GATA Motif on the Erythropoietin Gene Promoter Is Essential for Repression of Ectopic Constitutive Erythropoietin Production.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3139-3139
Author(s):  
Naoshi Obara ◽  
Norio Suzuki ◽  
Kim Ki-Bom ◽  
Shigehiko Imagawa ◽  
Toshiro Nagasawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Central Vein ◽  
Specific Expression ◽  
Neuronal Markers ◽  
Nucleotide Mutation ◽  
Gata Motif

Abstract In response to anemia erythropoietin ( Epo) gene transcription is markedly induced in the kidney and liver. This regulation has been ascribed for the promoter GATA motif and 3′ enhancer. To elucidate how the Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of renal cortex and hepatocytes surrounding the central vein. Surprisingly, the renal Epo-producing cells were identified with neuron-like morphology and expression of neuronal markers. We also examined the regulatory mechanism of Epo gene using the transgenes in which mutations had been introduced in the GATA promoter motif, since we have reported the motif as a negative regulatory element for the inducible Epo gene expression. A single nucleotide mutation in the motif resulted in constitutive ectopic expression of GFP in the renal distal tubules, collecting ducts and certain epithelial cells of other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in the distal tubular cells, these factors are likely to constitutively repress ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell-type specific expression of the Epo gene.

Repression via the GATA box is essential for tissue-specific erythropoietin gene expression

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5223-5232 ◽  
Author(s):  
Naoshi Obara ◽  
Norio Suzuki ◽  
Kibom Kim ◽  
Toshiro Nagasawa ◽  
Shigehiko Imagawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Marker Genes ◽  
Central Vein ◽  
Specific Expression ◽  
Neuronal Marker ◽  
Nucleotide Mutation ◽  
Gata Box

Abstract In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type–specific expression of the Epo gene.

Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos

Development ◽  
2001 ◽  
Vol 128 (12) ◽  
pp. 2341-2350
Author(s):  
Makoto Kobayashi ◽  
Keizo Nishikawa ◽  
Masayuki Yamamoto
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Regulatory Region ◽  
Ectopic Expression ◽  
Transgenic Fish ◽  
Functional Domain ◽  
Cis Acting ◽  
Gfp Reporter ◽  
Gata Motif

Expression of gata1 is regulated through multiple cis-acting GATA motifs. To elucidate regulatory mechanisms of the gata1 gene, we have used zebrafish. To this end, we isolated and analyzed zebrafish gata1 genomic DNA, which resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA. Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA1 overexpression and was dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct revealed that both the N- and C-terminal zinc-finger domains of GATA1, hence intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation.

scholarly journals Potential Autoregulation of Transcription Factor PU.1 by an Upstream Regulatory Element

2005 ◽  
Vol 25 (7) ◽  
pp. 2832-2845 ◽  
Author(s):  
Yutaka Okuno ◽  
Gang Huang ◽  
Frank Rosenbauer ◽  
Erica K. Evans ◽  
Hanna S. Radomska ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Reporter Gene ◽  
Regulatory Element ◽  
Mammalian Species ◽  
Critical Role ◽  
Homologous Region ◽  
Specific Expression ◽  
Upstream Regulatory Element

ABSTRACT Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb −14 site confers lineage-specific reporter gene expression in vivo. This kb −14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.

scholarly journals A study of somatolactin actions by ectopic expression in transgenic zebrafish larvae

2010 ◽  
Vol 45 (5) ◽  
pp. 301-315 ◽  
Author(s):  
Guohui Wan ◽  
King Ming Chan
Keyword(s):  
Fatty Acid Synthase ◽  
Fluorescent Protein ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Gene Promoter ◽  
Transgenic Fish ◽  
Fish Growth ◽  
Marker Genes ◽  
Transgenic Zebrafish

Somatolactin (SL) is a fish-specific hormone that belongs to the prolactin (PRL) and GH family. Recently, two forms of SL, SLα and SLβ, have been found in some species, and may have different actions and functions. To investigate the role of SL in fish growth and metabolism, we generated transgenic fish founders with ectopic expression of SLα and SLβ to study the physiological functions and actions of these SLs among several marker genes. We fused the cDNAs encoding the precursor SLs in frame to a zebrafish β-actin gene promoter to generate transgenic zebrafish lines that were coinjected with a green fluorescent protein (GFP) driven by the same promoter. The transgenic zebrafish were selected based on GFP expression and confirmed by genomic PCR, Southern blot analysis, and transgene expression. Investigations into the expression of marker genes in larvae on different pathways using real-time PCR have provided a general understanding of the actions of SLs. This study found that the overexpression of SLα and SLβ in vivo significantly enhanced the transcription of IGFs, insulin, leptin, sterol regulatory element binding protein 1, and fatty acid synthase, as well as the expression level of vitellogenin and proopiomelanocortin, while causing reduced levels of catalase and glutathione S-transferase in the larvae of transgenic zebrafish.

Smooth muscle expression of Cre recombinase and eGFP in transgenic mice

2002 ◽  
Vol 10 (3) ◽  
pp. 211-215 ◽  
Author(s):  
H.-B. Xin ◽  
K.-Y. Deng ◽  
M. Rishniw ◽  
G. Ji ◽  
M. I. Kotlikoff
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Transgenic Mice ◽  
Gene Function ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Smooth Muscles ◽  
Cre Recombinase ◽  
Specific Expression

We report the generation of transgenic mice designed to facilitate the study of vascular and nonvascular smooth muscle biology in vivo. The smooth muscle myosin heavy chain (smMHC) promoter was used to direct expression of a bicistronic transgene consisting of Cre recombinase and enhanced green fluorescent protein (eGFP) coding sequences. Animals expressing the transgene display strong fluorescence confined to vascular and nonvascular smooth muscle. Enzymatic dissociation of smooth muscle yields viable, fluorescent cells that can be studied as single cells or sorted by FACS for gene expression studies. smMHC/Cre/eGFP mice were crossed with ROSA26/lacZ reporter mice to determine Cre recombinase activity; Cre recombinase was expressed in all smooth muscles in adult mice, and there was an excellent overlap between expression of the recombinase and eGFP. Initial smooth muscle-specific expression of fluorescence and Cre recombinase was detected on embryonic day 12.5. These mice will be useful to define smooth muscle gene function in vivo in mice, for the study of gene function in single, live cells, and for the determination of gene expression in vascular and nonvascular smooth muscle.

scholarly journals Phosphorylation within Intrinsic Disordered Region Discriminates Histone Variant macroH2A1 Splicing Isoforms—macroH2A1.1 and macroH2A1.2

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 659
Author(s):  
Sebastiano Giallongo ◽  
Oriana Lo Re ◽  
Gabriela Lochmanová ◽  
Luca Parca ◽  
Francesco Petrizzelli ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Element ◽  
Histone Variants ◽  
Histone H2a ◽  
Human Hepatoma Cells ◽  
Nucleosome Core ◽  
Intrinsically Disordered ◽  
Splicing Isoforms ◽  
The Impact

Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.

1986 ◽  
Vol 6 (11) ◽  
pp. 4130-4132 ◽  
Author(s):  
S Hayashi ◽  
H Kondoh
Keyword(s):  
Gene Expression ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Fragments ◽  
Specific Expression ◽  
Mouse Cells ◽  
Gc Box ◽  
Simplex Virus

Expression of the chicken delta-crystallin gene 1 injected into the nuclei of mouse cells is lens specific. Coinjection of GC box-containing DNA fragments from delta-crystallin, simian virus 40 early, and herpes simplex virus type 1 tk promoters effectively suppressed delta-crystallin expression in the lens, but coinjection with DNA fragments not containing the GC box did not. This suppression was likely due to the competition of an Sp1-like transcription factor(s) and indicates involvement of the apparently ubiquitous factor(s) in the tissue-specific expression of the delta-crystallin gene.

Targeting gene expression to the head: the Drosophila orthodenticle gene is a direct target of the Bicoid morphogen

Development ◽  
1998 ◽  
Vol 125 (21) ◽  
pp. 4185-4193 ◽  
Author(s):  
Q. Gao ◽  
R. Finkelstein
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Repeated Sequence ◽  
Drosophila Embryo ◽  
Sequence Motif ◽  
Specific Expression ◽  
Gap Gene

The Bicoid (Bcd) morphogen establishes the head and thorax of the Drosophila embryo. Bcd activates the transcription of identified target genes in the thoracic segments, but its mechanism of action in the head remains poorly understood. It has been proposed that Bcd directly activates the cephalic gap genes, which are the first zygotic genes to be expressed in the head primordium. It has also been suggested that the affinity of Bcd-binding sites in the promoters of Bcd target genes determines the posterior extent of their expression (the Gene X model). However, both these hypotheses remain untested. Here, we show that a small regulatory region upstream of the cephalic gap gene orthodenticle (otd) is sufficient to recapitulate early otd expression in the head primordium. This region contains two control elements, each capable of driving otd-like expression. The first element has consensus Bcd target sites that bind Bcd in vitro and are necessary for head-specific expression. As predicted by the Gene X model, this element has a relatively low affinity for Bcd. Surprisingly, the second regulatory element has no Bcd sites. Instead, it contains a repeated sequence motif similar to a regulatory element found in the promoters of otd-related genes in vertebrates. Our study is the first demonstration that a cephalic gap gene is directly regulated by Bcd. However, it also shows that zygotic gene expression can be targeted to the head primordium without direct Bcd regulation.

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