Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice

AbstractGenes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements.SCN1A,which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations inSCN1Aare associated with epilepsy, including Dravet Syndrome (DS). The majority of DS patients harbor coding mutations causingSCN1Ahaploinsufficiency, however putative causal non-coding promoter mutations have been identified. To model the functional role of potentially redundantScn1apromoters, we focused on the non-codingScn1a1b regulatory region, previously described as a non-canonical alternative transcriptional start site. Mice harboring deletion of the extended evolutionarily-conserved 1b non-coding interval exhibited surprisingly severe reduction ofScn1aand NaV1.1 expression in brain with accompanying seizures and behavioral deficits. This identified the 1b region as a critical disease-relevant regulatory element and provides evidence that non-canonical and apparently redundant promoters can have essential function.

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Identification of Trophoblast-Specific Regulatory Elements in the Mouse Placental Lactogen II Gene

Molecular Endocrinology ◽

10.1210/mend.12.3.0078 ◽

1998 ◽

Vol 12 (3) ◽

pp. 418-427 ◽

Cited By ~ 7

Author(s):

Jiandie Lin ◽

Daniel I. H. Linzer

Keyword(s):

Giant Cell ◽

Regulatory Element ◽

Transcriptional Start Site ◽

Regulatory Region ◽

Giant Cells ◽

Regulatory Elements ◽

Positive Element ◽

Placental Lactogen ◽

Specific Gene ◽

Trophoblast Giant Cells

Abstract Placental lactogen II, the major ligand for the PRL receptor during the second half of gestation in rodents, is synthesized specifically by placental trophoblast giant cells. A transient transgenic analysis has been used to localize the giant cell-specific regulatory region within the mouse placental lactogen II gene to sequences between −1340 and −2019 upstream of the transcriptional start site. More precise mapping of the regulatory elements has been accomplished by transfection of promoter constructs into Rcho-1 trophoblast cells, resulting in the characterization of two positive regulatory elements in the −1471 to −1340 region; two other regulatory elements have been implicated but not further characterized, a negative regulatory element between −2019 and −1778 and another positive element within the region from −1340 to −569. Both of the characterized positive regulatory elements are recognized by factors that are enriched in differentiated giant cells compared with proliferative trophoblasts, and these factors are either absent or at low levels in fibroblasts. The complexes that form on the two elements are distinct and neither element competes with the other for factor binding, thus implicating at least two different regulatory elements in late-gestational trophoblast giant cell-specific gene expression.

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Organization of the murine and human interleukin-7 receptor genes: two mRNAs generated by differential splicing and presence of a type I-interferon-inducible promoter

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3052-3059.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3052-3059

Author(s):

C M Pleiman ◽

S D Gimpel ◽

L S Park ◽

H Harada ◽

T Taniguchi ◽

...

Keyword(s):

Cell Line ◽

Fusion Gene ◽

Regulatory Element ◽

Transcriptional Start Site ◽

Chloramphenicol Acetyltransferase ◽

Mouse Fibroblast ◽

Regulatory Sequence ◽

Type I ◽

Differential Splicing ◽

Interleukin 7

To better understand the regulation of interleukin-7 receptor (IL-7R) expression, we have pursued a detailed analysis of the structure of the murine and human IL-7R genes. The genes consist of eight exons, the sizes of which are conserved in mouse and human cells, spread out over 24 kbp (murine) and 19 kbp (human). A differential splicing event results in an mRNA encoding a secreted form of the human IL-7R gene. Primer extension and S1 nuclease analysis show a single transcriptional start site for the murine IL-7R gene. The 5'-flanking region of the murine IL-7R gene contains TATA- and CAAT-like sequences. The promoter region also contains a functional interferon regulatory element, to which the interferon-induced nuclear factors IRF-1 and IRF-2 are capable of binding and which is able to confer interferon-inducible expression on a heterologous gene. There are also potential binding sites for the transcription factors AP-1 and AP-2 as well as multiple glucocorticoid response elements. A fusion gene containing 2.5 kb of murine IL-7R 5' regulatory sequence linked to the bacterial chloramphenicol acetyltransferase gene directed expression of chloramphenicol acetyltransferase activity in murine pre-B-cell line 70Z/3 but not in the mouse fibroblast cell line NIH 3T3. Comparison of the murine and human IL-7R exon/intron boundaries with those of other hematopoietin receptor superfamily members whose exon/intron boundaries are also known reveals a conserved evolutionary structure.

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Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate

Development ◽

10.1242/dev.125.14.2735 ◽

1998 ◽

Vol 125 (14) ◽

pp. 2735-2746 ◽

Cited By ~ 3

Author(s):

D.H. Rowitch ◽

Y. Echelard ◽

P.S. Danielian ◽

K. Gellner ◽

S. Brenner ◽

...

Keyword(s):

Base Pair ◽

Regulatory Element ◽

Regulatory Region ◽

Neural Plate ◽

Homologous Region ◽

Regulatory Sequence ◽

Embryonic Mouse ◽

Cis Acting ◽

Evolutionarily Conserved

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(−/−) embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.

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A Pair of Highly Conserved Two-Component Systems Participates in the Regulation of the Hypervariable FIR Proteins in Different Legionella Species

Journal of Bacteriology ◽

10.1128/jb.01742-06 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3382-3391 ◽

Cited By ~ 10

Author(s):

Michal Feldman ◽

Gil Segal

Keyword(s):

Binding Site ◽

Legionella Pneumophila ◽

Response Regulator ◽

Regulatory Element ◽

Expression System ◽

Regulatory Region ◽

Regulatory Elements ◽

Mobility Shift ◽

Type Iv ◽

Essential Components

ABSTRACT Legionella pneumophila and other pathogenic Legionella species multiply inside protozoa and human macrophages by using the Icm/Dot type IV secretion system. The IcmQ protein, which possesses pore-forming activity, and IcmR, which functions as its chaperone, are two essential components of this system. It was previously shown that in 29 Legionella species, a large hypervariable-gene family (fir genes) is located upstream from a conserved icmQ gene, but although nonhomologous, the FIR proteins were found to function similarly together with their corresponding IcmQ proteins. Alignment of the regulatory regions of 29 fir genes revealed that they can be divided into three regulatory groups; the first group contains a binding site for the CpxR response regulator, which was previously shown to regulate the L. pneumophila fir gene (icmR); the second group, which includes most of the fir genes, contains the CpxR binding site and an additional regulatory element that was identified here as a PmrA binding site; and the third group contains only the PmrA binding site. Analysis of the regulatory region of two fir genes, which included substitutions in the CpxR and PmrA consensus sequences, a controlled expression system, as well as examination of direct binding with mobility shift assays, revealed that both CpxR and PmrA positively regulate the expression of the fir genes that contain both regulatory elements. The change in the regulation of the fir genes that occurred during the course of evolution might be required for the adaptation of the different Legionella species to their specific environmental hosts.

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The Divergently Transcribed Streptococcus parasanguis Virulence-Associated fimA Operon Encoding an Mn2+-Responsive Metal Transporter and pepO Encoding a Zinc Metallopeptidase Are Not Coordinately Regulated

Infection and Immunity ◽

10.1128/iai.70.10.5706-5714.2002 ◽

2002 ◽

Vol 70 (10) ◽

pp. 5706-5714 ◽

Cited By ~ 21

Author(s):

Joyce Oetjen ◽

Paula Fives-Taylor ◽

Eunice H. Froeliger

Keyword(s):

Transcriptional Start Site ◽

Regulatory Region ◽

Regulatory Elements ◽

Divalent Metal Ions ◽

Metal Transporters ◽

Metal Transporter ◽

Gram Positive Bacteria ◽

Growth Deficiency ◽

Domain Mapping ◽

Transcriptional Start Sites

ABSTRACT The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. The fimA operon is responsive to changes in Mn2+ concentration, but the pepO promoter is not. A S. parasanguis fimA mutant showed a growth deficiency under conditions of limiting Mn2+. This deficiency was not alleviated by compensation with either Mg2+ or Fe3+. Wild-type S. parasanguis could take up Mn2+ and Fe3+, while the fimA mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn2+ and Fe3+. FimA expression itself was shown to be responsive to Mn2+ concentration, but not to availability of Fe3+ or Mg2+.

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Repression and activation of the Drosophila dopa decarboxylase gene in glia.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5659 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5659-5666 ◽

Cited By ~ 6

Author(s):

G S Mastick ◽

S B Scholnick

Keyword(s):

Germ Line ◽

Transcriptional Start Site ◽

Regulatory Region ◽

Regulatory Elements ◽

Dopa Decarboxylase ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Cell Activator ◽

Germ Line Transformation

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

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Cloning and Functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.)

10.1101/2020.11.13.381327 ◽

2020 ◽

Author(s):

Guiying Tang ◽

Pingli Xu ◽

Pengxiang Li ◽

Jieqiong Zhu ◽

Guangxia Chen ◽

...

Keyword(s):

Transcriptional Start Site ◽

Functional Characterization ◽

Regulatory Region ◽

Regulatory Elements ◽

Histochemical Staining ◽

Expression Vectors ◽

Upstream Sequence ◽

Functional Region ◽

Chromosomal Walking ◽

Storage Reserve

AbstractLEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor, which controls several aspects of embryo and postembryo development, including embryo morphogenesis, storage reserve accumulation and skotomorphogenesis. Herein, using the method of chromosomal walking, a 2707bp upstream sequence from the ATG initiation codon site of AhLEC1A which is a homolog of Arabidopsis LEC1 was isolated in peanut. Its transcriptional start site confirmed by 5’ RACE was located at 82 nt from 5’ upstream of ATG. The bioinformatics analysis revealed that there existed many tissue-specific elements and light responsive motifs in its promoter. To identify the functional region of the AhLEC1A promoter, seven plant expression vectors expressing the GUS (β-glucuronidase) gene, driven by 5’ terminal series deleted fragments of AhLEC1A promoter, were constructed and transformed into Arabidopsis. Results of GUS histochemical staining showed that the regulatory region containing 82bp of 5’ UTR and 2228bp promoter could facilitate GUS to express preferentially in the embryos at different development periods of Arabidopsis. Taken together, it was inferred that the expression of AhLEC1A during seed development of peanut might be controlled positively by several seed-specific regulatory elements, as well as negatively by some other regulatory elements inhibiting its expression in other organs. Moreover, the GUS expression pattern of transgenic seedlings in darkness and in light was relevant to the light-responsive elements scattered in AhLEC1A promoter segment, implying that these light-responsive elements harbored in the AhLEC1A promoter regulate skotomorphogenesis of peanut seeds, and AhLEC1A expression was inhibited after the germinated seedlings were transferred from darkness to light.

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Repression and activation of the Drosophila dopa decarboxylase gene in glia

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5659-5666.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5659-5666

Author(s):

G S Mastick ◽

S B Scholnick

Keyword(s):

Germ Line ◽

Transcriptional Start Site ◽

Regulatory Region ◽

Regulatory Elements ◽

Dopa Decarboxylase ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Cell Activator ◽

Germ Line Transformation

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

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Organization of the murine and human interleukin-7 receptor genes: two mRNAs generated by differential splicing and presence of a type I-interferon-inducible promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3052 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3052-3059 ◽

Cited By ~ 53

Author(s):

C M Pleiman ◽

S D Gimpel ◽

L S Park ◽

H Harada ◽

T Taniguchi ◽

...

Keyword(s):

Cell Line ◽

Fusion Gene ◽

Regulatory Element ◽

Transcriptional Start Site ◽

Chloramphenicol Acetyltransferase ◽

Mouse Fibroblast ◽

Regulatory Sequence ◽

Type I ◽

Differential Splicing ◽

Interleukin 7

To better understand the regulation of interleukin-7 receptor (IL-7R) expression, we have pursued a detailed analysis of the structure of the murine and human IL-7R genes. The genes consist of eight exons, the sizes of which are conserved in mouse and human cells, spread out over 24 kbp (murine) and 19 kbp (human). A differential splicing event results in an mRNA encoding a secreted form of the human IL-7R gene. Primer extension and S1 nuclease analysis show a single transcriptional start site for the murine IL-7R gene. The 5'-flanking region of the murine IL-7R gene contains TATA- and CAAT-like sequences. The promoter region also contains a functional interferon regulatory element, to which the interferon-induced nuclear factors IRF-1 and IRF-2 are capable of binding and which is able to confer interferon-inducible expression on a heterologous gene. There are also potential binding sites for the transcription factors AP-1 and AP-2 as well as multiple glucocorticoid response elements. A fusion gene containing 2.5 kb of murine IL-7R 5' regulatory sequence linked to the bacterial chloramphenicol acetyltransferase gene directed expression of chloramphenicol acetyltransferase activity in murine pre-B-cell line 70Z/3 but not in the mouse fibroblast cell line NIH 3T3. Comparison of the murine and human IL-7R exon/intron boundaries with those of other hematopoietin receptor superfamily members whose exon/intron boundaries are also known reveals a conserved evolutionary structure.

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