scholarly journals Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation.

1997 ◽  
Vol 17 (2) ◽  
pp. 612-619 ◽  
Author(s):  
R C Iannello ◽  
J Young ◽  
S Sumarsono ◽  
M J Tymms ◽  
H H Dahl ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Promoter Activity ◽  
Core Promoter ◽  
Pyruvate Dehydrogenase Complex ◽  
Cpg Methylation ◽  
Proximal Promoter ◽  
Specific Gene ◽  
Specific Expression ◽  
Cpg Dinucleotides ◽  
The Core

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.

The −5 A/G single-nucleotide polymorphism in the core promoter region of MT2A and its effect on allele-specific gene expression and Cd, Zn and Cu levels in laryngeal cancer

2014 ◽  
Vol 280 (2) ◽  
pp. 256-263 ◽  
Author(s):  
Katarzyna Starska ◽  
Anna Krześlak ◽  
Ewa Forma ◽  
Jurek Olszewski ◽  
Alina Morawiec-Sztandera ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Nucleotide Polymorphism ◽  
Promoter Region ◽  
Core Promoter ◽  
Specific Gene ◽  
Nucleotide Polymorphism ◽  
Core Promoter Region ◽  
Single Nucleotide ◽  
The Core ◽  
Allele Specific

scholarly journals Identification and characterization of cis-regulatory elements for photoreceptor type-specific transcription in zebrafish

2019 ◽  
Author(s):  
Wei Fang ◽  
Yi Wen ◽  
Xiangyun Wei
Keyword(s):  
Core Promoter ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Protein Coding ◽  
Core Promoters ◽  
Protein Coding Genes ◽  
The Core ◽  
Cell Type Specific ◽  
Identification And Characterization

AbstractTissue-specific or cell type-specific transcription of protein-coding genes is controlled by both trans-regulatory elements (TREs) and cis-regulatory elements (CREs). However, it is challenging to identify TREs and CREs, which are unknown for most genes. Here, we describe a protocol for identifying two types of transcription-activating CREs—core promoters and enhancers—of zebrafish photoreceptor type-specific genes. This protocol is composed of three phases: bioinformatic prediction, experimental validation, and characterization of the CREs. To better illustrate the principles and logic of this protocol, we exemplify it with the discovery of the core promoter and enhancer of the mpp5b apical polarity gene (also known as ponli), whose red, green, and blue (RGB) cone-specific transcription requires its enhancer, a member of the rainbow enhancer family. While exemplified with an RGB cone-specific gene, this protocol is general and can be used to identify the core promoters and enhancers of other protein-coding genes.

scholarly journals Tamoxifen-inducible cardiac-specific Cre transgenic mouse using VIPR2 intron

2020 ◽  
Vol 36 (1) ◽  
Author(s):  
Hyun Jung Chin ◽  
So-young Lee ◽  
Daekee Lee
Keyword(s):  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Gene Function ◽  
Regulatory Region ◽  
Genetically Engineered ◽  
Specific Gene ◽  
Specific Expression ◽  
Genetically Engineered Mouse Models ◽  
Reporter Mice ◽  
Vasoactive Intestinal Peptide Receptor

Abstract Genetically engineered mouse models through gene deletion are useful tools for analyzing gene function. To delete a gene in a certain tissue temporally, tissue-specific and tamoxifen-inducible Cre transgenic mice are generally used. Here, we generated transgenic mouse with cardiac-specific expression of Cre recombinase fused to a mutant estrogen ligand-binding domain (ERT2) on both N-terminal and C-terminal under the regulatory region of human vasoactive intestinal peptide receptor 2 (VIPR2) intron and Hsp68 promoter (VIPR2-ERT2CreERT2). In VIPR2-ERT2CreERT2 transgenic mice, mRNA for Cre gene was highly expressed in the heart. To further reveal heart-specific Cre expression, VIPR2-ERT2CreERT2 mice mated with ROSA26-lacZ reporter mice were examined by X-gal staining. Results of X-gal staining revealed that Cre-dependent recombination occurred only in the heart after treatment with tamoxifen. Taken together, these results demonstrate that VIPR2-ERT2CreERT2 transgenic mouse is a useful model to unveil a specific gene function in the heart.

scholarly journals Binding of Phosphorylated Sp1 Protein to Tandem Sp1 Binding Sites Regulates α2 Integrin Gene Core Promoter Activity

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 678-689 ◽  
Author(s):  
Mary M. Zutter ◽  
Ellen E. Ryan ◽  
Audrey D. Painter
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Binding Sites ◽  
Promoter Activity ◽  
Core Promoter ◽  
K562 Cells ◽  
The Core ◽  
Nuclear Extracts ◽  
Integrin Gene ◽  
Α2β1 Integrin

Abstract The α2β1 integrin, a collagen/laminin receptor, is expressed by a variety of cell types, including epithelial cells, mesenchymal cells, and hematopoietic cells. To understand the molecular mechanisms that regulate expression of the α2β1 integrin in cells with megakaryocytic differentiation, we characterized the 5′ flanking region of the α2 integrin gene and identified three distinct regulatory regions, including a core promoter, a silencer, and megakaryocyte enhancers in the distal 5′ flank (Zutter et al, Blood 96:3006, 1995 and Zutter et al, J Biol Chem 269:463, 1994). We now focus on the core promoter of the α2 integrin gene located between bp −30 and −92 that is required for transcriptional activity of the α2 integrin gene. Sequence analysis identified two Sp1 consensus sites and a potential AP2 site. Gel retardation assays showed that nuclear proteins from uninduced K562 cells and K562 cells induced to become megakaryocytic bound specifically to the core promoter region (bp −30 to bp −92) producing two DNA-protein complexes. In addition, nuclear extracts from cells induced along the megakaryocyte lineage produced a selective increase in the slower migrating complex. Site-directed mutagenesis of the 5′, the 3′, or both Sp1 binding sites suggested that both Sp1 binding sites are required for full promoter activity and for DNA-protein complex formation. DNA footprinting also showed specific protection of the 5′ Sp1 site by nuclear extracts from uninduced K562 cells and protection of both the 5′ and the 3′ Sp1 sites by nuclear extracts from induced K562 cells. Sp1 protein-DNA complex formation was dependent on Sp1 phosphorylation. The faster migrating DNA-protein complex was enhanced by dephosphorylation; the slower migrating DNA-protein complex was diminished or lost.

Regulation of lineage-specific transcription of the sucrase-isomaltase gene in transgenic mice and cell lines

1995 ◽  
Vol 269 (6) ◽  
pp. G925-G939 ◽  
Author(s):  
A. J. Markowitz ◽  
G. D. Wu ◽  
A. Bader ◽  
Z. Cui ◽  
L. Chen ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cell Lines ◽  
Reporter Gene ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Transgenic Animals ◽  
Intestinal Cell ◽  
Specific Gene ◽  
Specific Expression ◽  
Endogenous Gene

Sucrase-isomaltase (SI), a gene expressed exclusively in absorptive enterocytes, was used to examine the molecular mechanisms that regulate cell-specific gene expression in the intestinal epithelium. Transgenic mice were made with a construct containing nucleotides -8,500 to +54 of the mouse SI gene linked to a human growth hormone reporter gene. In adult transgenic animals, high-level transgene expression was limited to the small intestine, with low levels of ectopic expression in the colon. In contrast to the endogenous gene that is expressed only in enterocytes, the transgene was expressed in all four cell lineages, including enterocytes, enteroendocrine, goblet, and Paneth cells. To examine this process of lineage-specific expression further we studied Caco-2 and COLO DM cell lines, which model enterocytes and enteroendocrine cells, respectively. Reminiscent of results in transgenic animals, only Caco-2 cells transcribed the endogenous SI gene, whereas both Caco-2 and COLO DM cells supported transcription from chimeric SI reporter gene constructs. Taken together, these data suggest that each intestinal cell lineage has the cellular machinery to transcribe the SI gene. Moreover, these findings imply that transcription is normally repressed in nonenterocytic cells, possibly via a transcriptional silencer residing outside of the region of the SI gene examined in these studies.

scholarly journals Hepatocyte-specific expression of the hepatitis B virus core promoter depends on both positive and negative regulation.

1993 ◽  
Vol 13 (1) ◽  
pp. 443-448 ◽  
Author(s):  
W Guo ◽  
M Chen ◽  
T S Yen ◽  
J H Ou
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Mammalian Cells ◽  
Core Promoter ◽  
Regulatory Sequence ◽  
Cell Type Specificity ◽  
Specific Expression ◽  
The Core ◽  
Upstream Regulatory Sequence ◽  
B Virus

The core promoter of hepatitis B virus shows hepatocyte specificity, which is largely dependent on an upstream regulatory sequence that overlaps with viral enhancer II. Footprint analyses by numerous groups have shown binding by cellular proteins over a large stretch of DNA in this region, but the identity of these proteins and their role in core promoter function remain largely unknown. We present data showing that the transcription factor HNF-4 is one such factor, as it activates the core promoter approximately 20-fold via a binding site within the upstream regulatory sequence. Since HNF-4 is enriched in hepatocytes, its involvement at least partially explains the hepatocyte specificity of this promoter. In addition, however, we have found a region upstream of the HNF-4 site that suppresses activation by HNF-4 in HeLa cells but not in hepatoma cells. Therefore, the cell type specificity of the core promoter appears to result from a combination of activation by one or more factors specifically enriched in hepatocytes and repression by some other factor(s) present in nonhepatocytes, and it may provide a convenient model system for studying this type of tissue-specific transcriptional regulation in mammalian cells.

Possible involvement of the calreticulin gene in the evolution of cognition in humans

2011 ◽  
Vol 26 (S2) ◽  
pp. 810-810
Author(s):  
T. Farokhashtiani ◽  
A. Mirabzadeh ◽  
M. Ohadi
Keyword(s):  
Schizoaffective Disorder ◽  
Core Promoter ◽  
Promoter Sequence ◽  
Type I ◽  
Specific Expression ◽  
The Core ◽  
Human Promoter ◽  
Genetic Overlap ◽  
Higher Order Functions ◽  
The Brain

The chromosomal region, 19p 13.2, has been suggested to be linked with schizoaffective disorder (Hamshere et al. 2005). This region harbors the calreticulin gene, protein encoded by which is an endoplasmic Ca+2-binding molecular chaperone. Development-dependent, tissue-specific expression of this gene in the gray matter coincides with the expression of psychoses phenotypes. We have recently reported instances of mutations within the core promoter (−48 G>C, −205 C>T) and coding sequence [exon 5 (c: 682 C>T, pro228ser)] of the gene in schizoaffective disorder. In view of the mounting evidence on the genetic overlap in the psychiatric spectrum, we investigated this gene in 386 patients afflicted with schizophrenia, schizoaffective disorder, major affective disorder and 600 controls by PCR/SSCA. We found that a unique mutation within the core promoter of the gene, located at a conserved genomic block, co-occurring with four cases of psychoses including schizophrenia, schizoaffective disorder and bipolar disorder type I. This unique mutation reverts the human promoter sequence to the ancestral types observed in chimpanzee, rhesus Macaque and mouse, implying that the genomic block harboring this nucleotide may be involved in the evolution of human-specific higher-order functions of the brain (i.e. cognition), that are ubiquitously impaired in psychoses. Our findings propose that calreticulin is not only a promising candidate in the spectrum of psychoses, but also, a gene that may be important in the process of human-unique higher-level functions of the brain.

Binding of Phosphorylated Sp1 Protein to Tandem Sp1 Binding Sites Regulates α2 Integrin Gene Core Promoter Activity

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 678-689 ◽  
Author(s):  
Mary M. Zutter ◽  
Ellen E. Ryan ◽  
Audrey D. Painter
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Binding Sites ◽  
Promoter Activity ◽  
Core Promoter ◽  
K562 Cells ◽  
The Core ◽  
Nuclear Extracts ◽  
Integrin Gene ◽  
Α2β1 Integrin

The α2β1 integrin, a collagen/laminin receptor, is expressed by a variety of cell types, including epithelial cells, mesenchymal cells, and hematopoietic cells. To understand the molecular mechanisms that regulate expression of the α2β1 integrin in cells with megakaryocytic differentiation, we characterized the 5′ flanking region of the α2 integrin gene and identified three distinct regulatory regions, including a core promoter, a silencer, and megakaryocyte enhancers in the distal 5′ flank (Zutter et al, Blood 96:3006, 1995 and Zutter et al, J Biol Chem 269:463, 1994). We now focus on the core promoter of the α2 integrin gene located between bp −30 and −92 that is required for transcriptional activity of the α2 integrin gene. Sequence analysis identified two Sp1 consensus sites and a potential AP2 site. Gel retardation assays showed that nuclear proteins from uninduced K562 cells and K562 cells induced to become megakaryocytic bound specifically to the core promoter region (bp −30 to bp −92) producing two DNA-protein complexes. In addition, nuclear extracts from cells induced along the megakaryocyte lineage produced a selective increase in the slower migrating complex. Site-directed mutagenesis of the 5′, the 3′, or both Sp1 binding sites suggested that both Sp1 binding sites are required for full promoter activity and for DNA-protein complex formation. DNA footprinting also showed specific protection of the 5′ Sp1 site by nuclear extracts from uninduced K562 cells and protection of both the 5′ and the 3′ Sp1 sites by nuclear extracts from induced K562 cells. Sp1 protein-DNA complex formation was dependent on Sp1 phosphorylation. The faster migrating DNA-protein complex was enhanced by dephosphorylation; the slower migrating DNA-protein complex was diminished or lost.

scholarly journals A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9

2015 ◽  
Vol 113 (2) ◽  
pp. 338-343 ◽  
Author(s):  
Kelli J. Carroll ◽  
Catherine A. Makarewich ◽  
John McAnally ◽  
Douglas M. Anderson ◽  
Lorena Zentilin ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Gene Deletion ◽  
Disease Modeling ◽  
Embryonic Lethality ◽  
Null Alleles ◽  
Specific Gene ◽  
Specific Expression ◽  
Guide Rna ◽  
Cardiac Gene ◽  
Genomic Editing

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.

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