Regulation of Pax6 expression is conserved between mice and flies

Development ◽  
1999 ◽  
Vol 126 (2) ◽  
pp. 383-395 ◽  
Author(s):  
P.X. Xu ◽  
X. Zhang ◽  
S. Heaney ◽  
A. Yoon ◽  
A.M. Michelson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
System Development ◽  
Regulatory Element ◽  
Amacrine Cells ◽  
Pax6 Expression ◽  
Specific Expression ◽  
Cis Acting ◽  
Evolutionarily Conserved ◽  
Visual System Development ◽  
Lens Placode

Pax6 plays a key role in visual system development throughout the metazoa and the function of Pax6 is evolutionarily conserved. However, the regulation of Pax6 expression during eye development is largely unknown. We have identified two physically distinct promoters in mouse Pax6, P0 and P1, that direct differential Pax6 expression in the developing eye. P0-initiated transcripts predominate in lens placode and corneal and conjunctival epithelia, whereas P1-initiated transcripts are expressed in lens placode, optic vesicle and CNS, and only weakly in corneal and conjunctival epithelia. To further investigate their tissue-specific expression, a series of constructs for each promoter were examined in transgenic mice. We identified three different regulatory regions which direct distinct domains of Pax6 expression in the eye. A regulatory element upstream of the Pax6 P0 promoter is required for expression in a subpopulation of retinal progenitors and in the developing pancreas, while a second regulatory element upstream of the Pax6 P1 promoter is sufficient to direct expression in a subset of post-mitotic, non-terminally differentiated photoreceptors. A third element in Pax6 intron 4, when combined with either the P0 or P1 promoter, accurately directs expression in amacrine cells, ciliary body and iris. These results indicate that the complex expression pattern of Pax6 is differentially regulated by two promoters acting in combination with multiple cis-acting elements. We have also tested whether the regulatory mechanisms that direct Pax6 ocular expression are conserved between mice and flies. Remarkably, when inserted upstream of either the mouse Pax6 P1 or P0 promoter, an eye-enhancer region of the Drosophila eyeless gene, a Pax6 homolog, directs eye- and CNS-specific expression in transgenic mice that accurately reproduces features of endogenous Pax6 expression. These results suggest that in addition to conservation of Pax6 function, the upstream regulation of Pax6 has also been conserved during evolution.

scholarly journals The transcriptional tissue specificity of the human proα1(I) collagen gene is determined by a negative cis-regulatory element in the promoter

1992 ◽  
Vol 286 (1) ◽  
pp. 179-185 ◽  
Author(s):  
C P Simkevich ◽  
J P Thompson ◽  
H Poppleton ◽  
R Raghow
Keyword(s):  
Tissue Specificity ◽  
Regulatory Element ◽  
Mesenchymal Cell ◽  
Cell Types ◽  
Regulatory Elements ◽  
Negative Influence ◽  
Collagen Gene ◽  
Specific Expression ◽  
Cis Acting ◽  
First Intron

The transcriptional activity of plasmid pCOL-KT, in which human pro alpha 1 (I) collagen gene upstream sequences up to -804 and most of the first intron (+474 to +1440) drive expression of the chloramphenicol acetyltransferase (CAT) gene [Thompson, Simkevich, Holness, Kang & Raghow (1991) J. Biol. Chem. 266, 2549-2556], was tested in a number of mesenchymal and non-mesenchymal cells. We observed that pCOL-KT was readily expressed in fibroblasts of human (IMR-90 and HFL-1), murine (NIH 3T3) and avian (SL-29) origin and in a human rhabdomyosarcoma cell line (A204), but failed to be expressed in human erythroleukaemia (K562) and rat pheochromocytoma (PC12) cells, indicating that the regulatory elements required for appropriate tissue-specific expression of the human pro alpha 1 (I) collagen gene were present in pCOL-KT. To delineate the nature of cis-acting sequences which determine the tissue specificity of pro alpha 1 (I) collagen gene expression, functional consequences of deletions in the promoter and first intron of pCOL-KT were tested in various cell types by transient expression assays. Cis elements in the promoter-proximal and intronic sequences displayed either a positive or a negative influence depending on the cell type. Thus deletion of fragments using EcoRV (nt -625 to -442 deleted), XbaI (-804 to -331) or SstII (+670 to +1440) resulted in 2-10-fold decreased expression in A204 and HFL-1 cells. The negative influences of deletions in the promoter-proximal sequences was apparently considerably relieved by deleting sequences in the first intron, and the constructs containing the EcoRV/SstII or XbaI/SstII double deletions were expressed to a much greater extent than either of the single deletion constructs. In contrast, the XbaI* deletion (nt -804 to -609), either alone or in combination with the intronic deletion, resulted in very high expression in all cells regardless of their collagen phenotype; the XbaI*/(-SstII) construct, which contained the intronic SstII fragment (+670 to +1440) in the reverse orientation, was not expressed in either mesenchymal or nonmesenchymal cells. Based on these results, we conclude that orientation-dependent interactions between negatively acting 5′-upstream sequences and the first intron determine the mesenchymal cell specificity of human pro alpha 1 (I) collagen gene transcription.

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 An internal regulatory element controls troponin I gene expression.

1989 ◽  
Vol 9 (4) ◽  
pp. 1397-1405 ◽  
Author(s):  
K E Yutzey ◽  
R L Kline ◽  
S F Konieczny
Keyword(s):  
Transcriptional Activation ◽  
Troponin I ◽  
Protein Gene ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Contractile Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
Specific Expression Pattern

During skeletal myogenesis, approximately 20 contractile proteins and related gene products temporally accumulate as the cells fuse to form multinucleated muscle fibers. In most instances, the contractile protein genes are regulated transcriptionally, which suggests that a common molecular mechanism may coordinate the expression of this diverse and evolutionarily unrelated gene set. Recent studies have examined the muscle-specific cis-acting elements associated with numerous contractile protein genes. All of the identified regulatory elements are positioned in the 5'-flanking regions, usually within 1,500 base pairs of the transcription start site. Surprisingly, a DNA consensus sequence that is common to each contractile protein gene has not been identified. In contrast to the results of these earlier studies, we have found that the 5'-flanking region of the quail troponin I (TnI) gene is not sufficient to permit the normal myofiber transcriptional activation of the gene. Instead, the TnI gene utilizes a unique internal regulatory element that is responsible for the correct myofiber-specific expression pattern associated with the TnI gene. This is the first example in which a contractile protein gene has been shown to rely primarily on an internal regulatory element to elicit transcriptional activation during myogenesis. The diversity of regulatory elements associated with the contractile protein genes suggests that the temporal expression of the genes may involve individual cis-trans regulatory components specific for each gene.

scholarly journals Reconstitution of human Fc gamma RIII cell type specificity in transgenic mice.

1996 ◽  
Vol 183 (3) ◽  
pp. 1259-1263 ◽  
Author(s):  
M Li ◽  
U Wirthmueller ◽  
J V Ravetch
Keyword(s):  
Transgenic Mice ◽  
Nk Cells ◽  
Gene Promoter ◽  
Homologous Region ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Specific Expression ◽  
Cis Acting ◽  
Sequence Elements ◽  
Flanking Sequences

The human low affinity receptors for the Fc domain of immunoglobulin G, Fc gamma RIII, are encoded by two genes (IIIA and IIIB) which share >95% sequence identity in both coding and flanking sequences. Despite this extraordinary sequence conservation, IIIA is expressed in natural killer (NK) cells and macrophages and is absent in neutrophils, whereas IIIB is expressed only in neutrophils. To determine the molecular basis for this differential expression, we have generated transgenic mice using the genomic sequences of IIIA and IIIB. IIIA and IIIB transgenic mice show faithful reconstitution of this human pattern of cell type specificity. To determine the cis acting sequence elements that confer this specificity, we constructed chimeric genes in which 5.8 kb of 5' sequences of the IIIB gene has been replaced with a homologous region from the IIIA gene, and conversely, IIIA 5' sequences have been substituted for the analogous region of the IIIB gene. Promoter swap transgenic mice that carry IIIA 5' flanking sequences express Fc gamma RIII in macrophages and NK cells. In contrast, promoter swap transgenic mice that contain IIIB 5' sequences express Fc gamma RIII in neutrophils only. These studies define the elements conferring the cell type-specific expression of the human Fc gamma RIII genes within the 5' flanking sequences and first intron of the human Fc gamma RIIIA and Fc gamma RIIIB genes.

Promoter assay using transgenic mice reveals the presence of cis-acting elements governing tissue- and site-specific expression of Col11a2

1996 ◽  
Vol 15 (3) ◽  
pp. 194-195
Author(s):  
N. Tsumaki ◽  
M. Sugimoto ◽  
Y. Matsui ◽  
K. Nakata ◽  
T. Ochi ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Specific Expression ◽  
Promoter Assay ◽  
Site Specific ◽  
Cis Acting

scholarly journals Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice.

1992 ◽  
Vol 12 (3) ◽  
pp. 1007-1020 ◽  
Author(s):  
M K Short ◽  
D E Clouthier ◽  
I M Schaefer ◽  
R E Hammer ◽  
M A Magnuson ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Transgenic Mice ◽  
Human Growth Hormone ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Human Growth ◽  
Fusion Genes ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Specific Manner

The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in multiple tissues and is regulated in a complex tissue-specific manner. To map the cis-acting DNA elements that direct this tissue-specific expression, we made transgenic mice containing truncated PEPCK-human growth hormone (hGH) fusion genes. The transgenes contained PEPCK promoter fragments with 5' endpoints at -2088, -888, -600, -402, and -207 bp, while the 3' endpoint was at +69 bp. Immunohistochemical analysis showed that the -2088 transgene was expressed in the correct cell types (hepatocytes, proximal tubular epithelium of the kidney, villar epithelium of the small intestine, epithelium of the colon, smooth muscle of the vagina and lungs, ductal epithelium of the sublingual gland, and white and brown adipocytes). Solution hybridization of hGH mRNA expressed from the transgenes indicated that white and brown fat-specific elements are located distally (-2088 to -888 bp) and that liver-, gut-, and kidney-specific elements are located proximally (-600 to +69 bp). However, elements outside of the region tested are necessary for the correct developmental pattern and level of PEPCK expression in kidney. Both the -2088 and -402 transgenes responded in a tissue-specific manner to dietary stimuli, and the -2088 transgene responded to glucocorticoid stimuli. Thus, different tissues utilize distinct cell-specific cis-acting elements to direct and regulate the PEPCK gene.

Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements

1990 ◽  
Vol 10 (3) ◽  
pp. 930-938
Author(s):  
G L Semenza ◽  
R C Dureza ◽  
M D Traystman ◽  
J D Gearhart ◽  
S E Antonarakis
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Cobalt Chloride ◽  
Fetal Liver ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Flanking Sequence ◽  
Rnase Protection ◽  
Cis Acting

Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.

Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice

1991 ◽  
Vol 11 (7) ◽  
pp. 3564-3572 ◽  
Author(s):  
J W Chamberlain ◽  
H A Vasavada ◽  
S Ganguly ◽  
S M Weissman
Keyword(s):  
Major Histocompatibility Complex ◽  
Transgenic Mice ◽  
Major Histocompatibility Complex Class ◽  
Class I ◽  
Complex Class ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Tissue Specific Expression ◽  
Histocompatibility Complex

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice

1987 ◽  
Vol 7 (10) ◽  
pp. 3749-3758
Author(s):  
V da C Soares ◽  
R M Gubits ◽  
P Feigelson ◽  
F Costantini
Keyword(s):  
Transgenic Mice ◽  
Gene Family ◽  
Hormonal Regulation ◽  
Germ Line ◽  
Regulatory Elements ◽  
Preputial Gland ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Globulin Gene

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.

Developmental expression of SI is regulated in transgenic mice by an evolutionarily conserved promoter

1997 ◽  
Vol 273 (1) ◽  
pp. G83-G92 ◽  
Author(s):  
J. Tung ◽  
A. J. Markowitz ◽  
D. G. Silberg ◽  
P. G. Traber
Keyword(s):  
Transgenic Mice ◽  
Gene Promoter ◽  
Developmental Expression ◽  
Major Transitions ◽  
Cis Acting ◽  
The Third ◽  
Evolutionarily Conserved ◽  
Mouse Intestine ◽  
Low Levels ◽  
Direct Induction

Developmental expression of the sucrase-isomaltase (SI) gene in the mouse intestine involves two major transitions that correspond to critical developmental events. Low levels of SI mRNA were first identified in day 16.5 fetal mouse intestine, immediately after the transition from stratified endoderm to a columnar epithelium organized in nascent villi. Low levels were maintained until the third week of life, when induction of SI mRNA to adult levels was observed coincident with the time of weaning. The mechanism of this pattern of SI gene expression was studied in transgenic mice using a reporter gene construct containing an SI gene promoter that is evolutionarily conserved between mouse and human (nucleotides -201 to +54 of the mouse SI gene). This promoter included the necessary regulatory information to direct transcription to enterocytes in developmental and differentiation-dependent patterns that recapitulated the expression of the endogenous SI gene. However, transgenes lacked the ability to direct induction of precocious expression in suckling animals after administration of corticosteroids. These findings define a short SI gene promoter that contains cis-acting elements that are responsible for developmental and differentiation-dependent transcriptional regulation.

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