Evolutionary alterations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos

Development ◽  
1999 ◽  
Vol 126 (17) ◽  
pp. 3725-3734 ◽  
Author(s):  
H. Takahashi ◽  
Y. Mitani ◽  
G. Satoh ◽  
N. Satoh
Keyword(s):  
Reporter Gene ◽  
Transcription Initiation ◽  
Fusion Gene ◽  
Ectopic Expression ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Binding Motif ◽  
Specific Expression ◽  
Notochord Cells ◽  
Flanking Region

The Brachyury genes of two divergent ascidians, As-T of Halocynthia roretzi and Ci-Bra of Ciona intestinalis, are expressed exclusively in notochord precursor cells. A previous study showed that the notochord-specific expression of Ci-Bra is controlled by a minimal promoter that is composed of three distinct regions: a region responsible for repression of expression in non-notochord mesoderm cells, a region for activation of expression in notochord cells, and a region for activation of expression in non-notochord mesoderm cells, distal to proximal to the transcription initiation site, respectively. We examined various deletion constructs of the As-T/lacZ fusion gene and demonstrate that a module between −289 and −250 bp of the 5′-flanking region is responsible for notochord-specific expression of the reporter gene. Gel-shift assays suggested the binding of nuclear protein(s) to this module. The 5′-flanking region of As-T contains a potential T-binding motif (-ACCTAGGT-) around −160 bp. Deletion of this motif from the p(−289)As-T/lacZ diminished the reporter gene expression. In addition, coinjection of p(−289)As-T/lacZ and synthetic As-T mRNA resulted in ectopic expression of lacZ in non-notochord cells, suggesting that the T-binding motif is responsible for autoactivation of the gene. These findings revealed striking differences between the minimal promoters of As-T and Ci-Bra so far revealed, with respect to their notochord-specific expression. Furthermore, reciprocal injections of reporter gene constructs, namely As-T/lacZ into Ciona eggs and Ci-Bra/lacZ into Halocynthia eggs, suggest alterations in the cis-regulatory elements and trans-activation factors that have occurred during evolution of the two ascidian species.

scholarly journals Identification of an enhancer required for the expression of a mouse major urinary protein gene in the submaxillary gland.

1991 ◽  
Vol 11 (8) ◽  
pp. 4244-4252 ◽  
Author(s):  
H J Son ◽  
K Shahan ◽  
M Rodriguez ◽  
E Derman ◽  
F Costantini
Keyword(s):  
Transcription Initiation ◽  
Fusion Gene ◽  
Submaxillary Gland ◽  
Regulatory Elements ◽  
Specific Pattern ◽  
Regulatory Sequence ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
Dna Fragment

The MUP1.5b gene was previously found to be expressed specifically in the submaxillary gland and at high levels when introduced into mice as a transgene including 4.7 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. To localize regulatory elements responsible for this tissue-specific pattern of expression, we tested the expression of three additional MUP1.5b transgenes including various amounts of 5'-flanking DNA. These experiments indicated that sequences between -1.85 and -3.46 kb from the transcription initiation site were required for high-level expression in the submaxillary gland. The presence of regulatory elements in this region was also suggested by the detection of a DNase I-hypersensitive site, seen only in submaxillary gland nuclei, at position -2.5 kb upstream from the MUP1.5a gene, a member of the same MUP gene subfamily and virtually identical to the MUP1.5b gene. Further evidence for enhancer activity was provided by the ability of the 1.6-kb DNA fragment including sequences between -1.85 and -3.46 kb to stimulate the expression of an otherwise inactive MUP1.5b-chloramphenicol acetyltransferase fusion gene specifically in the submaxillary gland. The nucleotide sequence of this 1.6-kb DNA fragment was found to be identical for the MUP1.5a and MUP1.5b genes. Together, these results provide the first localization of a cis-acting regulatory sequence involved in the differential tissue-specific expression of the MUP gene family.

Identification of an enhancer required for the expression of a mouse major urinary protein gene in the submaxillary gland

1991 ◽  
Vol 11 (8) ◽  
pp. 4244-4252
Author(s):  
H J Son ◽  
K Shahan ◽  
M Rodriguez ◽  
E Derman ◽  
F Costantini
Keyword(s):  
Transcription Initiation ◽  
Fusion Gene ◽  
Submaxillary Gland ◽  
Regulatory Elements ◽  
Specific Pattern ◽  
Regulatory Sequence ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
Dna Fragment

The MUP1.5b gene was previously found to be expressed specifically in the submaxillary gland and at high levels when introduced into mice as a transgene including 4.7 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. To localize regulatory elements responsible for this tissue-specific pattern of expression, we tested the expression of three additional MUP1.5b transgenes including various amounts of 5'-flanking DNA. These experiments indicated that sequences between -1.85 and -3.46 kb from the transcription initiation site were required for high-level expression in the submaxillary gland. The presence of regulatory elements in this region was also suggested by the detection of a DNase I-hypersensitive site, seen only in submaxillary gland nuclei, at position -2.5 kb upstream from the MUP1.5a gene, a member of the same MUP gene subfamily and virtually identical to the MUP1.5b gene. Further evidence for enhancer activity was provided by the ability of the 1.6-kb DNA fragment including sequences between -1.85 and -3.46 kb to stimulate the expression of an otherwise inactive MUP1.5b-chloramphenicol acetyltransferase fusion gene specifically in the submaxillary gland. The nucleotide sequence of this 1.6-kb DNA fragment was found to be identical for the MUP1.5a and MUP1.5b genes. Together, these results provide the first localization of a cis-acting regulatory sequence involved in the differential tissue-specific expression of the MUP gene family.

scholarly journals Genetic Manipulation of the Adipose Tissue via Transgenesis

1995 ◽  
Author(s):  
Moshe Shani ◽  
C.P. Emerson
Keyword(s):  
Skeletal Muscle ◽  
Reporter Gene ◽  
Germ Line ◽  
Genetic Manipulation ◽  
Regulatory Gene ◽  
Ectopic Expression ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Specific Expression

The long term goal of this study was to reduce caloric and fat content of beef and other red meats by means of genetic modification of the animal such that fat would not be accumulated. This was attempted by introducing into the germ line myogenic regulatory genes that would convert fat tissue to skeletal muscle. We first determined the consequences of ectopic expression of the myogenic regulatory gene MyoD1. It was found that deregulation of MyoD1 did not result in ectopic skeletal muscle formation but rather led to embryonic lethalities, probably due to its role in the control of the cell cycle. This indicated that MyoD1 should be placed under stringent control to allow survival. Embryonic lethalities were also observed when the regulatory elements of the adipose-specific gene adipsin directed the expression of MyoD1 or myogenin cDNAs, suggesting that these sequences are probably not strong enough to confer tissue specificity. To determine the specificity of the control elements of another fat specific gene (adipocyte protein 2-aP2), we fused them to the bacterial b-galactosidase reporter gene and established stable transgenic strains. The expression of the reporter gene in none of the strains was adipose specific. Each strain displayed a unique pattern of expression in various cell lineages. Most exciting results were obtained in a transgenic strain in which cells migrating from the ventro-lateral edge of the dermomyotome of developing somites to populate the limb buds with myoblasts were specifically stained for lacZ. Since the control sequences of the adipsin or aP2 genes did not confer fat specificity in transgenic mice we have taken both molecular and genetic approaches as an initial effort to identify genes important in the conversion of a multipotential cell such as C3H10T1/2 cell to adipoblast. Several novel adipocyte cell lines have been established that differ in the expression of transcription factors of the C/EBP family known to be markers for adipocyte differentiation. These studies revealed that one of the genetic programming changes which occur during 10T1/2 conversion from multipotential cell to a committed adipoblast is the ability to linduce C/EBPa gene expression. It is expected that further analysis of this gene would identify elements which regulate this lineage-specific expression. Such elements might be good candidates in future attempts to convert adipoblasts to skeletal muscle cells in vivo.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Characterization of oocyte-expressed GDF9 gene in buffalo and mapping of its TSS and putative regulatory elements

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 115-124 ◽  
Author(s):  
B. Roy ◽  
S. Rajput ◽  
S. Raghav ◽  
P. Kumar ◽  
A. Verma ◽  
...  
Keyword(s):  
Core Promoter ◽  
Regulatory Elements ◽  
Vital Role ◽  
Minimal Promoter ◽  
Base Pairs ◽  
Specific Expression ◽  
Oocyte Competence ◽  
Tata Element ◽  
Putative Transcription Factor ◽  
E Boxes

SummaryIn spite of emerging evidence about the vital role of GDF9 in determination of oocyte competence, there is insufficient information about its regulation of oocyte-specific expression, particularly in livestock animals. Because of the distinct prominence of buffalo as a dairy animal, the present study was undertaken to isolate and characterize GDF9 cDNA using orthologous primers based on the bovine GDF9 sequence. GDF9 transcripts were found to be expressed in oocytes irrespective of their follicular origin, and shared a single transcription start site (TSS) at –57 base pairs (bp) upstream of ATG. Assignment of the TSS is consistent with the presence of a TATA element at –23 of the TSS mapped in this study. Localization of a buffalo-specific minimal promoter within 320 bp upstream of ATG was consolidated by identification of an E-box element at –113bp. Presence of putative transcription factor binding sites and other cis regulatory elements were analyzed at ~5 kb upstream of TSS. Various germ cell-specific cis-acting regulatory elements (BNCF, BRNF, NR2F, SORY, Foxh1, OCT1, LHXF etc.) have been identified in the 5′ flanking region of the buffalo GDF9 gene, including NOBOX DNA binding elements and consensuses E-boxes (CANNTG). Presence of two conserved E-boxes found on buffalo sequence at –520 and –718 positions deserves attention in view of its sequence deviation from other species. Two NOBOX binding elements (NBE) were detected at the –3471 and –203 positions. The fall of the NBE within the putative minimal promoter territory of buffalo GDF9 and its unique non-core binding sequence could have a possible role in the control of the core promoter activity.

scholarly journals Specific expression of the human CD4 gene in mature CD4+ CD8- and immature CD4+ CD8+ T cells and in macrophages of transgenic mice

1994 ◽  
Vol 14 (2) ◽  
pp. 1084-1094
Author(s):  
Z Hanna ◽  
C Simard ◽  
A Laperrière ◽  
P Jolicoeur
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Transcription Initiation ◽  
Critical Role ◽  
Regulatory Elements ◽  
T Cell Subsets ◽  
Double Positive ◽  
Specific Expression

The CD4 protein plays a critical role in the development and function of the immune system. To gain more insight into the mechanism of expression of the human CD4 gene, we cloned 42.2 kbp of genomic sequences comprising the CD4 gene and its surrounding sequences. Studies with transgenic mice revealed that a 12.6-kbp fragment of the human CD4 gene (comprising 2.6 kbp of 5' sequences upstream of the transcription initiation site, the first two exons and introns, and part of exon 3) contains the sequences required to support the appropriate expression in murine mature CD4+ CD8- T cells and macrophages but not in immature double-positive CD4+ CD8+ T cells. Expression in CD4+ CD8+ T cells was found to require additional regulatory elements present in a T-cell enhancer fragment recently identified for the murine CD4 gene (S. Sawada and D. R. Littman, Mol. Cell. Biol. 11:5506-5515, 1991). These results suggest that expression of CD4 in mature and immature T-cell subsets may be controlled by distinct and independent regulatory elements. Alternatively, specific regulatory elements may control the expression of CD4 at different levels in mature and immature T-cell subsets. Our data also indicate that mouse macrophages contain the regulatory factors necessary to transcribe the human CD4 gene.

scholarly journals Characterization of two atypical promoters and alternate mRNA processing in the mouse Thy-1.2 glycoprotein gene.

1986 ◽  
Vol 6 (8) ◽  
pp. 2923-2931 ◽  
Author(s):  
H A Ingraham ◽  
G A Evans
Keyword(s):  
Genomic Sequence ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Lymphoid Cells ◽  
Gene Encoding ◽  
Specific Expression ◽  
Transcriptional Initiation ◽  
Glycoprotein Gene ◽  
Cell Type Specific Expression ◽  
Flanking Region

The promoter and 5' flanking region of the mouse Thy-1.2 glycoprotein gene were characterized by DNA sequencing, primer extension analysis, and deletion analysis. Transcriptional initiation sites were identified which corresponded to two separate exons upstream of the portion of the gene encoding the Thy-1.2 glycoprotein. We demonstrated that the mouse Thy-1.2 gene was transcribed from two atypical promoters separated by 260 base pairs in the genomic sequence. These promoters contained neither TATAAG nor GGPyCCAATCT homologous sequences but defined a conserved nonamer CTCCCTGCT at -48 from each initiation site. Two Thy-1.2 mRNA species of 1,835 and 1,939 nucleotides, differing in the 5' untranslated region of the mRNA, were thus transcribed from the single Thy-1.2 gene by mRNA splicing to the same downstream exon. Recombinant genomes in which the bacterial chloramphenicol acetyltransferase gene was expressed from either of the two Thy-1.2 promoters demonstrated that each promoter functioned independently and did not direct cell-specific expression in lymphoid cells. The 5' flanking region of the Thy-1.2 gene upstream of -68 could be eliminated without altering cell-type-specific expression. This suggests that regulatory elements responsible for tissue and developmental stage-specific expression of the Thy-1.2 gene are not present in the 5' flanking DNA but may reside downstream of the promoters.

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.

PLAG1 and USF2 Regulate Primitive Hematopoietic Expression of Musashi-2

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3583-3583
Author(s):  
Muluken S Belew ◽  
Stefan Rentas ◽  
Laura de Rooij ◽  
Kristin J Hope
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Lines ◽  
Regulatory Elements ◽  
Hematopoietic Cell ◽  
Minimal Promoter ◽  
Luciferase Reporter ◽  
Specific Expression ◽  
Self Renewal ◽  
Model Cell

Abstract The Musashi-2 (MSI2) RNA binding protein is now recognized as a key regulator of hematopoietic stem cells (HSCs). Its expression is most elevated in the primitive HSC compartment and progressively decreases with differentiation. In mouse models of CML, ectopic expression of MSI2 drives progression from the chronic to the blast crisis state while in the human context its aberrantly high expression correlates with more aggressive CML disease states and is associated with poor prognosis in AML. These studies suggest that the precise molecular regulation of MSI2 gene expression may be among the critical mechanisms underlying balanced HSC self-renewal and differentiation and as a result, the prevention of leukemic transformation/progression. Despite the clear importance of understanding how Msi2 maintains an appropriate stem cell-specific expression level, very little is understood of the transcription factors (TFs) that mediate this. To define those factors that govern MSI2 expression and function specifically in the HSC compartment we undertook a systematic approach to map and define relevant regulatory elements of the MSI2 minimal promoter. We dissected a 3.5 kb region 5' upstream of MSI2's translational start site (TSS) shared between mouse and human and thus having the greatest potential of containing regulatory elements key to a conserved MSI2 stem-cell-specific gene expression program. Progressive 5'-terminal deletions of this region cloned upstream of a luciferase reporter gene and transfected into K562 and 293T model cell lines allowed us to define a minimal conserved promoter region from -588 to -203 bp upstream of the TSS that reports accurately on endogenous MSI2 expression. Coupled with in silico prediction of TF that bind this region, systematic TF binding site mutagenesis and luciferase reporter assays in model cell lines identified USF2 and PLAG1 as TFs whose direct binding to the MSI2 minimal promoter direct reporter activity. Loss and gain of function studies in K562 cells confirm that these factors co-regulate the transactivation of endogenous MSI2. Moreover we show in the most relevant primary human CD34+ hematopoietic cell context that these factors bind the MSI2 minimal promoter. While USF2 is a ubiquitously expressed TF across the hematopoietic hierarchy, the uniquely restricted expression of PLAG1 within only the most primitive of hematopoietic cells suggests that it specifically contributes to the heightened stem cell-specific expression of MSI2. Consistent with its role as a key driver of MSI2 and thus an enforcer of its pro-self-renewal functions, we found that overexpression of PLAG1 in human Lin-CD34+ cord blood cells enhanced MSI2 transcription and increased total Colony Forming Unit (CFU) output and re-plating efficiency of primitive CFU progenitors. PLAG1 overexpression also offered a pro-survival advantage to these cells as evidenced by a more than two-fold reduction in Annexin V positive cells compared to negative controls. We have thus described important transcriptional circuitry that governs stem-cell specific expression of MSI2 while at the same time functionally validated PLAG1 as a novel factor capable of modulating primitive hematopoietic cell self-renewal and survival. Disclosures No relevant conflicts of interest to declare.

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