The rice OsLTP6 gene promoter directs anther-specific expression by a combination of positive and negative regulatory elements

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of β-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.

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The GC-box is critical for high level expression of the testis-specific Hsp70.2/Hst70 gene.

Acta Biochimica Polonica ◽

10.18388/abp.2007_3275 ◽

2007 ◽

Vol 54 (1) ◽

pp. 107-112 ◽

Cited By ~ 2

Author(s):

Wiesława Widłak ◽

Natalia Vydra ◽

Volha Dudaladava ◽

Dorota Scieglińska ◽

Bolesław Winiarski ◽

...

Keyword(s):

Regulatory Element ◽

Gene Promoter ◽

Regulatory Elements ◽

Specific Expression ◽

Transcription Start Sites ◽

Nuclear Extracts ◽

Efficient Expression ◽

Gc Box ◽

Specific Expression Pattern ◽

High Level

The Hsp70.2/Hst70 gene, which belongs to the 70 kDa heat-shock protein (HSP) family, is expressed specifically in primary spermatocytes and spermatids. The regulatory elements required for a high level of testis-specific expression of the gene are placed between the two major transcription start sites T1 and T2 (approximately 350 and 115 bp upstream of the starting ATG codon). Here we have shown that sequences proximal to the exon1/intron splicing site in the 5' untranslated region of the Hsp70.2/Hst70 gene, which include a highly conserved element called box B, are required for efficient expression of the chloramphenicol acetyltransferase reporter gene in testes of transgenic mice. However, in spite of the drastically reduced overall activity, the stage-specific expression pattern of the transgene was preserved after removal of these sequences. We have also shown that GC-box located downstream of the box B (approximately 210 bp upstream of the starting ATG codon) is indispensable for efficient expression of the Hsp70.2/Hst70 gene promoter in spermatogenic cells. The GC-box specifically binds proteins present in nuclear extracts from testes (putatively Sp1-like factors). A change in the pattern of such GC-box-interacting factors corresponds to activation of the Hsp70.2/Hst70 gene, confirming the importance of this regulatory element.

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Multiple negative elements in a gene that codes for an extracellular matrix protein, collagen X, restrict expression to hypertrophic chondrocytes.

The Journal of Cell Biology ◽

10.1083/jcb.121.5.1173 ◽

1993 ◽

Vol 121 (5) ◽

pp. 1173-1179 ◽

Cited By ~ 50

Author(s):

P Lu Valle ◽

M Iwamoto ◽

P Fanning ◽

M Pacifici ◽

B R Olsen

Keyword(s):

Matrix Protein ◽

Skeletal Development ◽

Primary Cultures ◽

Gene Promoter ◽

Cell Types ◽

Regulatory Elements ◽

Hypertrophic Chondrocytes ◽

Extracellular Matrix Protein ◽

Specific Expression ◽

X Gene

During skeletal development, chondrocytes go through several stages of differentiation. The last stage, chondrocyte hypertrophy, occurs in areas of endochondral ossification. Mature hypertrophic chondrocytes differ from immature chondrocytes in that they become postmitotic, increase their cellular volume up to eightfold, and synthesize a unique set of matrix molecules. One such molecule is a short collagenous protein, collagen X. Previous studies have shown that collagen X is not expressed by other cell types and that its specific expression in hypertrophic chondrocytes is controlled by transcriptional mechanisms. To define these mechanisms, plasmid constructs containing the chicken collagen X gene promoter and 5' flanking regions fused to a reporter gene (chloramphenicol acetyl transferase, CAT) were transfected into primary cultures of collagen X-expressing and nonexpressing cells. A construct containing a short (558 bp) promoter exhibited high levels of CAT activity in all cell types (fibroblasts, immature, and hypertrophic chondrocytes). Adding a 4.2-kb fragment of 5' flanking DNA to this construct resulted in a dramatic reduction of CAT activity in fibroblasts and immature chondrocytes, but had no effect in hypertrophic chondrocytes. Addition of three subfragments of the 4.2-kb fragment to the initial construct, either individually or in various combinations, showed that all subfragments reduced CAT activity somewhat in non-collagen X-expressing cells, and that their effects were additive. Unrelated DNA had no effect on CAT activity. The results suggest that multiple, diffuse upstream negative regulatory elements act in an additive manner to restrict transcription of the collagen X gene to hypertrophic chondrocytes.

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Tissue-specific versus pleiotropic enhancers within the bric-a-brac tandem gene duplicates display differential regulatory activity and evolutionary conservation

10.1101/2021.03.25.436949 ◽

2021 ◽

Author(s):

Henri-Marc G BOURBON ◽

Mikhail Benetah ◽

Emmanuelle Guillou ◽

Luis Humberto MOJICA VAZQUEZ ◽

Aissette BAANANNOU ◽

...

Keyword(s):

Regulatory Networks ◽

De Novo ◽

Critical Role ◽

Specific Interaction ◽

Gene Promoter ◽

Regulatory Elements ◽

Specific Expression ◽

Animal Evolution ◽

Leg Axis ◽

Gene Reporter Assay

During animal evolution, de novo emergence and modifications of pre-existing transcriptional enhancers have contributed to biological innovations, by implementing gene regulatory networks. The Drosophila melanogaster bric-a-brac ( bab ) complex, comprising the tandem paralogous genes bab1 - 2 , provides a paradigm to address how enhancers contribute and co-evolve to regulate jointly or differentially duplicated genes. We previously characterized an intergenic enhancer (named LAE) governing bab2 expression in leg and antennal tissues. We show here that LAE activity also regulates bab1 . CRISPR/Cas9-mediated LAE excision reveals its critical role for bab2 -specific expression along the proximo-distal leg axis, likely through paralog-specific interaction with the bab2 gene promoter. Furthermore, LAE appears involved but not strictly required for bab1 - 2 co-expression in leg tissues. Phenotypic rescue experiments, chromatin features and a gene reporter assay reveal a large “pleiotropic” bab1 enhancer (termed BER) including a series of cis -regulatory elements active in the leg, antennal, wing, haltere and gonadal tissues. Phylogenomics analyses indicate that (i) bab2 originates from bab1 duplication within the Muscomorpha sublineage, (ii) LAE and bab1 promoter sequences have been evolutionarily-fixed early on within the Brachycera lineage, while (iii) BER elements have been conserved more recently among muscomorphans. Lastly, we identified conserved binding sites for transcription factors known or prone to regulate directly the paralogous bab genes in diverse developmental contexts. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution.

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Structure of the 5′ region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites

Biochemical Journal ◽

10.1042/bj3590129 ◽

2001 ◽

Vol 359 (1) ◽

pp. 129-137 ◽

Cited By ~ 3

Author(s):

Dorota ŚCIEGLIŃSKA ◽

Wiesława WID̄ŁAK ◽

Witold KONOPKA ◽

Matti POUTANEN ◽

Nafis RAHMAN ◽

...

Keyword(s):

Transcription Initiation ◽

Gene Promoter ◽

Regulatory Elements ◽

Transcription Unit ◽

Northern Blotting ◽

Start Codon ◽

Specific Expression ◽

Rnase Protection ◽

Exon 1 ◽

Testis Specific Expression

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5′ region of the transcription unit of these genes as well as localization of the ‘cis’ elements conferring their testis-specific expression gave contradictory results [Widłak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191–200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310–321]. In the present paper we solve these controversies and show that the 5′ untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5′ UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on ‘cis’ regulatory elements localized within exon 1 and the intron sequences.

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Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.3.f602 ◽

1998 ◽

Vol 274 (3) ◽

pp. F602-F610 ◽

Cited By ~ 6

Author(s):

Shinichi Uchida ◽

Tatemitsu Rai ◽

Hiroshi Yatsushige ◽

Yoshihiro Matsumura ◽

Masanobu Kawasaki ◽

...

Keyword(s):

Cell Lines ◽

Chloride Channel ◽

Gene Promoter ◽

Simian Virus 40 ◽

Regulatory Elements ◽

Start Codon ◽

Luciferase Reporter ◽

Specific Expression ◽

Isolation And Characterization ◽

Flanking Region

The rat ClC-K1 chloride channel is a kidney-specific member of the ClC chloride channel family found exclusively in the thin ascending limb of Henle’s loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of ClC-K1, a genomic clone that contains the 5′-flanking region of the rat ClC-K1 gene was isolated. A single transcription start site was located 84 bp upstream of the start codon. The sequence of the proximal 5′-flanking region contained an activator protein (AP)-3 site, a glucocorticoid-responsive element, several AP-2 sites, and several E-boxes, but it lacked a TATA box. To functionally express the promoter, the ∼2.5-kb pair 5′-flanking region was ligated to a luciferase reporter gene and transfected into inner medullary (IM) cells, a stable ClC-K1-expressing cell line derived from the inner medulla of simian virus 40 transgenic mouse, and ClC-K1-nonexpressing cell lines. Luciferase activity was 7- to 24-fold greater in IM cells than those in nonexpressing cell lines, suggesting that the ∼2.5-kb fragment contained cis-acting regulatory elements for cell-specific expression of the ClC-K1 gene. Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 bp of the 5′-flanking region but was lost in the −29 construct, clearly demonstrating that the 22 bp from −51 to −30 have a major role in the cell-specific activity of the ClC-K1 promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGGGAGGGGAG), and gel-retardation analysis demonstrated the existence of a specific protein(s) binding to this element in IM cells. These results suggest that the novel purine-rich element may play a key role in the activity of the ClC-K1 gene promoter.

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BACH1, the master regulator of oxidative stress, has a dual effect on CFTR expression.

Biochemical Journal ◽

10.1042/bcj20210252 ◽

2021 ◽

Author(s):

Monali NandyMazumdar ◽

Alekh Paranjapye ◽

James Browne ◽

Shiyi Yin ◽

Shih-Hsing Leir ◽

...

Keyword(s):

Oxidative Stress ◽

Cellular Response ◽

Gene Promoter ◽

Regulatory Elements ◽

Airway Epithelial Cells ◽

Cftr Gene ◽

Airway Epithelial ◽

Dual Effect ◽

Master Regulator ◽

Specific Expression

The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a TAD in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB Domain and CNC Homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (~8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.

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Transcriptional regulation of the calcitonin receptor gene

Biochemical Society Transactions ◽

10.1042/bst0300423 ◽

2002 ◽

Vol 30 (4) ◽

pp. 423-427 ◽

Cited By ~ 3

Author(s):

M. D. Pondel ◽

C. Jagger ◽

C. Hebden ◽

G. Partington ◽

R. Mould

Keyword(s):

Transcriptional Regulation ◽

Transgenic Mice ◽

Receptor Gene ◽

Gene Promoter ◽

Peptide Hormone ◽

Regulatory Elements ◽

Transcriptional Analysis ◽

Human Calcitonin ◽

Calcitonin Receptor ◽

Specific Expression

Calcitonin is a 32-amino-acid peptide hormone that is produced by parafollicular cells of the thyroid gland in response to increased levels of extracellular calcium. In this article we review our previous and current research on the transcriptional regulation of the pig and human calcitonin receptor (CTR) gene. Initially, our experiments on the transcriptional analysis of the pig CTR gene in transgenic mice are examined, followed by our studies on the cloning of the human CTR (hCTR) gene promoter and its transcriptional activity in hCTR-positive cell lines. We further describe the activity of the hCTR promoter in transgenic mice and review current experiments to identify transcriptional regulatory elements and the transcription factors they interact with, which direct the tissue-specific expression pattern of hCTR promoter activity.

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Identification and characterization of PsDREB2 promoter involved in tissue-specific expression and abiotic stress response from Paeonia suffruticosa

PeerJ ◽

10.7717/peerj.7052 ◽

2019 ◽

Vol 7 ◽

pp. e7052

Author(s):

Huichun Liu ◽

Kaiyuan Zhu ◽

Chen Tan ◽

Jiaqiang Zhang ◽

Jianghua Zhou ◽

...

Keyword(s):

Low Temperature ◽

Transgenic Plants ◽

Gene Promoter ◽

Regulatory Elements ◽

Histochemical Staining ◽

High Salt ◽

Paeonia Suffruticosa ◽

Abiotic Stress Response ◽

Specific Expression ◽

Binding Factor

Dehydration-responsive element-binding factor 2 (DREB2) belongs to the C-repeat-binding factor (CBF)/DREB subfamily of proteins. In this study, a 2,245 bp PsDREB2 promoter fragment was isolated from the genome of Paeonia suffruticosa. The fragment was rich in A/T bases and contained TATA box sequences, abscisic acid (ABA)-response elements, and other cis-elements, such as MYB and CAAT box. The promoter was fused with the β-glucuronidase (GUS) reporter gene to generate an expression vector. Arabidopsis thaliana was transformed with a flower dipping method. Gus activity in different tissues and organs of transgenic plants was determined via histochemical staining and quantified via GUS fluorescence. The activity of promoter regulatory elements in transgenic plants under drought, low-temperature, high-salt, and ABA stresses was analyzed. The results showed that the PsDREB2 gene promoter was expressed in the roots, stems, leaves, flowers, and silique pods but not in the seeds of transgenic Arabidopsis. Furthermore, the promoter was induced by drought, low temperature, high salt, and ABA. Hence, the PsDREB2 promoter is tissue- and stress-specific and can be used in the genetic engineering of novel peony cultivars in the future.

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Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia

International Journal of Molecular Sciences ◽

10.3390/ijms22126450 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6450

Author(s):

Anita Wiśniewska ◽

Kamila Wojszko ◽

Elżbieta Różańska ◽

Klaudia Lenarczyk ◽

Karol Kuczerski ◽

...

Keyword(s):

Cell Metabolism ◽

Gene Promoter ◽

Promoter Sequence ◽

Regulatory Elements ◽

Heterodera Schachtii ◽

Myb Transcription Factor ◽

Parasitic Nematodes ◽

Regulatory Sequences ◽

Gene Encoding ◽

Constitutive Overexpression

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants’ responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

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