Proximal Regulatory Elements Research Articles

Both locus control region and proximal regulatory elements direct the developmental regulation of β-globin gene cluster

Using ligation-mediated PCR and in vivo footprinting methods to study the status of DNA-protein interaction at hypersensitive site 2 of locus control region and βmaj promoter of erythroid cells of fetal liver and adult bone marrow, we found that during different developmental periods, the status of DNA-protein interaction at both hypersensitive site 2 and βmaj promoter changed significantly, and indicated that locus control region might function through a looping mechanism to regulate the expression of downstream genes, and that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β-globin gene cluster participate in the regulation of developmental specificity. J. Cell. Biochem. 76:376–385, 2000. © 2000 Wiley-Liss, Inc.

Characterization of Proximal Transcription Regulatory Elements in the Rat Phospholamban Promoter

C. F. McTiernan, B. H. Lemster, C. S. Frye, D. C. Johns and A. M. Feldman. Characterization of Proximal Transcription Regulatory Elements in the Rat Phospholamban Promoter. Journal of Molecular and Cellular Cardiology (1999)31 , 2137–2153. Phospholamban is a major regulator of cardiac diastole, with alterations in expression associated with modified cardiac relaxation. To study transcriptional regulation of phospholamban expression, we made reporter constructs that expressed luciferase under control of putative promoter sequences from the rat phospholamban gene. When transfected into neonatal rat cardiomyocytes, constructs containing at least 159 nucleotides preceding the transcription start site were equally active, while truncation to −66/+64 removed all promoter activity. Constructs were more active in cardiomyocytes than in HeLa cells (which do not express phospholamban), but did not show absolute cell-type specificity of expression. Addition of sequences upstream to −4032, all of the intron (7.4 kb), or 3′UTR sequences (0.8 kb) did not enhance cell-specific expression. To focus on the basal promoter region (−159/−66), a series of deletion constructs were made that identified a novel 35 bp region (−159/−125; Phospholamban Promoter Element 1, PPE1) required for promoter activity in cardiomyocytes. Site-specific mutations identified nucleotides −150/−133 as containing most of the promoter-enhancing activity. While the rat PPE1 is highly conserved (>70%) in four other mammalian phospholamban genes, it does not contain previously characterized regulatory elements. In cardiomyocytes the PPE1 sequence markedly enhanced activity of the SV40 early promoter. A conserved CCAAT element (−83/−79) was also required for promoter activity in both cardiomyocytes and HeLa cells. Exonuclease III footprinting identified protein/DNA interactions in both the extended CCAAT box and PPE1 domains. Gel shift studies identified the CCAAT elements as binding CBF/NF-Y.

Interleukin 2 receptor signaling regulates the perforin gene through signal transducer and activator of transcription (Stat)5 activation of two enhancers.

Optimal T cell differentiation into effector cells with specialized functions requires the participation of cytokine receptor signals. In T helper cells, this process is controlled by chromatin changes and distal and proximal regulatory elements as well as specific transcription factors. Analogous events during cytotoxic T lymphocyte (CTL) differentiation remain to be identified. This process is known, however, to be crucially regulated by interleukin (IL)-2 receptor (R) signals. It is accompanied by the induction of perforin expression via a mechanism that does not entail proximal regulatory elements. In this report, transgenically expressed human perforin gene locus DNAs demonstrate that IL-2R signals target two IL-2-dependent enhancers approximately 15 and 1 kilobase upstream of the promoter. The most distal enhancer may also respond to TCR signals. In transient transfections, both enhancers required two identically spaced Stat-like elements for their activation, which was abolished by expression of a dominant negative signal transducer and activator of transcription (Stat)5 molecule, whereas a constitutively active Stat5 molecule bypassed the requirement for IL-2R signals. These results provide a molecular explanation for the activation of the perforin gene during CTL differentiation and complement the analysis of animals deficient in the activation of the IL-2R Stat signaling pathway by establishing perforin as a target gene.

Attenuation of plasminogen activator inhibitor type-1 promoter activity in serum-stimulated renal epithelial cells by a distal 5' flanking region.

Urokinase plasminogen activator (u-PA) and its fast acting type-1 inhibitor (PAI-1) localize to cellular focal adhesive structures and the adjoining proximal undersurface region, respectively (Kutz et al., J. Cell. Physiol. 176:8-18, 1997). PAI-1 may function in this locale to modulate pericellular proteolytic activity, cell-to-substrate adhesion, or matrix-dependent motility. While PAI-1 synthesis is regulated in an immediate-early response manner in growth "activated" renal cells coincident with cytoskeletal restructuring, adhesive influences both repress the amplitude and prolong the time course of serum-induced PAI-1 transcription (Ryan et al., Biochem. J. 314:1041-1046, 1996). To identify potential adhesion-responsive elements within the PAI-1 gene that function in this complex mode of expression control, reporter constructs containing defined directionally deleted PAI-1 5' genomic fragments cloned upstream of a CAT gene were employed in transient transfection assays. A 483-bp distal PAI-1 flanking segment (corresponding to nucleotides -2395 to -1912) conferred significant adhesion-dependent attenuation on serum-induced PAI-1 transcription. This 483-bp distal PAI-1 segment functioned as a repressor of reporter (CAT) activity under both adhesive and suspension culture conditions, however, when ligated upstream of either an SV40 promoter/enhancer or a minimal PAI-1 promoter. These data suggest that repressor elements located between -2395 and -1912 bp interact with more proximal adhesion-dependent regulatory elements to affect PAI-1 expression attenuation during serum stimulation of adherent renal epithelial cells.

Upstream elements bestow T-cell and haemopoietic progenitor-specific activity on the granzyme B promoter.

Cytotoxic T cells and early haemopoietic progenitors share the expression of a number of specific genes. Of these, granzyme B has attracted particular interest because of its role in inducing apoptosis during cytotoxic T cell-mediated target cell killing, and its potential role in the mobilisation and homeostasis of haemopoietic stem cells. Studies of granzyme B regulation should therefore yield valuable information concerning the molecular control of these processes, and also identify elements capable of directing gene expression to two cell types of relevance to gene therapy. Here we show that proximal regulatory elements already known to direct promoter activity in T cells are similarly active in haemopoietic progenitors. However, this activity is not strictly specific, since the promoter regions also direct low levels of reporter gene expression in fibroblasts. More importantly, we also report the presence of two previously unidentified clusters of DNaseI hypersensitive sites upstream from the murine granzyme B gene, and show that these regions impart both increased transcriptional activity and the appropriate cell type specificity on the granzyme B promoter. These upstream regulatory regions are therefore likely to play a key role in the coordination of granzyme B expression in vivo.

Regulation of E-Cadherin Gene Expression during Tumor Progression: The Role of a New Ets-Binding Site and the E-pal Element

A new regulatory region (−108 to −86), named CE, containing potential CRE- and Ets-binding sites has been identified in the murine E-cadherin promoter. The Ets-binding site (at −97 position) negatively modulates the activity of the E-cadherin promoter in expressing keratinocyte cell lines and was responsible for the specific retarded complexes obtained with the CE region. Analysis of the methylation status of the endogenous E-cadherin promoter indicated that silencing of E-cadherin expression in malignant keratinocytes cannot be explained by hypermethylation mechanisms. Furthermore, treatment with 5′-aza-2′-deoxycytidine was unable to induce the expression of E-cadherin in deficient keratinocytes. However,in vivofootprinting analysis of the endogenous E-cadherin promoter showed a very distinct pattern in expressing and nonexpressing keratinocytes. Extensive interactions in the previously postulated proximal regulatory elements and in the CE region were detected in expressing cells, while only some nucleotides of the E-pal element and of the CE region were protected in nonexpressing keratinocytes. These results indicate a complex regulation of the mouse E-cadherin promoter and support a model where the combination of positive (CCAAT-box and GC-rich region) and negative (E-pal element and CE region)cis-acting elements contribute to the final level of E-cadherin gene expression. In addition, our results show that downregulation of E-cadherin expression in transformed epidermal keratinocytes is mainly exerted through the interaction of repressor factor(s) with the E-pal element and to the lack of interaction of positive acting factors with the proximal regions.

Transcriptional activation of the murine CTP:phosphocholine cytidylyltransferase gene ( Ctpct): combined action of upstream stimulatory and inhibitory cis-acting elements

CTP:phosphocholine cytidylyltransferase plays a key role in regulating the rate of phosphatidylcholine biosynthesis. However, the proximal regulatory elements for the gene ( Ctpct) that encode this enzyme and the cognate transcription factors involved have not been characterized. Ctpct promoter activities were deduced from promoter deletion constructs linked to a luciferase reporter and transiently transfected into C3H10T1/2 and McArdle RH7777 cells. Positive regulatory elements were located between −130 and −52 bp from the transcription start site. Basal expression resided downstream between −52 and +38 bp. DNase I protection and electromobility-shift assays indicated that Sp1-related nuclear factors bind to a stimulatory, a possible inhibitory and minimal promoter element. Gel-shift assays confirmed that all three regulatory regions bound Sp1. Sp1 was further implicated when Sp1-deficient Drosophila cells were co-transfected with promoter-reporter constructs and an Sp1 construct. DNase I assays also indicated that the Ap1 binding elements could be occupied in the proximal activator and minimal promoter regions. Gel-shift assays demonstrated that the distal activator region could bind Ap1 and an unknown transcription factor. We conclude that Sp1, Ap1 and an unknown transcription factor have important roles in regulating expression of the Ctpct gene.

Negative regulation of rat hepatic aldehyde dehydrogenase 3 by glucocorticoids.

The expression of the aldehyde dehydrogenase 3 gene is known to be controlled by multiple regulatory processes. In liver, inducible expression appears to be mediated by two AhRE sequences which allow regulation of this gene by xenobiotic compounds which are ligands for the Ah receptor (Takimoto et al., 1994; this work). Constitutive expression of ALDH3 in tissues such as the cornea also involves the -3,500 region which contains an AhRE (Boesch et al., 1996; Boesch et al, 1998). However, the constellation of transcription factors which appear to interact with the AhRE in constitutively expressing corneal cells does not include either the Ah receptor nor the prototypical ARNT protein (Boesch et al., 1998). For both inducible and constitutive ALDH3 expression the more distal 5' flanking region sequences appear to interact with more proximal regulatory elements. Of particular interest is the region near -1 kb which includes the GC (-930 to -910) and cAMP (-1057 to -991) responsive elements as well as the 2 NF1 sites (-916 to -815), all of which appear to act as negative modulators of ALDH3 expression. A second putative ALDH3 negative regulatory region lies even more distal than -3,500 bp. To date, this region has been little studied, but appears to be involved in regulating both inducible and constitutive ALDH3 expression. This region may also be responsible for some of the tissue-specificity of ALDH3 expression. With respect to the work described here, in both isolated hepatocytes and HepG2 cells, no consistent negative regulation by glucocorticoids was observed in the basal expression of ALDH3. This indicates that the mechanism of GC-mediated negative regulation involves direct interference with ALDH3 gene activation mediated by the Ah receptor. Our results suggest a complex interplay between multiple transcription factors, including the GC and Ah receptors, regulates the hepatic expression of the ALDH3 gene. Active recruitment of transcription factors needed for gene transactivation, amelioration of the actions of negative regulatory trans-acting factors or cis-acting elements and/or chromatin remodeling may be required for achieve proper regulation of the aldehyde dehydrogenase 3 gene.

Regulation of the Activity of IFN-γ Promoter Elements During Th Cell Differentiation

AbstractBefore they can deliver their effector functions, CD4+ Th cells must differentiate into Th1 or Th2 subsets. We have prepared reporter transgenic mice that express the luciferase gene under the control of proximal (prox.IFN-γ) and distal (dist.IFN-γ) regulatory elements from the IFN-γ promoter to permit investigation of mechanisms that regulate IFN-γ gene transcription during Th cell differentiation. Precursor Th cells (pTh) contain high levels of cAMP response element binding protein-activation transcription factor-1 (CREB-ATF1) proteins that bind these promoter elements from the IFN-γ gene, and these cells fail to express promoter activity. Restimulated effector Th (eTh) cells have reduced levels of CREB-ATF1 proteins, their nuclear extracts exhibit reduced CREB-ATF1 binding and greater Jun and Jun-ATF2 binding to dist.IFN-γ, and eTh cells express promoter activity. CREB directly competes with effector T cell nuclear proteins for dist.IFN-γ binding, and overexpression of CREB inhibits both prox.IFN-γ- and dist.IFN-γ-directed transcription in Jurkat T cells. IL-12-stimulated Th1 differentiation increases dist.IFN-γ activity in restimulated eTh1 cells; eTh1 nuclear extracts form increased levels of Jun-ATF2-dist.IFN-γ complexes. Taken together, these data suggest that both de-repression and trans-activation contribute to the induction of IFN-γ gene transcription during Th1 differentiation.

Analysis of a ribosomal DNA intergenic spacer region from the yellow fever mosquito, Aedes aegypti.

We have sequenced the 1.8 kb intergenic spacer (IGS) region from an Aedes aegypti ribosomal DNA repeat and have identified conserved functional motifs shared with the related mosquito, Aedes albopictus. Despite the shorter length and greater homogeneity of the Ae. aegypti IGS region, the sequences of two potential RNA polymerase I core promoters and closely associated terminator elements were highly conserved. Primer extension analysis indicated that the predominant transcription initiation site in the Ae. aegypti rDNA repeat unit region lay at or near the A residue at nucleotide position 1003 in the 'upstream' RNA polymerase I promoter. This observation was supported by the higher sequence identity between the upstream promoters in Ae. aegypti and Ae. albopictus, relative to the downstream promoters. In contrast to strong similarities among proximal regulatory elements, the Ae. aegypti IGS sequence upstream of the transcription initiation site lacked the ordered array of contiguous approximately 200 nucleotide subrepeats previously found in the IGS of Ae. albopictus. In Ae. aegypti, only 4 approximately 50 nucleotide R subrepeats separated by unique sequences, followed by 2 approximately 50 nucleotide E subrepeats, occurred upstream of the transcription initiation site. Despite their differences in size and sequence, however, the four Ae. aegypti R subrepeats shared an internal structural organization that included a conserved core with 'spacer' promoters and recombinogenic elements similar to those in the longer Ae. albopictus subrepeats. These observations provide an important basis for further characterization of transcription specificity among mosquito RNA polymerase I promoters and associated regulatory elements, and contribute towards the eventual use of these elements in transgenic applications.

A 3' remote control region is a candidate to modulate Hoxb-8 expression boundaries.

Hox genes have been shown to play a key role in the acquisition of positional identity by precursors of embryonic axial, paraxial and limb structures. This function is thought to depend on the sequential, concerted expression of these genes in time and space. However the underlying molecular mechanisms of this collinear expression are still largely unknown. So far we had identified proximal regulatory elements driving expression of Hoxb-8/LacZ transgenes in Hox-like expression patterns with rostral boundaries more posterior than those of the endogenous gene. In this work we have analyzed 30 kb of 3' genomic sequences for Hoxb-8 regulatory activity in transgenic mice. We have identified a control region in the Hoxb-5/b-4 intergenic region that rostrally extends the Hoxb-8/LacZ expression domain into the posterior hindbrain. In combination with the Hoxb-8 minimal promoter, the 3' control region drives transgene expression with boundaries more anterior than those of Hoxb-8 in the neural tube. When combined with a 4.5 kb Hoxb-8 upstream sequence, where essential proximal regulatory sequences are located, the 3' control region drives transgene expression in a domain which seems to correspond to that of the endogenous Hoxb-8. By deletion analysis we have narrowed down to 550bp the regulatory activity interacting with the Hoxb-8 minimal promoter. We discuss the possibility that this remote 3' enhancer, which is the closest regulatory region found in the cluster to rostrally extend Hoxb-8/LacZ expression, could be involved in the regulation of Hoxb-8 and interact with the proximal control elements.

The Epstein-Barr virus EBNA-1 promoter Qp requires an initiator-like element

Expression of the Epstein-Barr virus (EBV) EBNA-1 protein within EBV-positive tumor cells and subpopulations of latently infected B lymphocytes in vivo is mediated by the promoter Qp. Previous studies have established that Qp is a TATA-less promoter whose activation requires only proximal regulatory elements and that it is negatively autoregulated through two EBNA-1 binding sites downstream of the transcription initiation sites. The objective of this study was to better define the properties of an essential positive regulatory element (QRE-2) adjacent to a major transcription start site of Qp and to evaluate the contributions of other potential regulatory elements proximal to the Qp start site. Using DNA affinity purification and UV cross-linking, we have identified the QRE-2-binding protein as a single polypeptide of approximately 40 kDa. The DNA-binding properties of this protein are clearly distinct from those of the TATA-binding protein, suggesting that in the absence of a TATA box, QRE-2 may function as an initiator element to direct assembly of TFIID near the transcription start site. Mutational analysis of potential regulatory elements, furthermore, indicated that the putative E2F binding sites within the EBNA-1 binding domain can exert a positive influence on Qp that is EBNA-1 independent, suggesting that these regulatory elements play an additional if not different role in Qp regulation than previously proposed. A model for the regulation of Qp consistent with the current and previous findings which provides for a simple but efficient mechanism of ensuring the EBNA-1 expression necessary to sustain long-term latency is presented.

Involvement of Nuclear Orphan Receptor NGFI-B in Transcriptional Activation of Salivary-specific R15 Gene by cAMP

Proline-rich proteins (PRPs) are selectively expressed in the acinar cells of the salivary glands and are inducible by beta-agonist isoproterenol and dietary tannins. In the previous studies of rat PRP gene, R15, the 5'-flanking region up to -1.7 kilobase pairs (kb), which was thought to contain the necessary proximal regulatory elements, failed to confer the catecholamine isoproterenol and dietary tannin inducibility to the transgene expression in the salivary glands of transgenic mice. Here we analyzed distal 5'-flanking region of R15 in order to understand the mechanisms of tissue-specific and inducible gene regulation. An upstream regulatory region located between -2.4 and -1.7 kb of the R15 5'-flanking region is demonstrated to be indispensable for the salivary-specific and inducible reporter gene expression in vivo, by transgenic approach. Element(s) within the 0.7-kb (-2.4 to -1.7) region that is able to cis-activate the expression of a heterologous reporter gene expression is further elucidated by transient transfection assays in vitro. Three distinct nuclear orphan receptor NGFI-B regulatory sequences are identified within a 184-base pair (bp) minimal control region extended from -1995 to -1812 nucleotides relative to the transcription start site. When reporter gene containing this 184-bp control region and heterologous promoter was cotransfected with the NGFI-B expression construct, a transactivation that mimics the effect of cAMP is observed in the parotid cells. Finally, mutations on all three identified NGFI-B binding sites and coexpression of a dominant negative mutant construct, pCMV-NGFI-B(Delta25-195), abolish this transactivation mediated by NGFI-B. In summary, these data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites.

Binding Specificity and Modulation of the ApoA-I Promoter Activity by Homo- and Heterodimers of Nuclear Receptors

Three proximal regulatory elements, AIB, AIC, and AID, of the apoA-I gene are necessary and sufficient for its hepatic expression in vivo and in vitro. DNA binding and competition assays showed that elements AIB and AID contain hormone response elements composed of imperfect direct repeats that support the binding of the hepatic nuclear factor-4, other nuclear orphan receptors, and the ligand-dependent nuclear receptors retinoic X receptor (RXRalpha), RXRalpha/RARalpha, and RXRalpha/T3Rbeta. Substitution mutations on repeats 1 and 2 in the hormone response sites of elements AIB and AID, respectively, abolished the binding of all nuclear receptors and reduced promoter activity to background levels, indicating the importance of both hormone response elements for the hepatic expression of the apoA-I gene. Cotransfection experiments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXRalpha homodimers transactivated the wild type promoter 150% of control, in the presence of 9-cis-retinoic acid (RA), whereas RXR alpha/T3R beta heterodimers repressed transcription to 60% of control, in the presence of T3. RXR alpha/RAR alpha and hepatic nuclear factor-4 did not affect the transcription, driven by the proximal apoA-I promoter. Potassium permanganate and dimethyl sulfate interference experiments showed that RXRalpha homodimers, RXRalpha/RARalpha, and RXRalpha/T3Rbeta heterodimers participate in protein-DNA interactions with 12, 13, and 11 out of the 14 nucleotides, respectively, that span repeats 1 and 2 and the spacer region separating them on the hormone response element of element AID. The binding of RXRalpha homodimers and RXRalpha/T3Rbeta heterodimers is associated with ligand-dependent activation by 9-cis-RA or repression by T3. Upon deletion or mutation of repeat 1, homodimeric binding of RXRalpha is lost whereas heterodimeric binding is retained. This heterodimeric binding to the mutated element AID is mediated solely by interactions with repeat 2 and one adjacent nucleotide and is confined to a heptameric core recognition motif. The interactions of the RXRalpha heterodimers with repeat 2 are associated with low levels of ligand-independent transcriptional activity. The findings suggest that the specific types of homo- and heterodimers of nuclear hormone receptors occupying the hormone response elements of apoA-I and the availability of the ligand may play an important role in the transcriptional regulation of the human apoA-I gene.

Activity of the Distal Positive Element of the Peripherin Gene Is Dependent on Proteins Binding to an Ets-like Recognition Site and a Novel Inverted Repeat Site

The peripherin gene, encoding a neuron-specific intermediate filament protein, is transcriptionally induced when PC12 cells begin to terminally differentiate into neurons in response to nerve growth factor. Previously we identified two regulatory sequences of the peripherin gene: a proximal negative element (centered at -173), which prevents peripherin expression in undifferentiated PC12 cells, and a distal positive region (-2660 to -2308) necessary for full induction of peripherin in differentiated PC12 cells (Thompson, M., Lee, E. Lawe, D., Gizang-Ginsberg, E., and Ziff, E. (1992) Mol. Cell. Biol. 12,2501-2513). Here we define a distal positive element (DPE, -2445 to -2337) within the distal positive region. Methylation interference footprinting of the DPE identified DNA-protein contact points at a novel inverted repeat sequence (AACCACTGGTT) and an Ets-like recognition sequence (CAGGAG). Functional analysis using site-directed mutagenesis demonstrates that both sites are necessary for the activity of the DPE. In addition, ternary complex formation at the DPE is dependent on both sites. Antibody competition assays confirm that an Ets family member participates in the DNA-protein complex. We have indirect evidence that the inverted repeat binding protein and the Ets-related protein interact directly with each other. Finally, we demonstrate that the DPE is constitutively active and that neuron-specific regulation of peripherin expression may be due to interaction with distal and proximal negative regulatory elements.

CACCC and GATA-1 sequences make the constitutively expressed alpha- globin gene erythroid-responsive in mouse erythroleukemia cells

Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter.

Antagonistic effects of transforming growth factor-beta on vitamin D3 enhancement of osteocalcin and osteopontin transcription: reduced interactions of vitamin D receptor/retinoid X receptor complexes with vitamin E response elements.

Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.8 cells, in which both genes are expressed, were transfected with reporter constructs driven by native (i.e. wild-type) rat OC and mouse OP promoters. TGF beta abrogated the 1,25-(OH)2D3 enhanced transcription of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions from proximal basal regulatory elements. These transcriptional effects were further investigated for contribution of the trans-activating factors, which interact with OC and OP VDREs, involving the vitamin D receptor (VDR) and retinoid X receptor (RXR). Gel mobility shift assays show that TGF beta significantly reduces induction of the heterodimers VDR/RXR complexes in 1,25-(OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analysis reveal that TGF beta does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGF beta on basal transcription. Our studies demonstrate that the inhibitory action of TGF beta on the 1,25-(OH)2D3 enhancement of OC and OP transcription in osteoblastic cells results from modulations of protein-DNA interactions at the OC and OP VDRE, which cannot be accounted for by changes in VDR protein levels. As OC and OP participate in bone turnover, our results provide insight into the contributions of TGF beta and 1,25-(OH)2D3 to VDR-mediated gene regulatory mechanism operative in bone formation and/or resorption events.

Complementation analyses for 45 mutations encompassing the pink-eyed dilution (p) locus of the mouse.

The homozygous and heterozygous phenotypes are described and characterized for 45 new pink-eyed dilution (p) locus mutations, most of them radiation-induced, that affect survival at various stages of mouse development. Cytogenetically detectable aberrations were found in three of the new p mutations (large deletion, inversion, translocation), with band 7C involved in each case. The complementation map developed from the study of 810 types of compound heterozygotes identifies five functional units: jls and jlm (two distinct juvenile-fitness functions, the latter associated with neuromuscular defects), pl-1 and pl-2 (associated with early-postimplantation and preimplantation death, respectively), and nl [neonatal lethality associated with cleft palate (the frequency of rare "escapers" from this defect varied with the genotype)]. Orientation of these units relative to genetic markers is as follows: centromere, Gas-2, pl-1, jls, jlm p, nl (equatable to cp 1 = Gabrb3); pl-2 probably resides in the c-deletion complex. pl-1 does not mask preimplantation lethals between Gas2 and p; and no genes affecting survival are located between p and cp1. The alleles specifying mottling or darker pigment (generically, pm and px, respectively) probably do not represent deletions of p-coding sequences but could be small rearrangements involving proximal regulatory elements.

The human I alpha 1 region contains a TGF-beta 1 responsive enhancer and a putative recombination hotspot.

It appears that the switch recombination machinery of a B lymphocyte targets preferentially unrearranged heavy chain genes that have been rendered transcriptionally active. Transcriptional activation of the 'germline' human C alpha 1 and C alpha 2 genes is triggered by TGF-beta 1 and is controlled by proximal positive and distal negative regulatory elements residing upstream of the alpha 1 and alpha 2 switch regions respectively. In this report we characterize the positive proximal regulatory elements and analyse their interaction with DNA binding proteins. Our data demonstrate that a 100 bp fragment that contains a cAMP responsive element (CRE)/activating transcription factor (ATF) motif, a putative Ets binding site and an element that is created by two previously described neighbouring direct repeats (DRE), can increase the basal level of transcription and confer TGF-beta 1 inducibility to a heterologous promoter in an orientation- and position-independent manner. Ubiquitously expressed DNA binding proteins interact specifically with the CRE/ATF, the Ets site and the DRE element. Additionally, nuclear proteins interact with sequences which are located downstream of this enhancer are not essential for transcription in the transient expression assays utilized; however, they contain motifs that have been previously implicated in regulating DNA recombination events. These motifs include a Chi motif and a Chi-like element previously found in the recombination hotspot region of the Bcl-2 proto-oncogene and close to chromosomal breakpoints in T-ALL lines. Our findings raise the possibility that the intervening region associated regulatory elements in addition to regulating the transcriptional activation of the Ig heavy chain genes could also facilitate the physical interaction of transcription and recombination controlling molecular mechanisms.

Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.