Target sequences for hunchback in a control region conferring Ultrabithorax expression boundaries

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1171-1179 ◽  
Author(s):  
C.C. Zhang ◽  
J. Muller ◽  
M. Hoch ◽  
H. Jackle ◽  
M. Bienz
Keyword(s):  
Long Range ◽  
Control Region ◽  
Binding Sites ◽  
Regulatory Sequences ◽  
Stripe Pattern ◽  
Anteroposterior Axis ◽  
Target Sequences ◽  
Beta Galactosidase ◽  
Control Regions

Boundaries of Ultrabithorax expression are mediated by long-range repression acting through the PBX or ABX control region. We show here that either of these control regions confers an early band of beta-galactosidase expression which is restricted along the anteroposterior axis of the blastoderm embryo. This band is succeeded by a stripe pattern with very similar anteroposterior limits. Dissection of the PBX control region demonstrates that the two patterns are conferred by distinct cis-regulatory sequences contained within separate PBX subfragments. We find several binding sites for hunchback protein within both PBX subfragments. Zygotic hunchback function is required to prevent ectopic PBX expression. Moreover, the PBX pattern is completely suppressed in embryos containing uniformly distributed maternal hunchback protein. Our results strongly suggest that hunchback protein directly binds to the PBX control region and acts as a repressor to specify the boundary positions of the PBX pattern.

scholarly journals Analysis of the Promoter of the ninaE Opsin Gene in Drosophila melanogaster

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 565-578 ◽  
Author(s):  
Drzislav Mismer ◽  
Gerald M Rubin
Keyword(s):  
Control Region ◽  
Photoreceptor Cells ◽  
P Element ◽  
Opsin Gene ◽  
Regulatory Sequences ◽  
Enhancer Element ◽  
Cis Acting ◽  
E Coli ◽  
Promoter Function ◽  
Control Regions

ABSTRACT We have analyzed the cis-acting regulatory sequences of the ninaE gene. This gene encodes the major Drosophila melanogasteropsin, the protein component of the primary chromophore of photoreceptor cells R1-R6 of the adult eye. DNA fragments containing the start point of transcription of the ninaE gene were fused to either the Escherichia coli chloramphenicol acetyltransferase or lacZ (β-galactosidase) gene and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the ninaE gene to confer the ninaE pattern of expression. Fragments containing between 2.8 kb and 215 bp of the sequences upstream of the start of transcription plus the first 67 bp of the untranslated leader were able to direct nearly wild-type expression. We have identified three separable control regions in the ninaE promoter. The first, which has the properties of an enhancer element, is located between nucleotides -501 and -219. The removal of this sequence had little effect on promoter function; this sequence appears to be redundant. However, it appears to be able to substitute for the second control region which is located between nucleotides -215 and -162, and which also affects the level of output from this promoter. Removal of these two control regions resulted in a 30-fold decrease in expression; however tissue specificity was not affected. The third control region, located downstream from nucleotide -120, appears to be absolutely necessary for promoter function in the absence of the first two regulatory sequences. Examination of larvae containing fusion genes expressing β-galactosidase suggests that the ninaE gene is also expressed in a subset of cells in the larval photoreceptor organ.

Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-dependent and -independent mechanisms

Development ◽  
1999 ◽  
Vol 126 (2) ◽  
pp. 281-292 ◽  
Author(s):  
D.J. Epstein ◽  
A.P. McMahon ◽  
A.L. Joyner
Keyword(s):  
Gene Expression ◽  
Sonic Hedgehog ◽  
Binding Sites ◽  
Transcriptional Regulators ◽  
Regulatory Sequences ◽  
Reporter Expression ◽  
Enhancer Activity ◽  
Ventral Midline ◽  
Genomic Clones ◽  
Anteroposterior Axis

The axial midline mesoderm and the ventral midline of the neural tube, the floor plate, share the property of being a source of the secreted protein, Sonic hedgehog (Shh), which has the capacity to induce a variety of ventral cell types along the length of the mouse CNS. To gain insight into the mechanisms by which Shh transcription is initiated in these tissues, we set out to identify the cis-acting sequences regulating Shh gene expression. As an approach, we have tested genomic clones encompassing 35 kb of the Shh locus for their ability to direct a lacZ reporter gene to the temporally and spatially restricted confines of the Shh expression domains in transgenic mice. Three enhancers were identified that directed lacZ expression to distinct regions along the anteroposterior axis including the ventral midline of the spinal cord, hindbrain, rostral midbrain and caudal diencephalon, suggesting that multiple transcriptional regulators are required to initiate Shh gene expression within the CNS. In addition, regulatory sequences were also identified that directed reporter expression to the notochord, albeit, under limited circumstances. Sequence analysis of the genomic clones responsible for enhancer activity from a variety of organisms, including mouse, chicken and human, have identified highly conserved binding sites for the hepatocyte nuclear factor 3 (Hnf3) family of transcriptional regulators in some, but not all, of the enhancers. Moreover, the generation of mutations in the Hnf3-binding sites showed their requirement in certain, but not all, aspects of Shh reporter expression. Taken together, our results support the existence of Hnf3-dependent and -independent mechanisms in the direct activation of Shh transcription within the CNS and axial mesoderm.

scholarly journals Locus Control Region Transcription Plays an Active Role in Long-Range Gene Activation

2006 ◽  
Vol 23 (4) ◽  
pp. 619
Author(s):  
Yugong Ho ◽  
Felice Elefant ◽  
Stephen A. Liebhaber ◽  
Nancy E. Cooke
Keyword(s):  
Long Range ◽  
Control Region ◽  
Gene Activation ◽  
Locus Control Region ◽  
Active Role

scholarly journals Requirement of an E1A-sensitive Coactivator for Long-range Transactivation by the β-Globin Locus Control Region

1999 ◽  
Vol 274 (38) ◽  
pp. 26850-26859 ◽  
Author(s):  
E. Camilla Forsberg ◽  
Kirby Johnson ◽  
Tatiana N. Zaboikina ◽  
Eric A. Mosser ◽  
Emery H. Bresnick
Keyword(s):  
Long Range ◽  
Control Region ◽  
Locus Control Region

scholarly journals Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

2005 ◽  
Vol 187 (20) ◽  
pp. 6928-6935 ◽  
Author(s):  
Valley Stewart ◽  
Peggy J. Bledsoe
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Haemophilus Influenzae ◽  
Control Region ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Class I ◽  
E Coli ◽  
Fnr Protein

ABSTRACT Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions −64.5 and −44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position −60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions −81.5 and −42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.

Positioning adjacent pair-rule stripes in the posterior Drosophila embryo

Development ◽  
1994 ◽  
Vol 120 (10) ◽  
pp. 2945-2955 ◽  
Author(s):  
J.A. Langeland ◽  
S.F. Attai ◽  
K. Vorwerk ◽  
S.B. Carroll
Keyword(s):  
Binding Sites ◽  
Drosophila Embryo ◽  
Regulatory Sequences ◽  
Posterior Border ◽  
Adjacent Pair ◽  
Gap Gene ◽  
Competitive Mechanism ◽  
Pair Rule ◽  
Drosophila Embryos ◽  
Do So

We present a genetic and molecular analysis of two hairy (h) pair-rule stripes in order to determine how gradients of gap proteins position adjacent stripes of gene expression in the posterior of Drosophila embryos. We have delimited regulatory sequences critical for the expression of h stripes 5 and 6 to 302 bp and 526 bp fragments, respectively, and assayed the expression of stripe-specific reporter constructs in several gap mutant backgrounds. We demonstrate that posterior stripe boundaries are established by gap protein repressors unique to each stripe: h stripe 5 is repressed by the giant (gt) protein on its posterior border and h stripe 6 is repressed by the hunchback (hb) protein on its posterior border. Interestingly, Kruppel (Kr) limits the anterior expression limits of both stripes and is the only gap gene to do so, indicating that stripes 5 and 6 may be coordinately positioned by the Kr repressor. In contrast to these very similar cases of spatial repression, stripes 5 and 6 appear to be activated by different mechanisms. Stripe 6 is critically dependent upon knirps (kni) for activation, while stripe 5 likely requires a combination of activating proteins (gap and non-gap). To begin a mechanistic understanding of stripe formation, we locate binding sites for the Kr protein in both stripe enhancers. The stripe 6 enhancer contains higher affinity Kr-binding sites than the stripe 5 enhancer, which may allow for the two stripes to be repressed at different Kr protein concentration thresholds. We also demonstrate that the kni activator binds to the stripe 6 enhancer and present evidence for a competitive mechanism of Kr repression of stripe 6.

Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of (alpha)5(beta)1 integrin expression

2000 ◽  
Vol 113 (18) ◽  
pp. 3173-3185 ◽  
Author(s):  
M.K. Duncan ◽  
Z. Kozmik ◽  
K. Cveklova ◽  
J. Piatigorsky ◽  
A. Cvekl
Keyword(s):  
Transgenic Mice ◽  
Binding Sites ◽  
Fiber Cell ◽  
Regulatory Sequences ◽  
Pax6 Gene ◽  
Fiber Cells ◽  
Nuclear Extracts ◽  
Shift Analysis ◽  
Beta 1 Integrin ◽  
Gel Shift

The PAX6 gene, a key regulator of eye development, produces two major proteins that differ in paired domain structure: PAX6 and PAX6(5a). It is known that an increase in the PAX6(5a) to PAX6 ratio leads to multiple ocular defects in humans. Here, transgenic mice were created that overexpress human PAX6(5a) in the lens. These mice develop cataracts with abnormalities in fiber cell shape as well as fiber cell/lens capsule and fiber cell/fiber cell interactions. While the structure of the actin cytoskeleton appeared relatively normal, the cataractous lens expresses increased amounts of paxillin and p120(ctn) as well as large aggregates of (alpha)5(beta)1 integrin in the dysgenic fiber cells. The elevated amounts of these proteins in the transgenic lens correlated well with elevated levels of their respective mRNAs. To investigate the role of Pax-6(5a) in the upregulation of these genes, a series of gel shift experiments using truncated proteins and consensus oligonucleotides demonstrated the complexity of Pax-6 and Pax-6(5a) binding to DNA, aiding our identification of potential binding sites in the human (α)5- and (beta)1-integrin promoters. Consequent gel shift analysis demonstrated that these putative regulatory sequences bind Pax-6 and/or Pax-6(5a) in lens nuclear extracts, suggesting that the human (alpha)5 and (beta)1 integrin promoters contain PAX6/PAX6(5a) binding sites and maybe directly regulated by this transcription factor in the transgenic lens. We conclude that these transgenic mice are good models to study a type of human cataract and for identifying batteries of genes that are directly or indirectly regulated by both forms of Pax-6.

Position independence and proper developmental control of gamma-globin gene expression require both a 5' locus control region and a downstream sequence element

1994 ◽  
Vol 14 (9) ◽  
pp. 6087-6096
Author(s):  
Q Li ◽  
J A Stamatoyannopoulos
Keyword(s):  
Transgenic Mice ◽  
Control Region ◽  
Locus Control Region ◽  
Regulatory Sequences ◽  
Globin Genes ◽  
Sequence Element ◽  
Position Effects ◽  
Developmental Control ◽  
Developmental Patterns ◽  
Gamma Globin

We have analyzed the expression of human gamma-globin genes during development in F2 progeny of transgenic mice carrying two types of constructs. In the first type, gamma-globin genes were linked individually to large (approximately 4-kb) sequence fragments spanning locus control region (LCR) hypersensitive site 2 (HS2) or HS3. These LCR fragments contained not only the core HS elements but also extensive evolutionarily conserved flanking sequences. The second type of construct contained tandem gamma- and beta-globin genes linked to identical HS2 or HS3 fragments. We show that gamma-globin expression in transgenic mice carrying HS2 gamma or HS3 gamma constructs is highly sensitive to position effects and that such effects override the cis regulatory elements present in these constructs to produce markedly different developmental patterns of gamma-globin expression in lines carrying the same transgene. In contrast, gamma-globin expression in both HS2 gamma beta and HS3 gamma beta mice is sheltered from position effects and the developmental patterns of gamma-globin expression in lines carrying the same transgene are identical and display stage-specific regulation. The results suggest that cis regulatory sequences required for proper developmental control of fetal globin expression in the presence of an LCR element reside downstream from the gamma genes.

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