Systematic characterisation of the rat and human CYP24A1 promoter.

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) ligands VDR (vitamin D receptor) and binds to the vitamin D response element (VDRE) located within target genes to regulate their transcription. Previously we showed that 1,25D-mediated rat CYP24A1 induction via the two critical VDREs is dependent on a short stretch of nucleotides called vitamin D stimulating element (VSE), located approximately 30bp upstream of VDRE-1 in the rat CYP24A1 promoter. We have now undertaken systematic analysis of the human CYP24A1 and rat CYP24A1 promoters to determine if the VSE is present in the human promoter. Using electrophoretic mobility shift and dual-luciferase reporter assays, we show that the VSE is absent in the human CYP24A1 promoter. In addition, we show that 1,25D-mediated induction of human CYP24A1 is dependant upon a promoter region spanning nucleotides -470 to -392 of the human CYP24A1 promoter.

Regulation of the 5'-flanking region of the human CYP27B1 gene in osteoblast cells.

Synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is catalysed by the enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). Regulation of CYP27B1 gene expression is poorly understood, particularly in non-renal tissues including bone where 1,25(OH)(2)D(3) is hypothesised to serve autocrine/paracrine roles. Transient transfection of ROS 17/2.8 osteoblast-like cells with reporter gene constructs containing deletions of the 5'-flanking region of the human CYP27B1 gene revealed a proximal promoter, enhancer region and strong upstream repressive region. Putative CCAAT and GC boxes, as well as Ets protein binding sites were shown to contribute to promoter and enhancer activities respectively in common with kidney and prostate cells. Inhibition of basal expression was largely attributed to a palindrome 5'-GTCTCAGAC-3' (-1015/-1007bp) that contains two putative canonical Smad binding elements. We conclude that repression of CYP27B1 gene expression may be a common event but the novel inhibitory elements we have identified may be unique to osteoblasts.

25-Hydroxyvitamin D requirement for maintaining skeletal health utilising a Sprague-Dawley rat model.

To study the role of vitamin D to optimise bone architecture, we have developed an animal model to investigate the effects of frank vitamin D-deficiency as well as graded depletion of circulating 25-hydroxyvitamin D(3) (25D) levels on the skeleton. Rats fed on dietary vitamin D levels from 0 to 500 ng/day achieved diet-dependent circulating levels of 25D ranging from 11 to 115 nmol/L. Levels of serum 1,25-dihydroxyvitamin D(3) (1,25D) increased as dietary vitamin D increased between 0 and 200 ng/day at which point a maximum level was achieved and retained with higher vitamin D intakes. The renal levels of 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) mRNA were highest in animal groups fed on vitamin D between 0 and 300 ng/day. In contrast, renal 25-hydroxyvitamin D 24-hydroxylase (CYP24) mRNA levels increased as dietary vitamin D increased achieving maximum levels in animals receiving 500 ng vitamin D/day. This animal model of vitamin D depletion is suitable to provide invaluable information on the serum levels of 25D and dietary calcium intake necessary for optimal bone structure. Such information is essential for developing nutritional recommendations to reduce the incidence of osteoporotic hip fractures.

Response of the 5'-flanking region of the human 25-hydroxyvitamin D 1alpha-hydroxylase gene to physiological stimuli using a transgenic mouse model.

The enzyme 25-hydroxyvitamin D 1alpha-hydroxylase, or CYP27B1, is the key enzyme in the two-step activation process of vitamin D to 1,25-dihydroxyvitamin D (1,25D). While a number of regulators of the renal CYP27B1 enzyme activity have been recognized for some years, their underlying molecular mechanisms remain largely unknown, and the DNA regions involved in the in vivo regulation of gene expression by these factors have not been delineated. We have generated a transgenic mouse line that expresses 1501 bp of 5' flanking region together with 44 bp of 5' untranslated region of the human CYP27B1 gene fused to the firefly luciferase reporter gene. Animals expressing the luciferase gene demonstrated that both luciferase protein and mRNA for CYP27B1 were localized to proximal convoluted tubule cells of the kidney. In 2-week-old animals, the expression of the transgene and the endogenous CYP27B1 mRNA levels in the kidney were highest and fell with increasing age. Both reporter gene expression and CYP27B1 mRNA levels were downregulated in response to increasing amounts of dietary calcium in a dose-dependent manner. Vitamin D deficiency resulted in an increase in both the reporter gene and CYP27B1 expression. Interestingly, the increase in CYP27B1 mRNA levels was substantially higher than the increase in reporter gene expression, suggesting either that there is a post-transcriptional mechanism that increases the amount of CYP27B1 mRNA or that other regulatory elements are required to maximize the effect of vitamin D deficiency. These findings demonstrate that the 1501 bp 5' flanking region of the CYP27B1 gene directs expression to the proximal convoluted tubules of the kidney and is responsible for increasing transcriptional activity when dietary calcium and vitamin D levels are depleted. It also responds in the kidney to the physiological regulators of development and ageing.

Calcitonin stimulates expression of the rat 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter in HEK-293 cells expressing calcitonin receptor: identification of signaling pathways.

Regulation of the gene for renal 25-hydroxyvitamin D-24-hydroxylase (CYP24) is important for controlling the level of circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We report here for the first time that the peptide hormone calcitonin significantly stimulates expression of a rat CYP24 promoter-luciferase construct in both transiently and stably transfected kidney HEK-293 cells. A GC box at -114/-101 and a CCAAT box at -62/-51 have been identified that underlie both basal expression of the CYP24 promoter and the calcitonin inductive response. Data from overexpression studies suggested that Sp1 and NF-Y are the proteins that function through the GC and CCAAT boxes respectively. ERK1/2 signaling pathways were not involved in the calcitonin-mediated response, since stimulation of the promoter was unaffected by the pharmacological ERK1/2 inhibitor PD98059 and by a dominant negative mutant of ERK1/2 (ERK1K71R). In contrast, calcitonin induction but not basal expression was dependent on protein kinase A and protein kinase C (PKC) activities with the inhibitors H89 and calphostin C lowering induction by 50-60%. The atypical PKC, PKCzeta contributes to calcitonin induction, but not to basal expression of the CYP24 promoter, since overexpression of a dominant negative clone PKCzetaK281 M lowered induction by 50%. Cotransfection of a dominant negative form of Ras resulted in calcitonin-mediated induction being reduced also by about 50%. A Ras-PKCzeta signaling pathway for calcitonin action is proposed, which acts through the GC box. The findings have been extrapolated to the in vivo situation where we suggest that induction of renal CYP24 by calcitonin could be important under hypercalcemic conditions thus contributing to the lowering of circulating 1,25(OH)2D3 levels.

Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase- polymerase chain reaction.

Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.

Vitamin D metabolism: new concepts and clinical implications.

The vitamin D endocrine system plays a primary role in the maintenance of calcium homeostasis as well as exerting a wider range of biological activities including the regulation of cellular differentiation and proliferation, immunity, and reproduction. Most of these latter activities have been demonstrated using in vitro techniques. A major issue is to place such in vitro findings into their physiological context. Vitamin D exerts its genomic effects through a nuclear gene transcription factor, the vitamin D receptor (VDR), while metabolism of vitamin D both to its biologically active form, as well as to its excretory product, plays a major role in determining biological activity at the tissue level. Considerable information has become available recently concerning the metabolism of vitamin D both in the kidney and in non-renal tissues. These data confirm the endocrine action of vitamin D through renal metabolism which provides 1,25 dihydroxyvitamin D (1,25D) to the circulation. The major organ responding to the endocrine action of 1,25D is the intestine where it controls absorption of calcium and phosphate. Preliminary information regarding the contribution of tissue-specific production of 1,25D to its paracrine/autocrine activity is now becoming available. In bone cells, these data provide evidence for the modulation of cell proliferation and stimulation of bone cell maturation. The relevance of these concepts to the clinical laboratory is discussed in the context of vitamin D insufficiency and the increased risk of hip fracture amongst the elderly.

A duplicated HNF-3 binding site in the CYP2H2 promoter underlies the weak phenobarbital induction response.

We are investigating induction of chicken cytochrome P450 genes by the sedative phenobarbital in chick embryo hepatocytes. The steady-state level of induced mRNA for the gene CYP2H1 is about 10-fold higher than that of a second gene, CYP2H2. Here, we show that a difference in drug-responsive enhancer activity does not underlie the differential response of these genes to phenobarbital since upstream enhancer regions are identical in these genes. The first 198 bp of CYP2H2 promoter sequence is identical to the CYP2H1 gene promoter, except that the functional HNF-3 binding site in the CYP2H1 promoter is replaced with a duplicated HNF-3 sequence in the CYP2H2 promoter. Transient expression analysis established that the promoter activity of the CYP2H2 gene was about ninefold lower than the CYP2H1 gene. Mutagenesis of either of the partially overlapping HNF-3 sites in the CYP2H2 gene substantially induced drug induction. Gel-shift analysis established that each of these HNF-3 sites bound HNF-3, most likely HNF-3beta. In-vitro footprint analysis demonstrated that all the identified sites in the CYP2H2 promoter bound protein except the duplicated HNF-3 region. However, protein binding was observed by in-vitro footprint analysis if either of the HNF-3 sites was mutated in the CYP2H2 promoter. Hence, duplication of the HNF-3 site in the CYP2H2 promoter does not allow binding of HNF-3 in the promoter context and may be predominantly, if not exclusively, responsible for the poor response of the CYP2H2 gene to phenobarbital.

The antiglucocorticoid RU486 inhibits phenobarbital induction of the chicken CYP2H1 gene in primary hepatocytes.

The cytochrome P450 gene CYP2H1 is highly induced by phenobarbital in chick embryo hepatocytes. Recent studies have established that the orphan nuclear receptor CAR plays a critical role in the induction mechanism. Here, we show that a high concentration of the potent glucocorticoid and progesterone receptor antagonist RU486 almost completely blocks phenobarbital-induced accumulation of CYP2H1 mRNA in hepatocytes yet has no effect on basal expression. In marked contrast, CYP2H1 mRNA induced by the phenobarbital-type inducers glutethimide and 2-allylisopropylacetamide is not affected by RU486. RU486 inhibition is not mediated through the glucocorticoid or progesterone receptors. Transient transfection studies showed that RU486 does not repress through activation of the orphan receptor PXR and subsequent competition with CAR for binding to the upstream drug-responsive 556-base-pair enhancer. Additionally, none of the known functional transcription factor binding sites found in the enhancer region was a target of RU486 inhibition. Using an artificial construct containing multiple CAR binding sites, we also established that RU486 has no direct effect on the activity of exogenously expressed CAR. There is no evidence that phenobarbital binds to CAR; we propose that RU486 inhibits phenobarbital induction, either by interfering with a phenobarbital-dependent mechanism responsible for nuclear import of CAR or with the metabolism of phenobarbital to the true inducer. Whether a novel nuclear receptor that binds RU486 at high concentrations plays a role in the inhibitory action of RU486 is an interesting possibility.

Overview of regulatory cytochrome P450 enzymes of the vitamin D pathway.

Cytochromes P450c1 and P450c24 are regulated hydroxylase enzymes that direct the bioactivation and metabolic degradation of vitamin D. The bioactivation pathway is regulated by cytochrome P450c1 through its synthesis of 1alpha,25(OH)(2)D(3), the hormonally active form of the vitamin. Expression of the P450c1 gene is regulated at the transcription level. Promoter regions within the P450c1 gene have been identified that respond to cAMP and 1alpha,25(OH)(2)D(3) during the respective up- and down-regulation of P450c1 gene expression. The diametric action of 1alpha,25(OH)(2)D(3) to up-regulate P450c24 gene expression is discussed in the context of two vitamin D response elements (VDREs) that are linked functionally to an adjoining Ets-binding site. It is apparent from sequence-derived data that the P450c1 and P450c24 enzymes share only 10-25% sequence identity, yet they display functionally similar domains that are conserved across the family of cytochrome P450 enzymes. Expression of E. coli recombinant P450c1 and P450c24 enzymes, and the substrate-binding parameters for P450c24 are discussed. Finally, the natural point mutations in human P540c1 from patients with pseudovitamin D-deficiency rickets (PDDR) are discussed in the context of the enzyme's structure and function.

Osteoblast gene expression in rat long bones: effects of ovariectomy and dihydrotestosterone on mRNA levels.

The steroid sex hormones exert major effects on bone formation although the molecular events associated with their activity remain unclear. We have investigated the effects of ovariectomy and dihydrotestosterone (DHT) administration to both sham-operated and ovariectomized (ovx) rats on the bone mRNA levels of osteoblast genes. Rats were randomly allocated to either sham or ovariectomy operations and were administered either vehicle or 40 mg/ kg body weight DHT by silastic tube implants at the time of operation for 8 weeks, at which time they were killed and total RNA was extracted from the long bones. Northern blot analysis indicated that the mRNA levels of the bone cell genes alpha1(I) collagen, alkaline phosphatase, osteocalcin, and osteopontin were markedly increased in ovx rats between 6- and 30-fold. DHT administration to ovary-intact, estrogen-sufficient rats increased the mRNA levels of alpha1(I) collagen, alkaline phosphatase, osteopontin, and osteocalcin between 3- and 9-fold. In contrast, DHT did not alter levels of these mRNA species in ovx rats. The data demonstrate that estrogen deficiency increased mRNA levels of genes expressed during osteoblast development and suggest an interplay between estrogen and androgen action in regulating the expression of a number of bone cell genes.

Regulation of rat cytochrome P450C24 (CYP24) gene expression. Evidence for functional cooperation of Ras-activated Ets transcription factors with the vitamin D receptor in 1,25-dihydroxyvitamin D(3)-mediated induction.

Transcription of the rat CYP24 gene is induced by 1, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) through two vitamin D response elements (VDREs). A functional Ras-dependent Ets-binding site (EBS) was located downstream from the proximal VDRE and was critical to 1,25(OH)(2)D(3)-mediated induction. Cotransfection of Ets-1 and Ets-2 stimulated induction, which was lost when the EBS was mutated. Multiple nuclear-protein complexes from COS-1 cells bound to the EBS in which three complexes were immunologically related to Ets-1. Transcriptional synergy was observed between the proximal VDRE and adjacent EBS as was the attendant formation of a ternary complex between vitamin D receptor- retinoid X receptor (VDR. RXR) and Ets-1. In the absence of 1,25-(OH)(2)D(3) or in the presence of an inactive proximal VDRE, the EBS failed to respond to exogenous Ets-1. However, Ets-1 increased basal expression when cotransfected with a mutant VDR. The inductive action of 1, 25-(OH)(2)D(3) was substantially increased by Ras, which was ablated by mutagenesis of the EBS or by expression of a mutated Ets-1 protein (T38A). EBS contribution to hormone induction was prevented by manumycin A, an inhibitor of Ras farnesylation. A fundamental role was established for transcriptional cooperation between Ras-activated Ets proteins and the VDR.RXR complex in mediating 1, 25-(OH)(2)D(3) action on the CYP24 promoter.

Regulation of erythroid 5-aminolevulinate synthase expression during erythropoiesis.

Erythroid tissue is the major site of heme production in the body. The synthesis of heme and globin chains is coordinated at both the transcriptional and post-transcriptional levels to ensure that virtually no free heme or globin protein accumulates. The key rate-controlling enzyme of the heme biosynthetic pathway is 5-aminolevulinate synthase (ALAS) and an erythroid-specific isoform (ALAS2) is up-regulated during erythropoiesis. Differentiation of embryonic stem cells with a disrupted ALAS2 gene has established that expression of this gene is critical for erythropoiesis and cannot be compensated by expression of the ubiquitous isoform of the enzyme (ALAS1). Interestingly, heme appears to be important for expression of globin and other late erythroid genes and for erythroid cell differentiation although the mechanism of this effect is not clear. Transcriptional control elements that regulate the human gene for ALAS2 have been identified both in the promoter and in intronic enhancer regions. Subsequent translation of the ALAS2 mRNA is dependent on an adequate iron supply. The mechanism by which transcription of the gene for ALAS2 is increased by erythropoietin late in erythropoiesis remains an interesting issue. Erythropoietin action may result in altered levels of critical erythroid transcription factors or modulate the phosphorylation/acetylation status of these factors. Defects in the coding region of the gene for ALAS2 underlie the disease state X-linked sideroblastic anemia. In this review, we focus on the regulation and function of erythroid-specific 5-aminolevulinate synthase during erythropoiesis and its role in the X-linked sideroblastic anemia.

Expression of 25(OH)D3 24-hydroxylase in distal nephron: coordinate regulation by 1,25(OH)2D3 and cAMP or PTH.

Previous studies using microdissected nephron segments reported that the exclusive site of renal 25-hydroxyvitamin D3-24-hydroxylase (24OHase) activity is the renal proximal convoluted tubule (PCT). We now report the presence of 24OHase mRNA, protein, and activity in cells that are devoid of markers of proximal tubules but express characteristics highly specific for the distal tubule. 24OHase mRNA was undetectable in vehicle-treated mouse distal convoluted tubule (DCT) cells but was markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. 24OHase protein and activity were also identified in DCT cells by Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or parathyroid hormone [PTH-(1-34); 10 nM] was found to potentiate the effect of 1, 25(OH)2D3 on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase expression in DCT cells suggests differential regulation of 24OHase expression in the PCT and DCT. In the presence of cAMP and 1, 25(OH)2D3, a four- to sixfold induction in vitamin D receptor (VDR) mRNA was observed. VDR protein, as determined by Western blot analysis, was also enhanced in the presence of cAMP. Transient transfection analysis in DCT cells with rat 24OHase promoter deletion constructs demonstrated that cAMP enhanced 1, 25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP response elements (CREs) in the 24OHase promoter. We conclude that 1) although the PCT is the major site of localization of 24OHase, 24OHase mRNA and activity can also be localized in the distal nephron; 2) both PTH and cAMP modulate the induction of 24OHase expression by 1,25(OH)2D3 in DCT cells in a manner different from that reported in the PCT; and 3) in DCT cells, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.

Analysis of a phenobarbital-responsive enhancer sequence located in the 5' flanking region of the chicken CYP2H1 gene: identification and characterization of functional protein-binding sites.

We previously identified in the chicken CYP2H1 gene an upstream enhancer domain (-5900/-1100) that responds to phenobarbital. Deletion and restriction enzyme analyses of this domain have now identified two separate enhancer regions that respond to phenobarbital (from -5900 to -4550 and from -1956 to -1400). We have focused here on the latter and in particular a resident 240-base pair (bp) restriction enzyme fragment that retains drug responsiveness. Using deletion analysis and in vitro DNase I footprinting, transcription factor binding sites have been located in the 240-bp fragment. The sites identified are an E-box-like element, a consensus hepatocyte nuclear factor 1 site, a CCAAT box motif, and a novel site. Mutagenesis demonstrated that each site contributed to enhancer activity, although there was a weaker contribution from the CCAAT box, and that no individual site was critical for responsiveness. In keeping with the tissue-restricted expression of the CYP2H1 gene, gel shift experiments established that the proteins binding to these enhancer sites are enriched in chicken liver, kidney, and small intestine. In vitro footprint experiments showed a stronger protection with liver nuclear extracts from drug-treated chickens compared with control extracts on the E-box-like element, the CCAAT box motif, and the novel binding site; however, the basis for this apparent increase in binding remains to be determined. The proteins binding to the 240-bp fragment are different from those recently reported to be required for the activity of the phenobarbital responsive enhancer domains of rodent CYP2 genes.

Repression of basal transcription by vitamin D receptor: evidence for interaction of unliganded vitamin D receptor with two receptor interaction domains in RIP13delta1.

Repression of basal transcription of a 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) responsive 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter construct as observed in kidney cells in the absence of ligand and this repression was dependent on a functional vitamin D response element (VDRE). Basal repression was also seen with a construct where a consensus DR-3-type VDRE was fused to the thymidine kinase promoter. Expression of a dominant negative vitamin D receptor (VDR) isoform that strongly bound to the VDRE motif in the CYP24 promoter ablated basal repression. This VDR isoform lacked sequence in the hinge- and ligand-binding domains implicating one or both of these domains in basal repression. It is well known that thyroid hormone and retinoic acid receptors silence basal transcription of target genes in the absence of ligands and this repressor function can be mediated by the nuclear receptor corepressor N-CoR. Two variants of N-CoR have been described, RIP13a and RIP13delta1. N-CoR and the variants contain two receptor interaction domains, ID-I and ID-II, which are identical except region ID-II in RIP13delta1 has an internal deletion. We have used the mammalian two hybrid system to investigate whether VDR, in the absence of ligand 1,25-(OH)2D3, can interact with these domains. The data showed that unliganded VDR does not interact with either ID-I or ID-II from RIP13a and RIP13delta1, but does interact strongly with a composite domain of ID-I and ID-II from RIP13delta1 (but not from RIP13a) and this strong interaction is abrogated in the presence of ligand. This finding implicates RIP13delta1 in VDR-dependent basal repression of the promoter constructs under investigation. However, over-expression of RIP13delta1 in kidney cell lines did not alter basal expression of the CYP24 promoter construct. It is concluded that either the level of endogenous RIP13delta1 in these kidney cells permits maximal repression or that repression occurs by a mechanism that is independent of RIP13delta1. Alternatively, repression may be dependent on RIP13delta1 but requires an additional cofactor that is limiting in these cells.

Identification and characterization of a conserved erythroid-specific enhancer located in intron 8 of the human 5-aminolevulinate synthase 2 gene.

Thirty five kilobases of sequence encompassing the human erythroid 5-aminolevulinate synthase (ALAS2) gene have been determined. Analysis revealed a very low GC content, few repetitive elements, and evidence for the insertion of a reverse-transcribed mRNA sequence and a neighboring gene. We have investigated whether introns 1, 3, and 8, which correspond to DNase I-hypersensitivity sites in the structurally related mouse ALAS2 gene, affect expression of the human ALAS2 promoter in transient expression assays. Whereas intron 3 was marginally inhibitory, introns 1 and 8 of the human gene stimulated promoter activity. Intron 8 harbored a strong erythroid-specific enhancer activity which was orientation-dependent. Deletion analysis of this region localized enhancer activity to a fragment of 239 base pairs. Transcription factor binding sites clustered within this region include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. These sites were also identified in the corresponding intron of both the murine and canine ALAS2 genes. Mutagenesis of these conserved sites in the human intron 8 sequence and transient expression analysis in erythroid cells established the functional importance of one GATA motif and two CACCC boxes. The GATA motif bound GATA-1 in vitro. The two functional CACCC boxes each bound Sp1 or a related protein in vitro, but binding of the erythroid Krüppel-like factor and the basic Krüppel-like factor could not be detected. The intron 8 enhancer region was not activated by GATA-1 together with Sp1 in transactivation experiments in COS-1 cells indicating the involvement of a related Sp1 protein or of another unidentified erythroid factor. Overall, these results demonstrate that a GATA-1-binding site and CACCC boxes located within the human ALAS2 intron 8 are critical for the erythroid-specific enhancer activity in transfected erythroid cells, and due to the conserved nature of these binding sites across species, it seems likely that these sites play a functional role in the tissue-restricted expression of the gene in vivo.

Transcriptional activation of cytochrome P450 genes by different classes of chemical inducers.

1. We review here the molecular mechanisms underlying the xenobiotic induction of genes encoding cytochrome P450 (CYP) enzymes in the liver and other tissues. We will focus on four major families of CYP genes. 2. Members of the CYP1 gene family are induced by polycyclic aromatic hydrocarbons and this process is mediated by the basic helix-loop-helix proteins: the Ah receptor and its heterodimeric partner Arnt. Considerable progress has been made in elucidating the molecular details of this induction process. 3. CYP4 genes are activated by peroxisomal proliferators, a group of structurally diverse chemicals that also induce peroxisome proliferation. The transcriptional response is dependent on the peroxisome proliferator-activated receptor and its partner RXR, both members of the nuclear receptor superfamily; their role in the induction process has been well characterized at the molecular level. 4. In contrast, the mechanism of gene induction of CYP2 genes by phenobarbital and other structurally diverse inducers is not well understood and a specific phenobarbital-responsive receptor has not been identified. 5. Induction of the CYP3 gene family by the glucocorticoid dexamethasone appears to involve the glucocorticoid receptor, but this receptor is not apparently required for induction by metapyrone and a complete molecular understanding of the induction processes is lacking at present.

Transcriptional regulation of the human erythroid 5-aminolevulinate synthase gene. Identification of promoter elements and role of regulatory proteins.

We have characterized the 5'-flanking region of the human erythroid-specific 5-amino levulinate synthase (ALAS) gene (the ALAS2 gene) and shown that the first 300 base pairs of promoter sequence gives maximal expression in erythroid cells. Transcription factor binding sites clustered within this promoter sequence include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. GATA sites at -126/-121 (on the noncoding strand) and -102/-97 were each recognized by GATA-1 protein in vitro using erythroid cell nuclear extracts. Promoter mutagenesis and transient expression assays in erythroid cells established that both GATA-1 binding sites were functional and exogenously expressed GATA-1 increased promoter activity through these sites in transactivation experiments. A noncanonical TATA sequence at the expected TATA box location (-30/-23) bound GATA-1- or TATA-binding protein (TBP) in vitro. Conversion of this sequence to a canonical TATA box reduced expression in erythroid cells, suggesting a specific role for GATA-1 at this site. However, expression was also markedly reduced when the -30/-23 sequence was converted to a consensus GATA-1 sequence (that did not bind TBP in vitro), suggesting that a functional interaction of both factors with this sequence is important. A sequence comprising two overlapping CACCC boxes at -59/-48 (on the noncoding strand) was demonstrated by mutagenesis to be functionally important. This CACCC sequence bound Sp1, erythroid Krüppel-like factor, and basic Krüppel-like factor in vitro, while in transactivation experiments erythroid Krüppel-like factor activated ALAS2 promoter expression through this sequence. A sequence at -49/-39 with a 9/11 match to the consensus for the erythroid specific factor NF-E2 was not functional. Promoter constructs with 5'-flanking sequence from 293 base pairs to 10.3 kilobase pairs expressed efficiently in COS-1 cells as well as in erythroid cells, indicating that an enhancer sequence located elsewhere or native chromatin structure may be required for the tissue-restricted expression of the gene in vivo.

Liver-enriched transcription factors, HNF-1, HNF-3, and C/EBP, are major contributors to the strong activity of the chicken CYP2H1 promoter in chick embryo hepatocytes.

Chicken CYP2H1 promoter constructs express strongly in chick embryo hepatocytes at a level comparable with that of Rous sarcoma viral promoter. We have identified the transcription factors responsible for the active CYP2H1 promoter. Binding sites for transcription factors were located within the first 160 bp of promoter sequence using promoter deletion experiments and DNase I footprint analysis. Sequence analysis revealed characteristic sites for the liver-enriched transcription factors of the HNF-1, HNF-3, and C/EBP families and for the ubiquitous factor, USF. Protein binding to these sites was established by gel mobility shift assays. Mutagenesis and transient transfection experiments demonstrated that these sites, in combination, were responsible for the strong promoter activity with a substantial contribution from HNF-1 and HNF-3. The promoter was also active in mammalian HepG2 and COS-1 cell lines where expression was dependent on the identified transcription factor binding sites but promoter activity in the HeLa cells was low. Transactivation experiments revealed that promoter expression could be activated through the appropriate binding sites by exogenously expressed rat HNF-1alpha or HNF-1beta, rat HNF-3alpha or HNF-3beta and chicken C/EBP alpha. Transcriptional synergism between HNF-1 and C/EBP was observed in these transactivation experiments. A Barbie box-like sequence overlapped the USF element but was not functional. The results demonstrate that liver-enriched transcription factors and USF direct strong expression of the CYP2H1 promoter in transiently transfected cells. By comparison, in vivo expression of this gene in uninduced chick embryo hepatocytes is low but markedly increased by phenobarbital. Drug induction may therefore substantially reflect derepression of this inherently active promoter.