Functional characterization of the human hypoxanthine phosphoribosyltransferase gene promoter: evidence for a negative regulatory element

1991 ◽  
Vol 11 (8) ◽  
pp. 4157-4164
Author(s):  
D E Rincón-Limas ◽  
D A Krueger ◽  
P I Patel
Keyword(s):  
Regulatory Element ◽  
Genomic Structure ◽  
Functional Characterization ◽  
Translation Initiation Site ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Negative Element ◽  
Cis Acting ◽  
Human Hprt Gene

The enzyme hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the metabolic salvage of the purine bases hypoxanthine and guanine. We previously characterized the genomic structure of the human HPRT gene and described its promoter sequence. In this report, we identify cis-acting transcriptional control regions of the human HPRT gene by linking various 5'-flanking sequences to the bacterial chloramphenicol acetyltransferase gene. The sequence from positions -219 to -122 relative to the translation initiation site is required for maximal expression of this gene, and it functions equally in both normal and reverse orientations. In addition, a cis-acting negative element is present in the region spanning from positions -570 to -388. This negative element can also repress promoters of heterologous genes, such as those of adenosine deaminase and dihydrofolate reductase, which are structurally and functionally similar to the human HPRT promoter. Furthermore, this repressor element functions independently of its orientation but appears to be distance dependent. In vivo competition assays demonstrated that the trans-acting factor(s) that binds to this negative element specifically inhibits human HPRT promoter activity. Taken together, these data localize cis-acting sequences important in the regulation of human HPRT gene expression and should allow the study of protein-DNA interactions which modulate the transcription of this gene.

scholarly journals Functional characterization of the human hypoxanthine phosphoribosyltransferase gene promoter: evidence for a negative regulatory element.

1991 ◽  
Vol 11 (8) ◽  
pp. 4157-4164 ◽  
Author(s):  
D E Rincón-Limas ◽  
D A Krueger ◽  
P I Patel
Keyword(s):  
Regulatory Element ◽  
Genomic Structure ◽  
Functional Characterization ◽  
Translation Initiation Site ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Negative Element ◽  
Cis Acting ◽  
Human Hprt Gene

The enzyme hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the metabolic salvage of the purine bases hypoxanthine and guanine. We previously characterized the genomic structure of the human HPRT gene and described its promoter sequence. In this report, we identify cis-acting transcriptional control regions of the human HPRT gene by linking various 5'-flanking sequences to the bacterial chloramphenicol acetyltransferase gene. The sequence from positions -219 to -122 relative to the translation initiation site is required for maximal expression of this gene, and it functions equally in both normal and reverse orientations. In addition, a cis-acting negative element is present in the region spanning from positions -570 to -388. This negative element can also repress promoters of heterologous genes, such as those of adenosine deaminase and dihydrofolate reductase, which are structurally and functionally similar to the human HPRT promoter. Furthermore, this repressor element functions independently of its orientation but appears to be distance dependent. In vivo competition assays demonstrated that the trans-acting factor(s) that binds to this negative element specifically inhibits human HPRT promoter activity. Taken together, these data localize cis-acting sequences important in the regulation of human HPRT gene expression and should allow the study of protein-DNA interactions which modulate the transcription of this gene.

Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2735-2746 ◽  
Author(s):  
D.H. Rowitch ◽  
Y. Echelard ◽  
P.S. Danielian ◽  
K. Gellner ◽  
S. Brenner ◽  
...  
Keyword(s):  
Base Pair ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Neural Plate ◽  
Homologous Region ◽  
Regulatory Sequence ◽  
Embryonic Mouse ◽  
Cis Acting ◽  
Evolutionarily Conserved

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(−/−) embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.

scholarly journals In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes.

1996 ◽  
Vol 16 (11) ◽  
pp. 6190-6199 ◽  
Author(s):  
M D Litt ◽  
I K Hornstra ◽  
T P Yang
Keyword(s):  
High Resolution ◽  
X Chromosome ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Genomic Sequencing ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Methylation Analysis ◽  
In Vivo Footprinting

To investigate potential mechanisms regulating the hypoxanthine phosphoribosyltransferase (HPRT) gene by X-chromosome inactivation, we performed in vivo footprinting and high-resolution DNA methylation analysis on the 5' region of the active and inactive mouse HPRT alleles and compared these results with those from the human HPRT gene. We found multiple footprinted sites on the active mouse HPRT allele and no footprints on the inactive allele. Comparison of the footprint patterns of the mouse and human HPRT genes demonstrated that the in vivo binding of regulatory proteins between these species is generally conserved but not identical. Detailed nucleotide sequence comparison of footprinted regions in the mouse and human genes revealed a novel 9-bp sequence associated with transcription factor binding near the transcription sites of both genes, suggesting the identification of a new conserved initiator element. Ligation-mediated PCR genomic sequencing showed that all CpG dinucleotides examined on the active allele are unmethylated, while the majority of CpGs on the inactive allele are methylated and interspersed with a few hypomethylated sites. This pattern of methylation on the inactive mouse allele is notably different from the unusual methylation pattern of the inactive human gene, which exhibited strong hypomethylation specifically at GC boxes. These studies, in conjunction with other genomic sequencing studies of X-linked genes, demonstrate that (i) the active alleles are essentially unmethylated, (ii) the inactive alleles are hypermethylated, and (iii) the high-resolution methylation patterns of the hypermethylated inactive alleles are not strictly conserved. There is no obvious correlation between the pattern of methylated sites on the inactive alleles and the pattern of binding sites for transcription factors on the active alleles. These results are discussed in relationship to potential mechanisms of transcriptional regulation by X-chromosome inactivation.

Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation

1992 ◽  
Vol 12 (12) ◽  
pp. 5345-5354
Author(s):  
I K Hornstra ◽  
T P Yang
Keyword(s):  
X Chromosome ◽  
Protein Interactions ◽  
Cultured Cells ◽  
Dimethyl Sulfate ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Active Allele

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.

scholarly journals Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation.

1992 ◽  
Vol 12 (12) ◽  
pp. 5345-5354 ◽  
Author(s):  
I K Hornstra ◽  
T P Yang
Keyword(s):  
X Chromosome ◽  
Protein Interactions ◽  
Cultured Cells ◽  
Dimethyl Sulfate ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Active Allele

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.

scholarly journals Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene.

1995 ◽  
Vol 15 (12) ◽  
pp. 6561-6571 ◽  
Author(s):  
D E Rincón-Limas ◽  
F Amaya-Manzanares ◽  
M L Niño-Rosales ◽  
Y Yu ◽  
T P Yang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Neuronal Differentiation ◽  
Complex I ◽  
Regulatory Element ◽  
Constitutive Expression ◽  
Site Directed Mutagenesis ◽  
Nuclear Proteins ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
The Brain

The hypoxanthine phosphoribosyltransferase (HPRT) gene is constitutively expressed at low levels in all tissues but at higher levels in the brain; the significance and mechanism of this differential expression are unknown. We previously identified a 182-bp element (hHPRT-NE) within the 5'-flanking region of the human HPRT (hHPRT) gene, which is involved not only in conferring neuronal specificity but also in repressing gene expression in nonneuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation. We also mapped the binding sites for both complexes to a 60-bp region (Ff; positions -510 to -451) which, when analyzed in transfection assays, functioned as a repressor element analogous to the full-length hHPRT-NE sequence. Methylation interference footprintings revealed a minimal unique DNA motif, 5'-GGAAGCC-3', as the binding site for nuclear proteins from both neuronal and nonneuronal sources. However, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the associations of these two complexes. Moreover, UV cross-linking experiments showed that both complexes are formed by the association of several different proteins. Taken together, these data suggest that differential interaction of DNA-binding factors with this regulatory element plays a crucial role in the brain-preferential expression of the gene, and they should lead to the isolation of transcriptional regulators important in neuronal expression of the HPRT gene.

scholarly journals Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 861-864 ◽  
Author(s):  
N Raich ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos ◽  
T Enver
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Negative Element ◽  
Cis Acting

The human epsilon-globin gene displays normal developmental regulation in transgenic mice; it is expressed only in embryonic and in definitive erythroid cells. We show here that deletion of a negative element located between -182 and -467 bp upstream of the epsilon-globin gene cap site results in continuation of epsilon gene expression in the definitive erythroblasts of the fetal liver and in the red blood cells of adult transgenic mice. These data provide direct in vivo evidence that cis acting silencing elements are involved in the developmental control of the epsilon-globin gene.

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