2 is a positive regulator of apoCIII gene transcription. Cotransfections with ATF-2 and HNF-4 expression plasmids resulted in additive transactivation of the apoCIII promoter. Furthermore, apoCIII promoter constructs bearing mutations in the D/E and I ATF-2 binding sites were efficiently transactivated by HNF-4, suggesting that these two factors contribute independently to the apoCIII promoter strength. Members of the Jun family (c-Jun, JunB, and JunD) caused a dose-dependent inhibition of the -890/+24 apoCIII promoter activity. A synthetic promoter containing the apoCIII enhancer in front of the minimal AdML promoter was also repressed by Jun. In contrast, apoCIII promoter segments lacking the enhancer region were transactivated by Jun. The findings suggest that homodimers of Jun or heterodimers of Jun with other AP-1 subunits could be responsible for the observed repression by interfering with the function(s) of the apoCIII enhancer. Repression by Jun could be reversed in the presence of ATF-2 and HNF-4, suggesting that ATF2 and possibly Jun/ATF-2 heterodimers exert a positive effect on apoCIII gene transcription, as opposed to Jun homodimers or heterodimers with other AP-1 members. These findings suggest a role for members of the Jun family and ATF-2 that participate in signal transduction pathways in basal or induced apoCIII promoter activity in cells of hepatic origin.

Characterization of the Aldolase B Intronic Enhancer

The aldolase B gene is transcribed at a high level in the liver, kidney, and small intestine. This high level of gene expression results from cooperation between a weak but liver-specific promoter and an intronic activator. A deletional study of this activator present in the first intron allowed us to ascribe the maximal enhancer function to a 400-base pair (bp) fragment (+1916 to + 2329). This enhancer is highly liver-specific and enhances the activity of heterologous minimal promoters in a position and distance-independent fashion in transiently transfected Hep G2 hepatoma cells. The aldolase B enhancer is composed of two domains, a 200-bp module (Ba) inactive by itself but which synergizes with another 200-bp module (Bb) that alone retains 25% of the total enhancer activity. The Bb sequence is 76% homologous between human and rat genes and contains several binding sites for liver-enriched nuclear factors. By electrophoretic mobility shift assays, we demonstrated that elements 5 and 7 bind hepatic nuclear factor 1 (HNF1), whereas element 2 binds hepatic nuclear factor 4 (HNF4). A functional analysis of the enhancer whose elements have been mutated demonstrated that mutation of any of the HNF1 sites totally suppressed enhancer activity, whereas mutation of the HNF4-binding site reduced it by 80%.

Regulation of a transcription factor network required for differentiation and metabolism.

Hepatocyte nuclear factors (HNFs) are a heterogeneous class of evolutionarily conserved transcription factors that are required for cellular differentiation and metabolism. Mutations in HNF-1alphaand HNF-4alpha genes impair insulin secretion and cause type 2 diabetes. Regulation of HNF-4/HNF-1 expression by HNF-3alpha and HNF-3beta was studied in embryoid bodies in which one or both HNF-3alpha or HNF-3beta alleles were inactivated. HNF-3beta positively regulated the expression of HNF-4alpha/HNF-1alpha and their downstream targets, implicating a role in diabetes. HNF-3beta was also necessary for expression of HNF-3alpha. In contrast, HNF-3alpha acts as a negative regulator of HNF-4alpha/HNF-1alpha demonstrating that HNF-3alpha and HNF-3beta have antagonistic transcriptional regulatory functions in vivo. HNF-3alpha does not appear to act as a classic biochemical repressor but rather exerts its negative effect by competing for HNF-3 binding sites with the more efficient activator HNF-3beta. In addition, the HNF-3alpha/HNF-3beta ratio is modulated by the presence of insulin, providing evidence that the HNF network may have important roles in mediating the action of insulin.

Winged helix hepatocyte nuclear factor 3 and POU-domain protein Brn-2/N-Oct-3 bind overlapping sites on the neuronal promoter of human aromatic l-amino acid decarboxylase gene

The neuronal promoter of human aromatic l-amino acid decarboxylase gene has been analysed to elucidate the mechanisms of neuron type-specific expression. The (−560/+92) promoter segment was sufficient to direct luciferase expression at a higher level in SK-N-BE neuroblastoma cells, than in CHP126 neuroepithelia, HepG2 hepatoma or SK-Hep1 epithelioma cells. Deletions experiments showed that this segment contained a neuronal-specific (element T1) and a SK-N-BE-specific (element N1) cis-activating sequences. Element T1 (−72/−36) bound Sp1 and NF-Y proteins, and unidentified neuronal-specific factors. Element N1 (−102/−72) bound cell-specific factors, identified as HNF-3, N-Oct-3/Brn-2 and N-Oct-2. HNF-3 proteins recognized the sequence TCAGTAAATA that matches the consensus motif. Oct-1, N-Oct-2 and N-Oct-3 bound the AAATAATGC sequence that overlaps the HNF-3 binding site. In addition, we show that the HNF-3 binding sites from aldolase C and HNF-3β gene promoters also bind N-Oct-2 and N-Oct-3 proteins. These data suggest a functional interplay of winged helix/forkhead and POU-domain transcription factors on a variety of neuronal gene promoters.

E1A represses apolipoprotein AI enhancer activity in liver cells through a pRb- and CBP-independent pathway.

The apolipoprotein AI (apoAI) promoter/enhancer contains multiple cis -acting elements on which a variety of hepatocyte-enriched and ubiquitous transcription factors function synergistically to regulate liver-specific transcription. Adenovirus E1A proteins repress tissue-specific gene expression and disrupt the differentiated state in a variety of cell types. In this study expression of E1A 12Sor 13S in hepatoblastoma HepG2 cells repressed apoAI enhancer activity 8-fold. Deletion mapping analysis showed that inhibition by E1A was mediated by the apoAI promoter site B. E1A selectively inhibited the ability of HNF3beta and HNF3alpha to transactivate reporter genes controlled by the apoAI site B and the HNF3 binding site from the transthyretin promoter. The E1A-mediated repression of HNF3 activity was not reversed by overexpression of HNF3beta nor did E1A alter nuclear HNF3beta protein levels or inhibit HNF3 binding to DNA in mobility shift assays. Overexpression of two cofactors known to interact with E1A, pRb and CBP failed to overcome inhibition of HNF3 activity. Similarly, mutations in E1A that disrupt its interaction with pRb or CBP did not compromise its ability to repress HNF3beta transcriptional activity. These data suggest that E1A inhibits HNF3 activity by inactivating a limiting cofactor(s) distinct from pRb or CBP.

Effect of developmental stage or differentiation modulators on functional expression of maxi-K channels in cultured chick hepatocytes

PLC-'/1 and maps to the distal portion of mouse chromosome 2. Reverse • L0708 transcriptase PCR assay utilizing Cded specific primers demonstrate Cded mRNAs in liver, heart, and brain tissues, throughout mid-embryonic mouse gestation. Analysis of the Cded promoter region shows high GC-content, high ratio of CpG/GpC, multiple GC-boxes, an absent TATA box, as well as AP2, and HNF1 binding sites. These characteristics are also found in several genes important in the regulation of cell growth or DNA synthesis, such as transforming growth factor-131, c-Ha-ras, nerve growth factor and epidermal growth factor receptor. Conclusions: The structural details described here are significant because Cded, by tethering other proteins with its PH domain, could play a vital role in liver development, particularly by involving the signal transduction process in hepatocyte formation.

Type I Protein C Deficiency Caused by Disruption of a Hepatocyte Nuclear Factor (HNF)-6/HNF-1 Binding Site in the Human Protein C Gene Promoter

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by proteolytic inactivation of factors Va and VIIIa. Individuals affected by protein C deficiency are at risk for venous thrombosis. One such affected individual was shown earlier to carry a -14 T --> C mutation in the promoter region of the protein C gene. It is shown here that the region around this mutation corresponds to a binding site for the transcription factor hepatocyte nuclear factor (HNF)-6 and that this site completely overlaps an HNF-1 binding site. HNF-6 and HNF-1 bound in a mutually exclusive manner. The -14 T --> C mutation reduced HNF-6 binding. In transient transfection experiments, HNF-6 transactivated the wild-type protein C promoter and introduction of the mutation abolished transactivation by HNF-6. Similar experiments showed that wild-type protein C promoter activity was reduced by cotransfection of an HNF-1 expression vector. This inhibiting effect of HNF-1 was reversed to a stimulatory effect when promoter sequences either upstream or downstream of the HNF-6/HNF-1 site were deleted. It is concluded that HNF-6 is a major determinant of protein C gene activity. Moreover, this is the first report describing the putative involvement of HNF-6 and of an HNF-6 binding site in human pathology.

Functional analysis of ground squirrel hepatitis virus enhancer II.

We have characterized a major regulatory element of ground squirrel hepatitis virus (GSHV) located within a 90-nucleotide fragment of the core promoter upstream sequences and have compared its organization to that of woodchuck hepatitis virus (WHV) enhancer II (We2). The GSHV element (Ge2) stimulates transcription from the viral core promoter and heterologous promoters in an orientation-independent manner but displays a lower level of activity than We2 in transient transfection assays in human hepatoma cells. The general organization of Ge2 into binding sites for the liver-enriched HNF-1 and HNF-4 proteins and for ubiquitous factors of the NF1 and Oct families was found to be mostly conserved with respect to the homologous We2 region. Accordingly, transactivation by HNF-1 and HNF-4 plays an essential role in the liver-specific transcriptional activity of both the GSHV and WHV core promoters. Distinctive features of the GSHV enhancer consist of its ability to bind C/EBP family factors in a central motif that overlaps with one of the two HNF-4 sites and its differential binding affinities for HNF-4.

Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

Differential regulation of the pre-C and pregenomic promoters of human hepatitis B virus by members of the nuclear receptor superfamily.

Synthesis of the pre-C and pregenomic RNAs of human hepatitis B virus (HBV) is directed by two overlapping yet separate promoters (X. Yu and J. E. Mertz, J. Virol. 70:8719-8726, 1996). Previously, we reported the identification of a binding site for the nuclear receptor hepatocyte nuclear factor 4 (HNF4) spanning the TATA box-like sequence of the pre-C promoter. This HNF4-binding site consists of an imperfect direct repeat of the consensus half-site sequence 5'-AGGTCA-3' separated by one nucleotide; i.e., it is a DR1 hormone response element (HRE). We show here that other receptors, including chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1), human testicular receptor 2 (TR2), and peroxisome proliferator-activated receptors (PPARs) as heterodimers with retinoid X receptors (RXRs), can also specifically bind this DR1 HRE. Synthesis of the pre-C and pregenomic RNAs was affected both in transfected hepatoma cells and in a cell-free transcription system by the binding of factors to this DR1 HRE. Interestingly, whereas some members of the hormone receptor superfamily differentially repressed synthesis of the pre-C RNA (e.g., HNF4 and TR2) or activated synthesis of the pregenomic RNA (e.g., PPARgamma-RXRalpha), other members (e.g., COUP-TF1) coordinately repressed synthesis of both the pre-C and pregenomic RNAs. Thus, HBV likely regulates its expression and replication in part via this DR1 HRE. These findings indicate that appropriate ligands to nuclear receptors may be useful in the treatment of HBV infection.

Expression of hepatocyte nuclear factor 6 in rat liver is sex-dependent and regulated by growth hormone.

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the CYP2C12 gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the CYP2C12 promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the CYP2C12 promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.

Characterization of the hepatitis B virus major surface antigen promoter hepatocyte nuclear factor 3 binding site.

Transcription of the HBV 2.1 kb RNAs is regulated by the major surface antigen promoter. Previously, transient transfection analysis identified regulatory sequence elements in this promoter located between -189 and +1 which govern the level of transcription from this promoter and appear to bind only ubiquitous transcription factors including NF1, Sp1 and NF-Y. However, in vivo transcription analysis in transgenic mice has demonstrated that the expression of the HBV 2.1 kb RNAs is largely restricted to hepatocytes. In this study, the presence of a functional HNF3 transcription factor binding site located between -231 and -240 in the major surface antigen promoter suggests that the in vivo liver-restricted expression of the 2.1 kb RNAs may be governed by this liver-enriched transcription factor. The identification of a functional HNF3 binding site upstream of the DNA polymerase open reading frame also supports the contention that transient transfection analysis may fail to detect all of the cis-acting regulatory sequence elements involved in modulating the level of transcription from the viral promoters.

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.

Transcriptional regulation by HNF-4 of the steroid 15α-hydroxylase P450 ( Cyp2a-4) gene in mouse liver

The mouse P450 gene Cyp2a-4 encodes the hepatic steroid 15α-hydroxylase. We have defined in the 5′-flanking sequence of Cyp2a-4 gene, a composite regulatory element ( −61AGACCAAAGTC CG GCCTTC −42) which contains a potential CpG methylation site at position −50. Gel-shift assays indicate that this element consists of overlapped binding sites for a hepatocyte-enriched transcription factor HNF-4 and a Sp1-like protein. Moreover, transcription of the Cyp2a-4 gene is activated by coexpression of HNF-4 in HepG2 cells. A mutation (C at −50 to A) abolishes the binding of HNF-4 to the element as well as the transcriptional activation by HNF-4. The methylated C at position −50, however, does not affect HNF-4 binding. Neither the mutation nor the methylation at position −50 affect the binding of Sp1-like protein to the element. It appears, therefore, that HNF-4 activates the hepatic transcription of Cyp2a-4 gene through its direct binding to the regulatory element regardless of the methylation at position −50.

Characterization of Hepatic-specific Regulatory Elements in the Promoter Region of the Human Cholesterol 7α-Hydroxylase Gene

Cholesterol 7alpha-hydroxylase is the rate-limiting enzyme in the degradation of cholesterol to bile salts and plays a central role in regulating cholesterol homeostasis. The mechanisms involved in the transcriptional control of the human gene are largely unknown. HepG2 cells represent an appropriate model system for the study of the regulation of the gene. To identify liver-specific DNA sequences in the promoter of the human CYP7 gene, we first examined the DNase I hypersensitivity in the 5'-region of the gene. An area of hypersensitivity was observed in the region from -50 to -200 of the human gene in nuclei from transcriptionally active HepG2 cells, but was absent in transcriptionally inactive HeLa cell nuclei or in free DNA. Various 5'-promoter deletion constructs were made and transfected into HepG2 cells. About 300 base pairs of upstream sequence are required for high level promoter activity of the human CYP7 gene in HepG2 cells. DNase I footprinting of the hypersensitive region revealed nine protected sequences. Gel retardation experiments demonstrated binding of HNF-3 to the segment from -80 to -70 and of hepatocyte nuclear factor HNF-4 (and ARP-1) to the segment from -148 to -127 of the human CYP7 promoter. Deletion of either of these sites depressed promoter activity in HepG2 cells. A third region from -313 to -285 is bound by members of the HNF-3 family and acts as an enhancer. Additionally, the segment from -197 to -173 binds a negative regulatory protein that is present in Chinese hamster ovary cell extracts and in HepG2 cell extracts. These experiments define the key control elements responsible for basal transcription of the human CYP7 gene in HepG2 cells.

Dietary fat, genes, and human health.

These studies show that a macronutrient like dietary fat plays an important role in gene expression. In the cases presented here, dietary fat regulates gene expression leading to changes in carbohydrate and lipid metabolism. The interesting outcome of these studies is the finding that the molecular targets for dietary fat action did not converge with the principal targets for hormonal regulation of gene transcription, like hormone receptors. Instead, PUFA-RF targets elements that play key ancillary roles in gene transcription. This is important because it shows how PUFA can interfere with hormone regulation of a specific gene without having generalized effect on overall hormonal control, i.e. PUFA effects are promoter-specific. How PUFA-RF interferes with gene transcription will require the isolation and characterization of PUFA-RF along with the tissue-specific factors targeted by PUFA-RF. A different story emerges when fatty acids activate PPAR. Based on the studies presented here and elsewhere, long chain-highly unsaturated fatty acids (like 20:5,n-3 and 22:6, n-3) or high levels of fat activate PPAR. PPAR directly activates genes like AOX, but also inhibits transcription of genes like S14, FAS, apolipoprotein CIII, transferrin. For S14, the mechanism of inhibition involves sequestration of RXR, a critical factor for T3 receptor binding to DNA. Thus, PPAR can have generalized effects on T3 action or on other nuclear receptors, like vit. D (VDR) and retinoic acid (RAR) receptors, that require RXR for action. For apolipoprotein CIII and transferrin, PPAR/RXR heterodimers compete for HNF-4 binding sites (DR + 1). In addition to HNF-4, COUP-TF, ARP-1 and RXR all bind the DR + 1 type motif. These factors are important for tissue-specific regulation of gene transcription. PPAR can potentially interfere with the transcription of multiple genes through disruption of nuclear receptor signaling leading to changes in phenotype. Clearly, more studies are required to assess the role PPAR plays in the fatty acid regulation of gene transcription and its contribution to chronic disease. Finally, it is clear that dietary fat has the potential to affect gene expression through multiple pathways. Depending on the gene examined, PUFA might augment or abrogate gene transcription which leads to specific phenotypic changes altering metabolism, differentiation or cell growth. These effects can be beneficial to the organism, such as the n-3 PUFA-mediated suppression of serum triglycerides or detrimental, like the saturated and n-6 PUFA-mediated promotion of insulin resistance. How such effects contribute to the onset or progression of specific neoplasia is unclear. However, studies in metabolism might provide important clues for this connection.