A GATA-dependent nkx-2.5 regulatory element activates early cardiac gene expression in transgenic mice

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4461-4470 ◽  
Author(s):  
R.D. Searcy ◽  
E.B. Vincent ◽  
C.M. Liberatore ◽  
K.E. Yutzey
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Expression Pattern ◽  
Binding Sites ◽  
Early Stage ◽  
Regulatory Element ◽  
Gene Activation ◽  
Flanking Sequence ◽  
Regulatory Factors ◽  
Developing Heart

nkx-2.5 is one of the first genes expressed in the developing heart of early stage vertebrate embryos. Cardiac expression of nkx-2.5 is maintained throughout development and nkx-2.5 also is expressed in the developing pharyngeal arches, spleen, thyroid and tongue. Genomic sequences flanking the mouse nkx-2.5 gene were analyzed for early developmental regulatory activity in transgenic mice. Approximately 3 kb of 5′ flanking sequence is sufficient to activate gene expression in the cardiac crescent as early as E7.25 and in limited regions of the developing heart at later stages. Expression also was detected in the developing spleen anlage at least 24 hours before the earliest reported spleen marker and in the pharyngeal pouches and their derivatives including the thyroid. The observed expression pattern from the −3 kb construct represents a subset of the endogenous nkx-2.5 expression pattern which is evidence for compartment-specific nkx-2.5 regulatory modules. A 505 bp regulatory element was identified that contains multiple GATA, NKE, bHLH, HMG and HOX consensus binding sites. This element is sufficient for gene activation in the cardiac crescent and in the heart outflow tract, pharynx and spleen when linked directly to lacZ or when positioned adjacent to the hsp68 promoter. Mutation of paired GATA sites within this element eliminates gene activation in the heart, pharynx and spleen primordia of transgenic embryos. The dependence of this nkx-2. 5 regulatory element on GATA sites for gene activity is evidence for a GATA-dependent regulatory mechanism controlling nkx-2.5 gene expression. The presence of consensus binding sites for other developmentally important regulatory factors within the 505 bp distal element suggests that combinatorial interactions between multiple regulatory factors are responsible for the initial activation of nkx-2.5 in the cardiac, thyroid and spleen primordia.

scholarly journals Phorbol Ester-Induced Human Cytomegalovirus Major Immediate-Early (MIE) Enhancer Activation through PKC-Delta, CREB, and NF-κB Desilences MIE Gene Expression in Quiescently Infected Human Pluripotent NTera2 Cells

2010 ◽  
Vol 84 (17) ◽  
pp. 8495-8508 ◽  
Author(s):  
Xiaoqiu Liu ◽  
Jinxiang Yuan ◽  
Allen W. Wu ◽  
Patrick W. McGonagill ◽  
Courtney S. Galle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Gene Activation ◽  
Dominant Negative ◽  
Immediate Early ◽  
Signaling Cascade ◽  
Pkc Delta

ABSTRACT The ways in which human cytomegalovirus (HCMV) major immediate-early (MIE) gene expression breaks silence from latency to initiate the viral replicative cycle are poorly understood. A delineation of the signaling cascades that desilence the HCMV MIE genes during viral quiescence in the human pluripotent N-Tera2 (NT2) cell model provides insight into the molecular mechanisms underlying HCMV reactivation. In this model, we show that phorbol 12-myristate 13-acetate (PMA) immediately activates the expression of HCMV MIE RNA and protein and greatly increases the MIE-positive (MIE+) NT2 cell population density; levels of Oct4 (pluripotent cell marker) and HCMV genome penetration are unchanged. Decreasing PKC-delta activity (pharmacological, dominant-negative, or RNA interference [RNAi] method) attenuates PMA-activated MIE gene expression. MIE gene activation coincides with PKC-delta Thr505 phosphorylation. Mutations in MIE enhancer binding sites for either CREB (cyclic AMP [cAMP] response element [CRE]) or NF-κB (κB) partially block PMA-activated MIE gene expression; the ETS binding site is negligibly involved, and κB does not confer MIE gene activation by vasoactive intestinal peptide (VIP). The PMA response is also partially attenuated by the RNAi-mediated depletion of the CREB or NF-κB subunit RelA or p50; it is not diminished by TORC2 knockdown or accompanied by TORC2 dephosphorylation. Mutations in both CRE and κB fully abolish PMA-activated MIE gene expression. Thus, PMA stimulates a PKC-delta-dependent, TORC2-independent signaling cascade that acts through cellular CREB and NF-κB, as well as their cognate binding sites in the MIE enhancer, to immediately desilence HCMV MIE genes. This signaling cascade is distinctly different from that elicited by VIP.

scholarly journals Nuclear factor I and mammary gland factor (STAT5) play a critical role in regulating rat whey acidic protein gene expression in transgenic mice.

1995 ◽  
Vol 15 (4) ◽  
pp. 2063-2070 ◽  
Author(s):  
S Li ◽  
J M Rosen
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Transgene Expression ◽  
Protein Gene ◽  
Whey Acidic Protein ◽  
Nuclear Factor ◽  
Factor I ◽  
Nuclear Factor I

The rat whey acidic protein (WAP) gene contains a mammary gland-specific and hormonally regulated DNase I-hypersensitive site 830 to 720 bp 5' to the site of transcription initiation. We have reported previously that nuclear factor I (NFI) binding at a palindromic site and binding at a half-site are the major DNA-protein interactions detected within this tissue-specific nuclease-hypersensitive region. We now show that point mutations introduced into these NFI-binding sites dramatically affect WAP gene expression in transgenic mice. Transgene expression was totally abrogated when the palindromic NFI site or both binding sites were mutated, suggesting that NFI is a key regulator of WAP gene expression. In addition, a recognition site for mammary gland factor (STAT5), which mediates prolactin induction of milk protein gene expression, was also identified immediately proximal to the NFI-binding sites. Mutation of this site reduced transgene expression by approximately 90% per gene copy, but did not alter tissue specificity. These results suggest that regulation of WAP gene expression is determined by the cooperative interactions among several enhancers that constitute a composite response element.

scholarly journals Identification of a Ty1 regulatory sequence responsive to STE7 and STE12.

1988 ◽  
Vol 8 (6) ◽  
pp. 2545-2554 ◽  
Author(s):  
M Company ◽  
C Adler ◽  
B Errede
Keyword(s):  
Gene Expression ◽  
Complex Formation ◽  
Reporter Gene ◽  
Regulatory Element ◽  
Gene Activation ◽  
Cell Types ◽  
Reporter Gene Expression ◽  
Regulatory Sequence ◽  
Gene Products ◽  
Cell Extracts

Ty1 activation of gene expression observed in haploid cell types of Saccharomyces cerevisiae requires the STE7 and STE12 gene products. An activator sequence within Ty1 that is responsive to these two regulators has been defined. Complex formation between a factor in whole-cell extracts and the DNA regulatory element showed the same dependence on the STE7 and STE12 gene products as did reporter gene expression. Base pair substitutions within the binding site abolished the ability to form the factor-DNA complex and to activate gene expression. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for gene activation. Because the predicted protein for the STE7 gene product is homologous to protein kinases, we suggest that protein phosphorylation may directly or indirectly regulate formation of this DNA-protein complex.

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.

The murine cripto gene: expression during mesoderm induction and early heart morphogenesis

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1157-1168 ◽  
Author(s):  
R. Dono ◽  
L. Scalera ◽  
F. Pacifico ◽  
D. Acampora ◽  
M.G. Persico ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Adult Animal ◽  
Mesoderm Induction ◽  
Coding Region ◽  
Developing Heart ◽  
Human Counterpart ◽  
Epidermal Growth

The murine cripto gene encodes a 171-aminoacid epidermal growth factor-related protein, with 93% similarity to its human counterpart in the ‘EGF-like’ domain. The murine cripto mRNA contains two B1 repeats in its 3′ non-coding region and a 163-nucleotide homology to the human mRNA. The mouse cripto gene is expressed at low level in specific organs of the adult animal such as spleen, heart, lung and brain. In situ hybridization analysis during murine embryogenesis (day 6.2 to day 10.5) reveals a very restricted expression pattern. cripto transcripts are first detected in a few epiblastic cells at day 6.5. During gastrulation, the transcripts are expressed in the forming mesoderm and later during development cripto gene expression is restricted to the truncus arteriosus of the developing heart. This expression pattern suggests a role for cripto gene in the determination of the epiblastic cells that subsequently give rise to the mesoderm.

scholarly journals Analysis of dopamine transporter gene expression pattern − generation of DAT-iCre transgenic mice

FEBS Journal ◽  
2007 ◽  
Vol 274 (14) ◽  
pp. 3568-3577 ◽  
Author(s):  
Marc Turiault ◽  
Sébastien Parnaudeau ◽  
Aude Milet ◽  
Rosanna Parlato ◽  
Jean-Denis Rouzeau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Dopamine Transporter ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Pattern Generation ◽  
Transporter Gene ◽  
Dopamine Transporter Gene

Distal elements are critical for human CD34 expression in vivo

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4420-4426 ◽  
Author(s):  
Yutaka Okuno ◽  
Claudia S. Huettner ◽  
Hanna S. Radomska ◽  
Victoria Petkova ◽  
Hiromi Iwasaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Analysis ◽  
Transgenic Mice ◽  
Flanking Sequence ◽  
Hematopoietic Stem ◽  
Critical Elements ◽  
Human Cd34 ◽  
Stem And Progenitor Cells ◽  
Cd34 Expression

The elements regulating gene expression in hematopoietic stem cells are still poorly understood. We previously reported that a 141-kilobase (kb) human CD34 transgene confers properly regulated human CD34 expression in transgenic mice. A construct with only the human CD34 promoter and 3′ enhancer region is not sufficient, suggesting that critical distal elements are necessary for expression of the human CD34 gene. To further localize such elements, we analyzed deletion constructs of the human CD34 gene and evaluated their function in transgenic mice. Constructs harboring as little as 18 kb of 5′ and 26 kb of 3′ human CD34 flanking sequence conferred human expression in tissues of transgenic mice with a pattern similar to that of the 141-kb human transgene. In contrast, a construct harboring 10 kb of 5′ and 17 kb of 3′ human CD34 flanking sequence gave no expression. These data demonstrate that regions between 10 to 18 kb upstream and/or 17 to 26 kb downstream of the human CD34 gene contain critical elements for human CD34 expression in vivo. Further functional analysis of these regions in transgenic mice will be crucial for understanding CD34 gene expression in hematopoietic stem and progenitor cells.

scholarly journals BEST: A Novel Computational Approach for Comparing Gene Expression Patterns From Early Stages of Drosophila melanogaster Development

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 2037-2047
Author(s):  
Sudhir Kumar ◽  
Karthik Jayaraman ◽  
Sethuraman Panchanathan ◽  
Rajalakshmi Gurunathan ◽  
Ana Marti-Subirana ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Biology ◽  
Drosophila Melanogaster ◽  
Expression Pattern ◽  
Early Stage ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Computational Approach ◽  
Gene Expression Patterns ◽  
Early Stages

Abstract Embryonic gene expression patterns are an indispensable part of modern developmental biology. Currently, investigators must visually inspect numerous images containing embryonic expression patterns to identify spatially similar patterns for inferring potential genetic interactions. The lack of a computational approach to identify pattern similarities is an impediment to advancement in developmental biology research because of the rapidly increasing amount of available embryonic gene expression data. Therefore, we have developed computational approaches to automate the comparison of gene expression patterns contained in images of early stage Drosophila melanogaster embryos (prior to the beginning of germ-band elongation); similarities and differences in gene expression patterns in these early stages have extensive developmental effects. Here we describe a basic expression search tool (BEST) to retrieve best matching expression patterns for a given query expression pattern and a computational device for gene interaction inference using gene expression pattern images and information on the associated genotypes and probes. Analysis of a prototype collection of Drosophila gene expression pattern images is presented to demonstrate the utility of these methods in identifying biologically meaningful matches and inferring gene interactions by direct image content analysis. In particular, the use of BEST searches for gene expression patterns is akin to that of BLAST searches for finding similar sequences. These computational developmental biology methodologies are likely to make the great wealth of embryonic gene expression pattern data easily accessible and to accelerate the discovery of developmental networks.

scholarly journals Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009039
Author(s):  
Yi Kuang ◽  
Anna Pyo ◽  
Natanel Eafergan ◽  
Brittany Cain ◽  
Lisa M. Gutzwiller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Binding Sites ◽  
Target Gene ◽  
Gene Activation ◽  
Notch Intracellular Domain ◽  
Repressor Complex ◽  
Notch Activation

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

89 ZYGOTICALLY ACTIVATED GENES ARE SUPPRESSED IN MOUSE NUCLEAR-TRANSFERRED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 162
Author(s):  
T. Suzuki ◽  
N. Minami ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Early Stage ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Gene Activation ◽  
Blastocyst Stage ◽  
Success Rates ◽  
Cell Nuclei ◽  
Cell Stage

Mammalian oocytes have the ability to confer totipotency to terminally differentiated somatic cell nuclei. Viable cloned animals have been produced by somatic cell nuclear transfer (NT) into oocytes in many mammalian species including mouse. However, the success rates of the production were quite low in all species. Many studies have measured differences in gene expression between NT and fertilized embryos in relatively advanced stages of development such as pre- and post-natal stages or the blastocyst stage. In the mouse, major zygotic gene activation (ZGA) occurs at the 2-cell stage after fertilization and leads to the transition of gene regulation from maternal control to embryonic control. Suppression of the ZGA by a transcription inhibitor was shown to decrease the viability of embryos, and causes developmental arrest at the 2-cell stage. An abnormal ZGA may therefore affect the viability of NT embryos and cause further abnormalities in later embryonic development. In the present study, we compared gene expression patterns using differential display RT-PCR (DDRT-PCR) between the NT and IVF embryos at the 2-cell stage to detect some abnormalities affecting later development of NT embryos. The developmental rate of NT embryos to blastocysts (32.9%) was significantly lower than that of IVF (92.7%) or PA (92.8%). In addition, the cell numbers of NT embryos at the blastocyst stage (39.5 � 2.6; n = 19) were less than those of IVF (66.8 � 2.1; n = 30) or PA embryos (48.2 � 2.1; n = 30). Using these embryos, we first identified 4 genes that were differentially expressed between NT and IVF embryos at the 2-cell stage. Among the identified genes, Inpp5b and Chst12 were up-regulated, and MuERV-L and Dnaja2 were down-regulated in the NT embryos compared with IVF embryos. Further analysis showed that the expression of zygotically activated genes such as Interferon-γ, Dub-1, Spz1, DD2106, and DD2111 were not properly activated in NT embryos, suggesting that the cellular process involved in the control of the zygotic genome activation is not appropriately regulated. These results indicate that abnormal gene expression has already occurred at the early stage of pre-implantation development as a failure of nuclear reprogramming.

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