scholarly journals Two distinct motifs for Zic-r.a drive specific gene expression in two cell lineages

Development ◽  
2021 ◽  
Author(s):  
Izumi Oda-Ishii ◽  
Deli Yu ◽  
Yutaka Satou
Keyword(s):  
Binding Sites ◽  
In Vitro Selection ◽  
Regulatory Region ◽  
Specific Gene ◽  
Cell Lineages ◽  
Neural Genes ◽  
Canonical Motif ◽  
Ascidian Embryos ◽  
Direct Target

Zic-r.a, a maternal transcription factor, specifies posterior fate in ascidian embryos. However, its direct target, Tbx6-r.b, does not contain typical Zic-r.a binding sites in its regulatory region. Using an in vitro selection assay, we found that Zic-r.a binds to sites dissimilar from the canonical motif, by which it activates Tbx6-r.b in a sub-lineage of muscle cells. These sites with non-canonical motifs have weak affinity for Zic-r.a; therefore, it activates Tbx6-r.b only in cells expressing Zic-r.a abundantly. Meanwhile, we found that Zic-r.a expressed zygotically in late embryos activates neural genes through canonical sites. Because different zinc-finger domains of Zic-r.a are important for driving reporters with canonical and non-canonical sites, it is likely that the non-canonical motif is not a divergent version of the canonical motif. In other words, our data indicate that the non-canonical motif represents a motif distinct from the canonical motif. Thus, Zic-r.a recognizes two distinct motifs to activate two sets of genes at two timepoints in development.

Regulation of touch receptor differentiation by the Caenorhabditis elegans mec-3 and unc-86 genes

Development ◽  
1998 ◽  
Vol 125 (20) ◽  
pp. 4107-4119 ◽  
Author(s):  
A. Duggan ◽  
C. Ma ◽  
M. Chalfie
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Fate ◽  
Binding Sites ◽  
Ectopic Expression ◽  
The Body ◽  
Specific Gene ◽  
Homeodomain Protein ◽  
Activation Domains ◽  
Cell Lineages

The nematode Caenorhabditis elegans possesses six morphologically similar neurons that are responsible for sensing gentle touch to the body. Previous genetic studies identified genes that are necessary for the production and differentiation of these touch cells. In particular, unc-86 encodes a POU-type homeodomain protein needed for the production of the touch cells, while mec-3 encodes a LIM-type homeodomain protein needed for the differentiation of the touch cells. Molecular studies showed that MEC-3 and UNC-86 bind cooperatively to sites in the mec-3 promoter and can synergistically activate transcription from it in vitro. Here we show that UNC-86::MEC-3 hetero-oligomer-binding sites are also found in the promoters of two presumed targets of mec-3, the mec-4 and mec-7 genes, that are necessary for the function of the touch cells. These sites, which are well-conserved in the related nematode C. briggsae, are required for promoter activity. When one of the binding sites is cloned into a heterologous promoter, expression is found in the touch cells and two to four other cells that express mec-3 and unc-86. These data support a model in which touch-cell differentiation is specified, in part, by the UNC-86::MEC-3 hetero-oligomer and not by MEC-3 alone. Ectopic expression of mec-3, driven by a heat-shock promoter, also supports this hypothesis: the acquisition of touch-cell characteristics by several additional cells under these conditions required unc-86. Since the touch-cell lineages express UNC-86 before MEC-3, MEC-3 appears to modify the activity of UNC-86, leading to touch-cell-specific gene expression. Because both UNC-86 and MEC-3 have activation domains, the formation of the hetero-oligomer may create a strong activator. In the modification of UNC-86 function by MEC-3 in the touch cells, these studies provide an example of how the sequential activation of transcription factors can determine cell fate within particular cell lineages.

Sequence Analysis of Fis Binding Sites Obtained by in Vitro Selection

1997 ◽  
Vol 16 (5-6) ◽  
pp. 579-584
Author(s):  
Karsten Theis ◽  
Cornelia Bartsch ◽  
Wolfram Saenger
Keyword(s):  
Sequence Analysis ◽  
Binding Sites ◽  
In Vitro Selection

In vitro selection of high affinity HspR-binding sites within the genome of Helicobacter pylori

Gene ◽  
2002 ◽  
Vol 283 (1-2) ◽  
pp. 63-69 ◽  
Author(s):  
Isabel Delany ◽  
Gunther Spohn ◽  
Rino Rappuoli ◽  
Vincenzo Scarlato
Keyword(s):  
Helicobacter Pylori ◽  
Binding Sites ◽  
In Vitro Selection ◽  
High Affinity ◽  
Selection Of

scholarly journals In Vitro Selection of Integration Host Factor Binding Sites

1999 ◽  
Vol 181 (10) ◽  
pp. 3246-3255 ◽  
Author(s):  
Steven D. Goodman ◽  
Nerissa J. Velten ◽  
Qian Gao ◽  
Scott Robinson ◽  
Anca M. Segall
Keyword(s):  
Binding Sites ◽  
In Vitro Selection ◽  
Structural Characteristics ◽  
Integration Host Factor ◽  
Host Factor ◽  
Binding Domain ◽  
Physical Constraints ◽  
Individual Sequences ◽  
And Function

ABSTRACT Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3′ domain is conserved, the 5′ domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5′ domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5′ binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5′ binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.

beta3-tubulin is directly repressed by the engrailed protein in Drosophila

Development ◽  
1997 ◽  
Vol 124 (13) ◽  
pp. 2527-2536 ◽  
Author(s):  
N. Serrano ◽  
H.W. Brock ◽  
F. Maschat
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Transgenic Line ◽  
Target Genes ◽  
Regulatory Protein ◽  
Polytene Chromosomes ◽  
First Intron ◽  
Direct Target

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles during embryonic development. Although Engrailed is a transcription factor, little progress has been achieved in identifying its target genes. We report here the identification of an effector gene, the beta3-tubulin gene, as a direct target of Engrailed. The cytological location of beta3-tubulin, 60C, is a strong site of Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta3-tubulin-lacZ] construct shows an additional site of Engrailed binding at the location of the transgene. Molecular analysis allowed identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta3-tubulin locus. Engrailed binding sites identified in vitro are active in larvae. Furthermore, expression of beta3-tubulin is derepressed in the ectoderm of engrailed mutant embryos. Repression of beta3-tubulin by Engrailed is also obtained when Engrailed is ectopically expressed in embryonic mesoderm. Finally, two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta3-tubulin by Engrailed during embryogenesis.

The Xenopus homeobox gene twin mediates Wnt induction of goosecoid in establishment of Spemann's organizer

Development ◽  
1997 ◽  
Vol 124 (23) ◽  
pp. 4905-4916 ◽  
Author(s):  
M.N. Laurent ◽  
I.L. Blitz ◽  
C. Hashimoto ◽  
U. Rothbacher ◽  
K.W. Cho
Keyword(s):  
Reporter Gene ◽  
Binding Sites ◽  
Homeobox Gene ◽  
Dorsal Side ◽  
Expression Cloning ◽  
Specific Gene ◽  
Dependent Manner ◽  
Spemann's Organizer ◽  
Dorsal Axis

We describe the isolation of the Xenopus homeobox gene twin (Xtwn), which was identified in an expression cloning screen for molecules with dorsalizing activities. Injection of synthetic Xtwn mRNA restores a complete dorsal axis in embryos lacking dorsal structures and induces a complete secondary dorsal axis when ectopically expressed in normal embryos. The sequence homology, expression pattern and gain-of-function phenotype of Xtwn is most similar to the previously isolated Xenopus homeobox gene siamois (Xsia) suggesting that Xtwn and Xsia comprise a new subclass of homeobox genes important in dorsal axis specification. We find that Xtwn is able to activate the Spemann organizer-specific gene goosecoid (gsc) via direct binding to a region of the gsc promoter previously shown to mediate Wnt induction. Since Xtwn expression is strongly induced in ectodermal (animal cap) cells in response to overexpression of a dorsalizing Wnt molecule, we examined the possibility that Xtwn might be a direct target of a Wnt signal transduction cascade. First, we demonstrate that purified LEF1 protein can interact, in vitro, with consensus LEF1/TCF3-binding sites found within the Xtwn promoter. Second, these binding sites were shown to be required for Wnt-mediated induction of a Xtwn reporter gene containing these sites. As LEF1/TCF3 family transcription factors have previously been shown to directly mediate Wnt signaling, these results suggest that Xtwn induction by Wnt may be direct. Finally, in UV-hyperventralized embryos, expression of endogenous Xtwn is confined to the vegetal pole and a Xtwn reporter gene is hyperinduced vegetally in a LEF1/TCF3-binding-site-dependent manner. These results suggest that cortical rotation distributes Wnt-like dorsal determinants to the dorsal side of the embryo, including the dorsal marginal zone, and that these determinants may directly establish Spemann's organizer in this region.

scholarly journals Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

2002 ◽  
Vol 68 (3) ◽  
pp. 1102-1108 ◽  
Author(s):  
Sau-Ching Wu ◽  
Sui-Lam Wong
Keyword(s):  
Bacillus Subtilis ◽  
Binding Sites ◽  
Culture Medium ◽  
Regulatory Region ◽  
Subtilis Strain ◽  
Biotin Deficiency ◽  
Poor Growth ◽  
Biotin Binding

ABSTRACT Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

scholarly journals Two distinct classes of FixJ binding sites defined by in vitro selection

FEBS Letters ◽  
2002 ◽  
Vol 517 (1-3) ◽  
pp. 185-189 ◽  
Author(s):  
Lionel Ferrières ◽  
Daniel Kahn
Keyword(s):  
Binding Sites ◽  
In Vitro Selection

The cathepsin L gene is a direct target of FOXO1 in skeletal muscle

2010 ◽  
Vol 427 (1) ◽  
pp. 171-178 ◽  
Author(s):  
Yoshihiro Yamazaki ◽  
Yasutomi Kamei ◽  
Satoshi Sugita ◽  
Fumiko Akaike ◽  
Sayaka Kanai ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Cathepsin L ◽  
Skeletal Muscle Atrophy ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Direct Target

FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominant-negative form of FOXO1 or in skeletal-muscle-specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy.

Sign in / Sign up

Export Citation Format

Share Document