Positive Transcriptional Element Research Articles

Transcriptional Regulation of the Salivary Histatin Gene: Finding of a Strong Positive Regulatory Element and Its Binding Protein

Histatins are salivary proteins found and expressed in human salivary glands. They play a role in the non-immune system of antimicrobial defense, for instance, against Candida albicans. The transcriptional regulatory sequences of the histatin gene, HIS1, have remained obscure for a long time. Here, we cloned the putative promoter from human genomic DNA and tested it in a luciferase reporter system. This promoter is much more active in salivary gland cells than in other cell types. Analysis of deletion mutants revealed that the region encompassing -2254 to -1748 is a strong positive transcriptional element, and its functional core sequence (termed HTN27 box) works in correct and reverse orientations in synergy with downstream sequences, the region spanning -680 to +28 and a proximal promoter. The plus single-stranded HTN27 box is specifically bound by a 100 kDa protein that is present in HSG cells, but not in HeLa cells. These findings indicate that the regulation of the histatin gene expression may be intricate, and it seems to have a cell-type preference in the salivary gland cells.

Frequent activation of CArG binding factor-A expression and binding in N-methyl-N-nitrosourea-induced rat mammary carcinomas

We previously identified a positive transcriptional element identical to human Ha-ras response element (HRE) within the promoter of the rat Ha-ras gene. We further identified CArG binding factor A (CBF-A), a member of heterogeneous nuclear ribonuclear protein (hnRNP) gene family, as a trans-acting factor that binds the HRE sequence with high affinity in rat mammary carcinoma cells. To determine if activation of CBF-A plays a role in tumor development in vivo , we investigated CBF-A expression and binding activity in rat mammary tumors induced by N-methyl-N-nitrosourea. We found that approximately 82% of tumors expressed CBF-A at levels that were 3-20 fold higher than detected in normal mammary gland. Moreover, elevated CBF-A protein levels were invariably associated with increased binding activity to the HRE. CBF-A mRNA levels in tumors were on average elevated only two fold as compared to normal mammary gland, indicating that increased CBF-A protein levels in tumors resulted from both translational and/or post-translational regulation. The level of CBF-A expression in mammary tumors was independent of Ha-ras mutational status. Together, these findings indicated that deregulation of CBF-A contributes to mammary carcinogenesis via a mechanism that is distinct from its hnRNP functions in binding and post-transcriptional regulation of RNA.

CArG binding factor A (CBF-A) is involved in transcriptional regulation of the rat Ha-ras promoter.

In the present study we identified a positive transcriptional element within the rat Ha-ras promoter previously known as Ha-ras response element (HRE) and identified a trans-acting factor that binds HRE sequences in rat mammary cells. To identify the binding protein we employed sequence specific DNA affinity chromatography. Amino acid sequence analysis of the affinity-purified proteins was performed by tandem mass spectroscopy. The results unexpectedly demonstrated that in rat mammary cells CArG box-binding factor A (CBF-A) is the major protein species that bind specifically to the rat and human HRE sequences with high affinity. The affinity of CBF-A binding to HRE was significantly higher than to the CArG box described as a recognition sequence for CBF-A protein. Transient transfection assays using reporter plasmids verified that mutations within the HRE that disrupt binding of CBF-A also reduced the activity of the rat Ha-ras promoter. Despite the fact that the HRE within the Ha-ras promoter resembles a binding site for Ets transcription factors, we did not detect the binding of Ets-related proteins to the rat HRE in BICR-M1Rk cells. We further demonstrated a correlation between the presence of HRE binding activity and induction of Ha-ras mRNA expression following serum stimulation in the mammary carcinoma cell line. Taken together, our results suggest that CBF-A may play an important role in transcriptional regulation of Ha-ras promoter activity during normal mammary cell growth and carcinogenesis.

An antisilencer element is involved in the transcriptional regulation of the human vimentin gene.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. The promoter of the human vimentin gene was previously reported to contain two positive-acting regions, separated by a negative region (Rittling, S.R., Baserga, R., 1987. Functional analysis and growth factor regulation of the human vimentin promoter. Mol. Cell. Biol. 7, 3908-3915). Here, detailed studies reveal two additional regulatory elements, a new positive transcriptional element located between -717 and -757, and a new repressor element at -780 to -821. In transient transfections, the positive-acting element is able to completely override the effect of different silencer elements when fused to a heterologous promoter. However, this element does not enhance gene activity when the silencer element is absent and thus cannot be viewed as a true enhancer. Since it appears to overcome the effect of a silencer element, we refer to it as an antisilencer element. Gel mobility shift assays, UV-cross-linking experiments, and Southwestern blots reveal that a 105-kDa protein specifically binds to this region.

Identification of Upstream Regulatory Elements That Repress Expression of Adult Beta-like Globin Genes in a Primitive Erythroid Environment

ABSTRACT: Our investigations have focused on localizing cis-elements responsible for the down regulation of the adult beta-like globin genes (delta and beta) in immature, or primitive erythroid tissues. We studied their activity after transfection into K562 cells, an erythroleukemia cell line with an embryonic-fetal phenotype. Analyzed DNA sequences included delta and beta 5′ flanking regions extending from approximately -500 to +50bp (promoter regions), truncated delta and beta 5′ flanking regions extending from approximately -250 to +50 bp, and chimeric promoter constructions, which consisted of a distal delta or beta fragment fused to a proximal beta or delta sequence. In CAT reporter constructions no appreciable level of CAT activity was supported by the beta globin promoter, and only low level activity by the delta promoter. Truncation of the beta globin promoter led to a 2-3 fold increase in promoter activity. In contrast, deletion of the upstream portion of the delta promoter led to a 10 fold decrease in expression. Coupling of the upstream beta globin sequence from approximately -500 to -250 bp to the truncated delta promoter fragment led to complete extinction of transcriptional activity, consistent with a negative regulatory effect of the beta globin gene upstream element(s). Fusion of the upstream portion of the delta promoter to the truncated beta globin promoter yielded a modest increase in promoter strength relative to the truncated beta gene promoter, indicating the presence of a positive transcriptional element(s) in the upstream delta globin regulatory region. Site-directed mutagenesis of binding sites for the repressor proteins BP1 and BP2 in the upstream portion of the beta globin gene flanking region led to a 4-6 fold increase in promoter activity. DNase I footprinting of the upstream delta-globin region revealed protected sequences corresponding to consensus binding sites for GATA-1 and BP2. These results confirm that sequences in the upstream promoter region of the adult beta globin gene contribute to its factor-mediated suppression early in development and then may modulate its expression at a later stage.

Elements between the protein‐coding regions of the adjacent β4 and α3 acetylcholine receptor genes direct neuron‐specific expression in the central nervous system

The expression patterns of three clustered neuronal nicotinic acetylcholine receptor (nAchR) subunit genes ordered β4, α3, and α5 overlap extensively in the peripheral nervous system (PNS) but only partially in the central nervous system (CNS). We have begun to investigate cell type-specific cis elements regulating these genes by analyzing in both cell culture and transgenic mice, a 2.8-kb fragment (−2732/+47) containing the α3 promoter region, the β4/α3 intergenic region, and a portion of the β4 3′-untranslated exon. The −2732/+47 fragment is preferentially active in PC12 cells relative to nonneural cell lines. Deletion analysis revealed a cell type-specific positive transcriptional element positioned in the β4 3′-untranslated exon. The positive element is likely to be an enhancer and not a second α3 promoter, because no α3 exons are present in this region. Having shown in cell culture that cell-type specific cis elements are positioned between the β4 and α3 coding regions, we investigated the activity of −2732/+47 in vivo. Transgenic mice were generated, which carry the lacZ gene fused downstream of −2732/+47. Expression of the lacZ transgene is restricted to neurons of the CNS; no expression was detected in the PNS or in nonneural tissues. LacZ-positive cells were detected virtually exclusively in a subset of CNS nuclei that transcribe the endogenous α3 gene. Some overlap was seen with the β4 gene, but nearly none with the α5 gene. Our results demonstrate that cis elements positioned between the α3 and β4 coding regions are important for establishing part of the restricted CNS patterns of β4, α3, and α5 gene transcription. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 311–324, 1997

Elements between the protein-coding regions of the adjacent ?4 and ?3 acetylcholine receptor genes direct neuron-specific expression in the central nervous system

The expression patterns of three clustered neuronal nicotinic acetylcholine receptor (nAchR) subunit genes ordered beta 4, alpha 3, and alpha 5 overlap extensively in the peripheral nervous system (PNS) but only partially in the central nervous system (CNS). We have begun to investigate cell type-specific cis elements regulating these genes by analyzing in both cell culture and transgenic mice, a 2.8-kb fragment (-2732/+47) containing the alpha 3 promoter region, the beta 4/alpha 3 intergenic region, and a portion of the beta 4 3'-untranslated exon. The -2732/+47 fragment is preferentially active in PC12 cells relative to nonneural cell lines. Deletion analysis revealed a cell type-specific positive transcriptional element positioned in the beta 4 3'-untranslated exon. The positive element is likely to be an enhancer and not a second alpha 3 promoter, because no alpha 3 exons are present in this region. Having shown in cell culture that cell-type specific cis elements are positioned between the beta 4 and alpha 3 coding regions, we investigated the activity of -2732/+47 in vivo. Transgenic mice were generated, which carry the lacZ gene fused downstream of -2732/+47. Expression of the lacZ transgene is restricted to neurons of the CNS; no expression was detected in the PNS or in nonneural tissues. LacZ-positive cells were detected virtually exclusively in a subset of CNS nuclei that transcribe the endogenous alpha 3 gene. Some overlap was seen with the beta 4 gene, but nearly none with the alpha 5 gene. Our results demonstrate that cis elements positioned between the alpha 3 and beta 4 coding regions are important for establishing part of the restricted CNS patterns of beta 4, alpha 3, and alpha 5 gene transcription.

In VivoRole of the SP1 Site Neighboring Sterol-Responsive Element-1 in Controlling Low-Density Lipoprotein Receptor Gene Expression

Thein vivorole of the crucial Sp1 site neighboring sterol-responsive element-1 (SRE-1) in controlling LDL receptor gene expression in the presence or absence of sterols was examined. For this purpose theXenopus laevissystem was utilized as there are two different genes for LDL receptors in frogs which differ in their promoter region in the Sp1-binding sequence of repeat 3 present immediately adjacent to SRE-1. DNase I footprinting of promoters of both receptors showed differences in the affinity of this Sp1 site to purified transcription factor Sp1. Transcript levels of both LDL receptors were measured in livers of frogs on normal and cholesterol-enriched diets. Basal levels and extent of repression of LDL receptor gene on sterol administration were found to be dependent on the nature of the Sp1 site of repeat 3 underin vivoconditions. We conclude that this Sp1 site acts as a constitutive positive transcriptional element that forms a part of the active transcription complex irrespective of cellular sterol levels.

Identification of two positive transcriptional elements within the 91‐base pair promoter for mouse testis angiotensin converting enzyme (testis ACE)

Testis angiotensin-converting enzyme (testis ACE) is an isozyme of ACE only expressed by male germ cells during spermiogenesis. It is the result of a strong sperm-specific promoter found within the 12th intron of the somatic ACE gene. Previous studies have localized the boundaries of the mouse testis ACE promoter as being from -91 to -9, relative to the transcriptional start site, and have suggested two important DNA regulatory elements starting at positions -55 and -32. DNA constructs were made in which these motifs were either eliminated or substituted. Each construct was tested for its ability to promote transcription in vitro, using a rat testis nuclear extract. Disruption of either motif reduced in vitro transcription to about 30% of control levels, while mutations of both elements abolished transcription. Two sites were selected inside each motif and altered by point mutation. Each of four constructs, containing a mutation at -51, -48, -30, or -28, transcribed at 29% or less the efficiency of the parent construct. The DNA element at -55, TGAGGTCA, is homologous to a consensus cyclic AMP response element. The motif at -32, TCTTAT, is located at a position analogous to a TATA box. Substitution of the -32 motif with a consensus TATA box sequence, TATAAA, stimulated transcriptional activity about 3-fold. As measured by gel mobility shift, oligonucleotides encompassing the -32 motif and the consensus TATA box formed different DNA-protein complexes. However, the -32 motif oligonucleotide was recognized by nuclear proteins prepared from either liver or testis nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of the N- myc gene: Identification of positive regulatory element and its double- and single-stranded DNA binding proteins

The N- myc gene is amplified and overexpressed in neuroblastoma, retinoblastoma and small cell lung carcinoma, and is considered to be related to cell proliferation and/or differentiation. The transcriptional regulatory sequences of the c- myc gene have been already identified, while those of N- myc have remained obscure for a long time. In this report, we have identified several positive and negative transcriptional regulatory elements in the upstream region of the mouse N- myc gene. Among them, an activating sequence spanning −860 to −797 bp (63 bp) could be reduced to a functional core of 21 bp from −846 to −826. This sequence, termed N21 box, worked as a positive transcriptional element when linked directly upstream (but not downstream) of the putative N- myc promoter in HeLa, not in IMR32 cells. At least two proteins, of 42 kDa and 100 kDa, bound to the double-stranded N21 box, and were expressed in HeLa as well as in IMR32 cells. Moreover, the plus strand of N21 box could be specifically bound by a species of 42 kDa from either cell type and by a 37 kDa protein found only in HeLa cells. These proteins may be factors binding to positive transcriptional regulatory elements and may have a role in the regulation of N- myc expression.

Specific binding of heterogeneous ribonucleoprotein particle protein K to the human c-myc promoter, in vitro.

A homopurine/homopyrimidine-like sequence is found 100-150 base pairs upstream of the human c-myc promoter P1. This element, termed the CT-element, has been shown to augment expression from P1, and it serves as a positive transcriptional element when coupled to a heterologous promoter in vivo and in vitro. Synthetic oligonucleotides comprising this element were used to form DNA-protein complexes in electrophoretic mobility shift assays. By using conventional and affinity methods, 61- and 34-kDa proteins were shown to be associated with these complexes. Amino acid sequence analysis and immunological methods have identified these proteins as heterogeneous ribonucleoprotein particle (hnRNP) proteins K and A1. Surprisingly, hnRNP protein K binds to the pyrimidine-rich strand of the CT-element in a sequence-specific manner as well as to the double-stranded molecule. Cotransfection of vectors encoding hnRNP protein K in the sense or anti-sense orientations with reporter plasmids driven by wild-type or mutant CT-elements demonstrates that hnRNP protein K augments gene expression in a cis-element-dependent manner. Taken together, these results suggest that hnRNP protein K may play a role in the transcriptional regulation of the human c-myc gene.

Positive and negative cis-acting regulatory elements mediate expression of the mouse vascular smooth muscle alpha-actin gene.

Segments of the 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene were assayed for promoter activity in transfected mouse BC3H1 myogenic cells and AKR-2B embryonic fibroblasts. The region between -150 and -191 that functions as a positive transcriptional element in myogenic and fibroblastic cells contains a mammalian-specific inverted CC(A/T)6GG-type consensus sequence. Expression was restricted to fully differentiated myogenic cells when an additional sequence spanning -191 to -224 was included in reporter gene constructs. This 33-base pair (bp) negative regulatory element is 70% conserved between the mouse and human genes and contains a 10-bp motif at its 3' end that only partially resembles a CC(A/T)6GG element. Retention of a GGGA motif at the 3' boundary of the 33-bp region is sufficient to maintain full transcriptional repression in fibroblasts and is partly responsible for repression in undifferentiated myoblasts. Complete muscle tissue-restrictive expression requires an additional 8 bp from the CC(A/T)6GG-like element immediately 5' to the GGGA motif, since replacement of this region with an unrelated 10-bp sequence completely eliminated restrictive transcriptional behavior in undifferentiated myoblasts. The distal portion of the 5'-flanking region between -224 and -1074 contains six E-box motifs (CANNTG) and mediates high level transcription only in postconfluent BC3H1 myoblasts. Analysis of reporter gene constructs including either the proximal E-box at -240 or all six E-boxes indicate that the five distal E-boxes are not required for high level transcription. A 724-bp segment of the 5'-flanking region consisting of the proximal E-box flanked upstream by a mammalian-specific 352-bp region was sufficient for maximal transcriptional activation in postconfluent BC3H1 myoblasts. Deletion of the 352-bp region restricts the early transcriptional response to high cell density in temporal studies of promoter activity during BC3H1 myogenic cell differentiation.

Sterol-dependent repression of low density lipoprotein receptor promoter mediated by 16-base pair sequence adjacent to binding site for transcription factor Sp1.

A 42-base pair sequence from the 5' flanking region of the low density lipoprotein receptor gene was shown previously to confer sensitivity to sterol-mediated repression when inserted into the herpes simplex virus thymidine kinase promoter. This sequence contains two contiguous 16-base pair repeats, designated repeats 2 and 3, which differ from each other at four positions. In the current study we have analyzed separately the functions of repeats 2 and 3 by altering their sequences, inserting them into the -60 position of the thymidine kinase promoter, and introducing the hybrid promoters into hamster cells by transfection. These studies show that repeat 3 is a constitutive positive transcriptional element that acts in the absence or presence of sterols. Repeat 2 confers strong repression upon repeat 3 when sterols are present. In vitro DNase footprinting and gel retardation assays show that repeat 3, but not repeat 2, binds purified Sp1, a positive transcription factor. Mutants of repeat 3 that abolish transcriptional activity in vivo abolish Sp1 binding in vitro. We suggest that the low density lipoprotein receptor is regulated by a push-pull mechanism in which sterol-regulated binding of a protein to repeat 2 silences the activity of the adjacent Sp1-binding site in repeat 3.