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Results for “Enhancer Elements, Genetic”

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PubMed1998

Expression of the H2-Ea gene is modulated by a polymorphic transcriptional enhancer.

In all vertebrates the major histocompatibility complex (MHC) class II genes are polymorphic in their coding regions as well as in their promoter control elements. This polymorphism correlates with a variability in peptide binding and a variability in transcriptional activities. There is, however, one exception to this rule, which is the mouse H2-Ea gene or the corresponding human DRA gene. So far and for unkown reasons no polymorphism has been observed in these loci. We sequenced the distal transcriptional control elements of the H2-Ea, H2-Eb, and H2-Ab genes from the mouse haplotypes H2d, H2k, H2q, and H2z, and in contrast to the promoter and coding regions a sequence polymorphism can be detected which is limited to the H2-Ea gene. In transfection experiments this polymorphism can be seen to influence haplotype-specifically the transcriptional activities in B cells. This finding strongly suggests an evolutionary pressure towards a haplotype-specific expression pattern in all four MHC class II genes. The genetic differences in control elements of MHC class II genes may well contribute to differential immune reactivities and to immune disorders like allergies or autoimmune diseases.

Amino Acid Sequence↗

PubMed2005

A common sex-dependent mutation in a RET enhancer underlies Hirschsprung disease risk.

The identification of common variants that contribute to the genesis of human inherited disorders remains a significant challenge. Hirschsprung disease (HSCR) is a multifactorial, non-mendelian disorder in which rare high-penetrance coding sequence mutations in the receptor tyrosine kinase RET contribute to risk in combination with mutations at other genes. We have used family-based association studies to identify a disease interval, and integrated this with comparative and functional genomic analysis to prioritize conserved and functional elements within which mutations can be sought. We now show that a common non-coding RET variant within a conserved enhancer-like sequence in intron 1 is significantly associated with HSCR susceptibility and makes a 20-fold greater contribution to risk than rare alleles do. This mutation reduces in vitro enhancer activity markedly, has low penetrance, has different genetic effects in males and females, and explains several features of the complex inheritance pattern of HSCR. Thus, common low-penetrance variants, identified by association studies, can underlie both common and rare diseases.

Animals↗

PubMed2001

Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development.

Pax6 is a key transcription factor in eye development, particularly in lens development, but its molecular action has not been clarified. We demonstrate that Pax6 initiates lens development by forming a molecular complex with SOX2 on the lens-specific enhancer elements, e.g., the delta-crystallin minimal enhancer DC5. DC5 shows a limited similarity to the binding consensus sequence of Pax6 and is bound poorly by Pax6 alone. However, Pax6 binds cooperatively with SOX2 to the DC5 sequence, resulting in formation of a high-mobility form of ternary complex in vitro, which correlates with the enhancer activation in vivo. We observed Pax6 and SOX2-interdependent factor occupancy of DC5 in a chromatin environment in vivo, providing the molecular basis of synergistic activation by Pax6 and SOX2. Subtle alterations of the Pax6-binding-site sequence of DC5 or of the inter-binding-sites distance diminished the cooperative binding and caused formation of a non-functional low-mobility form complex, suggesting DNA sequence-guided and protein interaction-induced conformation change of the Pax6 protein. When ectopically expressed in embryo ectoderm, Pax6 and SOX2 in combination activate delta-crystallin gene and elicit lens placode development, indicating that the complex of Pax6 and SOX2 formed on specific DNA sequences is the genetic switch for initiation of lens differentiation.

Animals↗

PubMed2024

A mammalian tripartite enhancer cluster controls hypothalamic Pomc expression, food intake, and body weight.

Food intake and energy balance are tightly regulated by a group of hypothalamic arcuate neurons expressing the proopiomelanocortin (POMC) gene. In mammals, arcuate-specific POMC expression is driven by two cis-acting transcriptional enhancers known as nPE1 and nPE2. Because mutant mice lacking these two enhancers still showed hypothalamic Pomc mRNA, we searched for additional elements contributing to arcuate Pomc expression. By combining molecular evolution with reporter gene expression in transgenic zebrafish and mice, here, we identified a mammalian arcuate-specific Pomc enhancer that we named nPE3, carrying several binding sites also present in nPE1 and nPE2 for transcription factors known to activate neuronal Pomc expression, such as ISL1, NKX2.1, and ERα. We found that nPE3 originated in the lineage leading to placental mammals and remained under purifying selection in all mammalian orders, although it was lost in Simiiformes (monkeys, apes, and humans) following a unique segmental deletion event. Interestingly, ablation of nPE3 from the mouse genome led to a drastic reduction (>70%) in hypothalamic Pomc mRNA during development and only moderate (<33%) in adult mice. Comparison between double (nPE1 and nPE2) and triple (nPE1, nPE2, and nPE3) enhancer mutants revealed the relative contribution of nPE3 to hypothalamic Pomc expression and its importance in the control of food intake and adiposity in male and female mice. Altogether, these results demonstrate that nPE3 integrates a tripartite cluster of partially redundant enhancers that originated upon a triple convergent evolutionary process in mammals and that is critical for hypothalamic Pomc expression and body weight homeostasis.

Animals↗

PubMed2004

Characterization of a cardiac-specific enhancer, which directs {alpha}-cardiac actin gene transcription in the mouse adult heart.

Expression of the mouse alpha-cardiac actin gene in skeletal and cardiac muscle is regulated by enhancers lying 5' to the proximal promoter. Here we report the characterization of a cardiac-specific enhancer located within -2.354/-1.36 kbp of the gene, which is active in cardiocytes but not in C2 skeletal muscle cells. In vivo it directs reporter gene expression to the adult heart, where the proximal promoter alone is inactive. An 85-bp region within the enhancer is highly conserved between human and mouse and contains a central AT-rich site, which is essential for enhancer activity. This site binds myocyte enhancer factor (MEF)2 factors, principally MEF2D and MEF2A in cardiocyte nuclear extracts. These results are discussed in the context of MEF2 activity and of the regulation of the alpha-cardiac actin locus.

Actins↗

PubMed2004

Computational identification of developmental enhancers: conservation and function of transcription factor binding-site clusters in Drosophila melanogaster and Drosophila pseudoobscura.

BACKGROUND: The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. RESULTS: We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns. CONCLUSIONS: Measuring conservation of sequence features closely linked to function--such as binding-site clusterin--makes better use of comparative sequence data than commonly used methods that examine only sequence identity.

Animals↗

PubMed2003

Zic1 represses Math1 expression via interactions with the Math1 enhancer and modulation of Math1 autoregulation.

Math1 is a basic helix-loop-helix transcription factor expressed in progenitor cells that give rise to dorsal commissural interneurons in the spinal cord, granule cells of the cerebellum, and sensory cells in the inner ear and skin. Transcriptional regulation of this gene is tightly controlled both temporally and spatially during nervous system development. The signals that mediate this regulation are likely integrated at the Math1 enhancer, which is highly conserved among vertebrate species. We have identified the zinc-finger transcription factor Zic1 as a regulator of Math1 expression. Zic1 binds a novel conserved site within the Math1 enhancer, and represses both the expression of endogenous Cath1 (chicken homolog of Math1) and the activity of a Math1 enhancer driven lacZ reporter when expressed in chick neural tubes. Repression by Zic1 blocks the autoregulatory activity of Math1 itself. Although previous reports have shown that Zic1 and Math1 are both induced by BMP signaling, these genes appear to have opposing functions, as Math1 acts to promote neuronal differentiation in the chick neural tube and excess Zic1 appears to block differentiation. Zic1-mediated repression of Cath1 transcription may modulate the temporal switch between the progenitor state and differentiating dorsal cell types during neural tube development.

Animals↗

PubMed1999

Different contributions of pannier and wingless to the patterning of the dorsal mesothorax of Drosophila.

In Drosophila, the GATA family transcription factor Pannier and the Wnt secreted protein Wingless are known to be important for the patterning of the notum, a part of the dorsal mesothorax of the fly. Thus, both proteins are necessary for the development of the dorsocentral mechanosensory bristles, although their roles in this process have not been clarified. Here, we show that Pannier directly activates the proneural genes achaete and scute by binding to the enhancer responsible for the expression of these genes in the dorsocentral proneural cluster. Moreover, the boundary of the expression domain of Pannier appears to delimit the proneural cluster laterally, while antagonism of Pannier function by the Zn-finger protein U-shaped sets its limit dorsally. So, Pannier and U-shaped provide positional information for the patterning of the dorsocentral cluster. In contrast and contrary to previous suggestions, Wingless does not play a similar role, since the levels and vectorial orientation of its concentration gradient in the dorsocentral area can be greatly modified without affecting the position of the dorsocentral cluster. Thus, Wingless has only a permissive role on dorsocentral achaete-scute expression. We also provide evidence indicating that Pannier and U-shaped are main effectors of the regulation of wingless expression in the presumptive notum.

Animals↗

PubMed2000

Investigating the function of follicular subpopulations during Drosophila oogenesis through hormone-dependent enhancer-targeted cell ablation.

Although it is known that the establishment of polarity during Drosophila oogenesis is initiated by signalling from the oocyte to the overlying follicle cells, much less is understood about the role of specific follicular subpopulations. One powerful approach for addressing this question, toxigenic cell ablation of specific subpopulations, has not previously been applicable to studying follicular subpopulations because many of the genes and Gal4 enhancer trap insertions that are expressed in the ovary are also expressed at earlier times in development. To overcome this problem, we have utilized a fusion protein between Gal4 and the human estrogen receptor to achieve hormone-dependent, tissue-specific gene expression of UAS-linked transgenes in flies. We used this system to study the role of the polar subpopulations of follicle cells during oogenesis by expressing within them a modified form of diphtheria toxin that causes cell death. Our results confirmed previous functions ascribed to these cells, and also demonstrated a previously undescribed role for the border cells in facilitating the migration of the anterior Fasciclin III-expressing polar pair cells to the edge of the oocyte.

Animals↗

PubMed2005

A T lymphocyte-specific transcription complex containing RUNX1 activates MHC class I expression.

MHC class I expression is subject to both tissue-specific and hormonal regulatory mechanisms. Consequently, levels of expression vary widely among tissues, with the highest levels of class I occurring in the lymphoid compartment, in T cells and B cells. Although the high class I expression in B cells is known to involve the B cell enhanceosome, the molecular basis for high constitutive class I expression in T cells has not been explored. T cell-specific genes, such as TCR genes, are regulated by a T cell enhanceosome consisting of RUNX1, CBFbeta, LEF1, and Aly. In this report, we demonstrate that MHC class I gene expression is enhanced by the T cell enhanceosome and results from a direct interaction of the RUNX1-containing complex with the class I gene in vivo. T cell enhanceosome activation of class I transcription is synergistic with CIITA-mediated activation and targets response elements distinct from those targeted by CIITA. These findings provide a molecular basis for the high levels of MHC class I in T cells.

Animals↗

PubMed2001

Strain-dependency of chromosomal abnormalities in lymphomas developed in E mu-myc transgenic mice.

We previously showed that B and T cell lymphoma development in Em (immunoglobulin heavy chain enhancer)-myc transgenic mice is dependent on the mouse strain. To determine whether any non-random chromosomal abnormality that was present was caused by variations in the lymphoma cell type or by a different genetic background, we crossed C3H transgenic mice with other inbred strains of mice, C57BL / 6 or BALB / c. Cytogenetic analysis showed a high frequency of non-random chromosomal aberrations, namely, duplication or amplification of part of chromosome 5 containing the transgene and trisomy of chromosome 1, 6, or 12 in the genetic background of C3H x C57BL / 6 mouse and C3H x BALB / c mouse, respectively, regardless of cell type of lymphoma. These results suggest that non-random chromosomal abnormalities in lymphoma cells are dependent on the genetic background of mouse, not on the tumor cell type in Em-myc transgenic mouse.

Animals↗

PubMed1994

Genes for mast-cell serine protease and their molecular evolution.

Trypsin-related serine proteases are encoded by a very large gene family in mammals. We describe here a comparative analysis of the genomic DNA sequences of mouse, rat, and human mast-cell-specific serine protease genes. Strong evidence was found for multiple exchanges of genetic information between closely related members of this gene family. The 5' regulatory regions of MMCP-1 and MMCP-L share a remarkably high degree of sequence identity (98%), starting 10 base pairs downstream of exon 1 and extending to the end of the presently sequenced region at position -1347 of the MMCP-1 gene. The remaining parts of the two genes share approximately 80% sequence identity. Evidence for at least two additional, but not so recent, exchanges was found in the 3' regions of the MMCP-4 and MMCP-L genes and in the 5' regions of the genes for MMCP-1 and MMCP-2. The 5' regulatory regions of all presently characterized mouse mast-cell-specific chymotrypsin-like serine protease genes exhibit over 88% sequence identity in the region from the transcription initiation site to approximately position -600. An exception is MMCP-5 which is the most distantly related member of this subfamily. The high degree of sequence similarities indicates a strong evolutionary homogenization of the 5' regulatory region, possibly by several gene conversion events. In addition, several insertions of genetic information have been identified in genes for mast-cell chymases and genes for T-cell granzymes. A number of these have been found to represent repetitive sequences, such as L1. The previously characterized tissue-specific enhancer element of the RMCP II gene was identified as a member of a middle repetitive sequence. A cDNA for a newly discovered pseudogene, closely related to the mouse mast cell chymases was isolated by polymerase chain reaction amplification from a mouse connective tissue-like mast cell line. The structure of this cDNA is presented. We also present the characterization of a novel spliced variant of MMCP-6 that contains an alternative 3' terminal exon (exon 6). The function of this variant, if any, is still unknown. A comparative analysis of amino acid sequence identities between different hematopoietic serine proteases shows that a high degree of sequence similarity does not always correlate with relateness in cleavage specificity. This indicates that the substrate specificity evolved with a higher evolutionary rate than the degree of overall amino acid sequence identity of these proteases.

Alternative Splicing↗

PubMed1996

Isolation and analysis of the yeast TEA1 gene, which encodes a zinc cluster Ty enhancer-binding protein.

A genetic screen for mutants that affect the activity of internal regulatory sequences of Ty retrotransposons led to the identification of a new gene encoding a DNA-binding protein that interacts with the downstream enhancer-like region of Ty1 elements. The TEA1 (Ty enhancer activator) gene sequence predicts a protein of 86.9 kDa whose N terminus contains a zinc cluster and dimerization motif typical of the Gal4-type family of DNA-binding proteins. The C terminus encodes an acidic domain with a net negative charge of -10 and the ability to mediate transcriptional activation. Like other zinc cluster proteins, purified Tea1 was found to bind to a partially palindromic CGGNxCCG repeat motif located in the Ty1 enhancer region. The Ty1 Tea1 binding site has a spacing of 10 and is located near binding sites for the DNA-binding proteins Rap1 and Mcm1. Analysis of the phenotype of tea1 deletion mutants confirmed that the TEA1 gene is required for activation from the internal Ty1 enhancer characterized in this study and makes a modest contribution to normal Ty1 levels in the cell. Hence, Tea1, like Rap1, is a member of a small family of downstream activators in Saccharomyces cerevisiae. Further analysis of the Tea1 protein and its interactions may provide insight into the mechanism of downstream activation in yeast cells.

Amino Acid Sequence↗

PubMed1993

Detection of genes that are differentially expressed during mouse embryogenesis by genetic trapping strategies.

OBJECTIVE: Our objective was to identify novel genes that are expressed in temporally and spatially restricted patterns during mouse embryonic development and organogenesis. STUDY DESIGN: Two genetic trapping reporter constructs that lack transcriptional regulatory sequences were introduced independently into transcriptionally active gene loci by electroporation into mouse embryonic stem cells. Patterns of host gene-reporter construct expression were investigated in differentiated embryonic stem cells, embryoid bodies, and chimeric embryos at various stages of development. RESULTS: Three patterns of host gene-reporter construct expression were observed from the developmental analysis of four vector-integrated cell lines. Reporter expression patterns reflecting developmental regulation, constitutive activity, and developmental inactivation of the host genes were observed. CONCLUSIONS: Two vector-integrated gene loci from cell lines CCE-1C1 and D3-B44 have expression patterns not previously described by genetic trapping. Molecular characterization of these interrupted genes will shed light on their developmental function.

Animals↗

PubMed1997

Importance of a c-Myb binding site for lymphomagenesis by the retrovirus SL3-3.

All murine leukemia viruses (MuLVs) and related type C retroviruses contain a highly conserved binding site for the Ets family of transcription factors within the enhancer sequences in the viral long terminal repeats (LTRs). The T-cell lymphomagenic MuLV SL3-3 (SL3-3) also contains a c-Myb binding site adjacent to the Ets site. The presence of this Myb site distinguishes SL3 from most other MuLVs. We tested the importance of these two sites for the lymphomagenicity of SL3-3. Mutation of the Ets site had little effect on viral pathogenicity, as it only slightly extended the latency period to disease onset. In contrast, mutation of the Myb site strongly inhibited pathogenicity, as only a minority of the inoculated mice developed tumors in the two mouse strains that were tested. All tumors that were induced by either mutant appeared to be lymphomas, and no evidence for reversion of either mutation was detected. The effects of the Ets and Myb site mutations on transcriptional activity of the SL3 LTR were tested by inserting the viral enhancer sequences into a plasmid containing the promoter region of the c-myc gene linked to a reporter gene. Mutation the Myb site almost eliminated enhancer activity in T lymphocytes, while mutation of the Ets site had smaller effects. Thus, the effects of the enhancer mutations on transcriptional activity in T cells paralleled their effects on viral lymphomagenicity. The absence of the c-Myb site in the LTR enhancer of the weakly lymphomagenic MuLV, Akv, likely contributes to the low pathogenicity of this virus relative to SL3-3. However, Moloney MuLV also lacks the Myb site in its LTR, although it induces T-cell lymphomas with a potency similar to that of SL3-3. Thus, it appears that SL3-3 and Moloney MuLV evolved genetic determinants of T-cell lymphomagenicity that are, at least in part, distinct.

Animals↗

PubMed1995

Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression.

The Hairy/Enhancer of split/Deadpan family of basic helix-loop-helix (bHLH) proteins function as transcriptional repressors. We have examined the mechanisms of repression used by the Hairy and E(SPL) proteins by assaying the antagonism between wild-type or altered Hairy/E(SPL) and Scute bHLH proteins during sex determination in Drosophila melanogaster. Domain swapping and mutagenesis of the Hairy and E(SPL) proteins show that three evolutionarily conserved domains are required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. We show that the Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. In addition, we show that a Xenopus Hairy homology conserves not only Hairy's structure but also its biological activity in our assays. We propose that transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein.

Amino Acid Sequence↗

PubMed2006

NKR-P1 biology: from prototype to missing self.

Natural killer (NK) cells represent lymphocytes of the innate immune system capable of recognizing and destroying a broad array of target cells, including tumors, virus-infected cells, antibodycoated cells, foreign transplants, and "stressed" cells. NK cells eliminate their targets through two main effector mechanisms, cytokine secretion and cell-mediated cytotoxicity, which in turn depend on detection of target cells through a complex integration of stimulatory and inhibitory receptor-ligand interactions. The NKR-P1 molecules were the first family of NK cell receptors identified, yet they have remained enigmatic in their contribution to self-nonself discrimination until recently. Here, we outline a brief history of the NKR-P1 receptor family, then examine recent data providing insight into their genetic regulation, signaling function, cognate ligands, and gene organization and diversity.

Animals↗

PubMed1995

Identification of fat-cell enhancer activity in Drosophila melanogaster using P-element enhancer traps.

To identify genes important in fat-cell metabolism and development, we have screened Drosophila stocks carrying an engineered transposable element that can reveal the presence of nearby enhancer elements. We have identified those "enhancer-trap lines" that contain transposable P elements integrated near fat-cell specific enhancer elements. We anticipate that the genes associated with these enhancers will provide information concerning fat-cell function and serve as target genes for studying fat-cell specific gene expression. Furthermore, the identification of enhancer-trap lines active in the developing fat cell should provide an entry point into the molecular and genetic analysis of early fat-cell development. Analysis of two lines has revealed that the transcription factors svp, a steroid-hormone receptor, and Kr, a zinc-finger protein, are present in the fat body; these factors are likely to be involved in fat-cell gene expression. In two other lines, beta-galactosidase was detected in a subset of adepithelial cells that may be the precursors to the adult fat cell. And finally, in a single line transgene activity is present in the progenitor cells of the embryonic fat body. The genes associated with these enhancer-trap lines may be involved in fat-cell development.

Animals↗