scholarly journals The human FLT1 regulatory element directs vascular expression and modulates angiogenesis pathways in vitro and in vivo

2021 ◽  
Author(s):  
Julian Stolper ◽  
Holly K. Voges ◽  
Michael See ◽  
Neda Rahmani Mehdiabadi ◽  
Gulrez Chahal ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Regulatory Element ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Homeodomain Protein ◽  
Cardiovascular Development ◽  
Vascular Expression

AbstractThere is growing evidence that mutations in non-coding cis-regulatory elements (CREs) disrupt proper development. However, little is known about human CREs that are crucial for cardiovascular development. To address this, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined a highly conserved CRE within the FLT1 locus termed enFLT1. We show that the human enFLT1 is an enhancer capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka. Deletion of the human enFLT1 enhancer (ΔenFLT1) triggered molecular perturbations in extracellular matrix organisation and blood vessel morphogenesis in vitro in endothelial cells derived from human embryonic stem cells and vascular defects in vivo in medaka. These findings highlight the crucial role of the human FLT1 enhancer and its function as a regulator and buffer of transcriptional regulation in cardiovascular development.

scholarly journals The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain.

1995 ◽  
Vol 15 (12) ◽  
pp. 7091-7097 ◽  
Author(s):  
B Peers ◽  
S Sharma ◽  
T Johnson ◽  
M Kamps ◽  
M Montminy
Keyword(s):  
Target Genes ◽  
Gene Selection ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Cooperative Binding ◽  
Homeodomain Protein ◽  
Homeodomain Proteins ◽  
Target Sites

A number of homeodomain proteins have been shown to regulate cellular development by stimulating the transcription of specific target genes. In contrast to their distinct activities in vivo, however, most homeodomain proteins bind indiscriminately to potential target sites in vitro, suggesting the involvement of cofactors which specify target site selection. One such cofactor, termed extradenticle, has been shown to influence segmental morphogenesis in Drosophila melanogaster by binding cooperatively with certain homeodomain proteins to target regulatory elements. Here we demonstrate that STF-1, an orphan homeodomain protein required for pancreatic development in mammals, binds cooperatively to DNA with Pbx, the mammalian homolog of extradenticle. Cooperative binding with Pbx requires a pentapeptide motif (FPWMK) which is well conserved among a large subset of homeodomain proteins. The FPMWK motif is not sufficient to confer Pbx cooperativity on other homeodomain proteins, however; the N-terminal arm of the STF-1 homeodomain is also essential. As cooperative binding with Pbx occurs on only a subset of potential STF-1 target sites, our results suggest that Pbx may specify target gene selection in the developing pancreas by forming heterodimeric complexes with STF-1.

scholarly journals Genome-wide analysis of cis-regulatory changes in the metabolic adaptation of cavefish

2020 ◽  
Author(s):  
Jaya Krishnan ◽  
Chris W. Seidel ◽  
Ning Zhang ◽  
Jake VanCampen ◽  
Robert Peuß ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Metabolic Adaptation ◽  
Enhancer Activity ◽  
Genome Wide ◽  
Cave Environment

AbstractChanges in cis-regulatory elements play important roles in adaptation and phenotypic evolution. However, their contribution to metabolic adaptation of organisms is less understood. Here we have utilized a unique vertebrate model, Astyanax mexicanus, different morphotypes of which survive in nutrient-rich surface and nutrient-deprived cave water to uncover gene regulatory networks in metabolic adaptation. We performed genome-wide epigenetic profiling in the liver tissue of one surface and two independently derived cave populations. We find that many cis-regulatory elements differ in their epigenetic status/chromatin accessibility between surface fish and cavefish, while the two independently derived cave populations have evolved remarkably similar regulatory signatures. These differentially accessible regions are associated with genes of key pathways related to lipid metabolism, circadian rhythm and immune system that are known to be altered in cavefish. Using in vitro and in vivo functional testing of the candidate cis-regulatory elements, we find that genetic changes within them cause quantitative expression differences. We characterized one cis-regulatory element in the hpdb gene and found a genomic deletion in cavefish that abolishes binding of the transcriptional repressor IRF2 in vitro and derepresses enhancer activity in reporter assays. Genetic experiments further validated a cis-mediated role of the enhancer and suggest a role of this deletion in the upregulation of hpdb in wild cavefish populations. Selection of this mutation in multiple independent cave populations supports its importance in the adaptation to the cave environment, providing novel molecular insights into the evolutionary trade-off between loss of pigmentation and adaptation to a food-deprived cave environment.

A Single Pitx1 Binding Site Is Essential for Activity of the LHβ Promoter in Transgenic Mice

2001 ◽  
Vol 15 (5) ◽  
pp. 734-746 ◽  
Author(s):  
Christine C. Quirk ◽  
Kristen L. Lozada ◽  
Ruth A. Keri ◽  
John H. Nilson
Keyword(s):  
Transgenic Mice ◽  
Binding Site ◽  
Cell Lines ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Vital Role ◽  
Proximal Region ◽  
Expression Vectors ◽  
Homeodomain Protein

Abstract Reproduction depends on regulated expression of the LHβ gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHβ promoter and make essential contributions to its activity as well as mediate responsiveness to GnRH. Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHβ gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LβT2), an LHβ promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GnRH. In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHβ promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHβ promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.

scholarly journals The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo

2021 ◽  
Author(s):  
Giuliano Crispatzu ◽  
Rizwan Rehimi ◽  
Tomas Pachano ◽  
Tore Bleckwehl ◽  
Sara de la Cruz Molina ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Regulatory Elements ◽  
Small Subset ◽  
Regulatory Sequences ◽  
Pluripotent Cells ◽  
Developmental Genes ◽  
Topological Features ◽  
Regulatory Properties

AbstractPoised enhancers (PEs) represent a limited and genetically distinct set of distal regulatory elements that control the induction of developmental genes in a hierarchical and non-redundant manner. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally identified and subsequently characterized using embryonic stem cells (ESC) as an in vitro differentiation system, it is currently unknown whether PEs are functionally conserved in vivo. Here, we generate and mine various types of genomic data to show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that, in mouse pluripotent cells, the interactions between PEs and their bivalent target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. proximity to CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation, preferentially within specific vertebrate clades, with only a small subset being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting evolutionary conserved PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential and non-redundant roles during the induction of major developmental genes in vivo.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4349-4358 ◽  
Author(s):  
J. Charite ◽  
W. de Graaff ◽  
D. Consten ◽  
M.J. Reijnen ◽  
J. Korving ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hox Genes ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Positional Information ◽  
Regulatory Elements ◽  
Gene Products ◽  
Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

scholarly journals Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

1989 ◽  
Vol 9 (12) ◽  
pp. 5315-5323 ◽  
Author(s):  
J Imbert ◽  
M Zafarullah ◽  
V C Culotta ◽  
L Gedamu ◽  
D Hamer
Keyword(s):  
Gene Transcription ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Direct Role ◽  
Transcription In Vitro ◽  
Mouse Cells ◽  
Affinity Reagent

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

Tunable PEGylation of branch-type PEI/DNA polyplexes with a compromise of low cytotoxicity and high transgene expression: in vitro and in vivo gene delivery

2017 ◽  
Vol 5 (24) ◽  
pp. 4732-4744 ◽  
Author(s):  
A. Venault ◽  
Y.-C. Huang ◽  
J. W. Lo ◽  
C.-J. Chou ◽  
A. Chinnathambi ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Transgene Expression ◽  
In Vivo Gene Delivery ◽  
Low Cytotoxicity ◽  
Branch Type ◽  
Delivery Efficiency

Although PEGylated polyplexes for gene delivery are widespread, there is a need for an in-depth investigation of the role of the PEGylation degree on the delivery efficiency of the systems.

Embryo biotechnology in the dog: a review

2010 ◽  
Vol 22 (7) ◽  
pp. 1049 ◽  
Author(s):  
Sylvie Chastant-Maillard ◽  
Martine Chebrout ◽  
Sandra Thoumire ◽  
Marie Saint-Dizier ◽  
Marc Chodkiewicz ◽  
...  
Keyword(s):  
Mammalian Species ◽  
Embryonic Stem ◽  
Reproductive Technologies ◽  
Reproductive Physiology ◽  
Biological Models ◽  
The Past ◽  
Fertilisation Rate

Canine embryos are a scarce biological material because of difficulties in collecting in vivo-produced embryos and the inability, to date, to produce canine embryos in vitro. The procedure for the transfer of in vivo-produced embryos has not been developed adequately, with only six attempts reported in the literature that have resulted in the birth of 45 puppies. In vitro, the fertilisation rate is particularly low (∼10%) and the incidence of polyspermy particularly high. So far, no puppy has been obtained from an in vitro-produced embryo. In contrast, cloning of somatic cells has been used successfully over the past 4 years, with the birth of 41 puppies reported in the literature, a yield that is comparable to that for other mammalian species. Over the same period, canine embryonic stem sells and transgenic cloned dogs have been obtained. Thus, the latest reproductive technologies are further advanced than in vitro embryo production. The lack of fundamental studies on the specific features of reproductive physiology and developmental biology in the canine is regrettable in view of the increasing role of dogs in our society and of the current demand for new biological models in biomedical technology.

scholarly journals Identification of regulatory elements required for Stra8 expression, in fetal ovarian germ cells of the mouse

Development ◽  
2021 ◽  
pp. dev.194977
Author(s):  
Chun-Wei Feng ◽  
Guillaume Burnet ◽  
Cassy M. Spiller ◽  
Fiona Ka Man Cheung ◽  
Kallayanee Chawengsaksophak ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Germ Cells ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Egfp Expression ◽  
Spatiotemporal Regulation ◽  
Targeted Mutation ◽  
Fetal Ovary

In mice, the entry of germ cells into meiosis critically depends on the expression of stimulated by retinoic acid gene 8 (Stra8). Stra8 is expressed specifically in pre-meiotic germ cells of females and males, at fetal and postnatal stages respectively, but the mechanistic details of its spatiotemporal regulation are yet to be defined. In particular, there has been considerable debate regarding whether retinoic acid is required, in vivo, to initiate Stra8 expression in the mouse fetal ovary. We show that the distinctive anterior-to-posterior pattern of Stra8 initiation, characteristic of germ cells in the fetal ovary, is faithfully recapitulated when 2.9 kb of the Stra8 promoter is used to drive eGFP expression. Using in vitro transfection assays of cut-down and mutant constructs we identified two functional retinoic acid responsive elements (RAREs) within this 2.9 kb regulatory element. We also show that the transcription factor DMRT1 enhances Stra8 expression, but only in the presence of RA and the most proximal RARE. Finally, we used CRISPR/Cas9-mediated targeted mutation studies to demonstrate that both RAREs are required for optimal Stra8 expression levels, in vivo.

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