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Results for “Enhancer Elements, Genetic”

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PubMed2025

BRD2 bridges TFIID and MOF-H4K16ac-containing nucleosomes to promote transcriptional initiation.

Members of the bromodomain and extraterminal domain (BET) protein family play a central role in transcription by RNA polymerase II (RNA Pol II). Small-molecule inhibitors that block interaction between BET bromodomains and acetylated histones have been developed for disease therapeutics. However, the BET protein BRD4 does not require bromodomains to perform its major transcriptional elongation control, and mechanisms by which other BET proteins regulate RNA Pol II remain insufficiently understood. Addressing the disparity between pan-BET degraders and BRD4-specific depletion, we report that the BET protein BRD2 generally functions to promote transcriptional initiation in a bromodomain-dependent manner at both promoters and enhancers in human cell lines. We demonstrate that BRD2 bromodomains preferentially bind to histone H4 harboring MOF-mediated H4K16ac, while the BRD2 C-terminal domain facilitates recruitment of TFIID. Our studies provide mechanistic insight into distinct roles for BRD2 and BRD4 in transcriptional initiation and elongation control for proper regulation of gene expression.

Humans↗

PubMed2006

Regulation of Drosophila friend of GATA gene, u-shaped, during hematopoiesis: a direct role for serpent and lozenge.

Friend of GATA proteins interact with GATA factors to regulate development in a variety of tissues. We analyzed cis- and trans-regulation of the Drosophila gene, u-shaped, to better understand the transcriptional control of this important gene family during hematopoiesis. Using overlapping genomic fragments driving tissue-specific reporter-gene (lacZ) expression, we identified two minimal hematopoietic enhancers within the 7.4 kb region upstream of the transcription start site. One enhancer was active in all classes of hemocytes, whereas the other was active in hemocyte precursors and plasmatocytes only. The GATA factor, Serpent, directly regulated the activity of both enhancers. However, activity in the crystal cell lineage not only required Serpent but also the RUNX homologue, Lozenge. This is the first demonstration of GATA and RUNX direct regulation of Friend of GATA gene expression and provides additional evidence for the combinatorial control of crystal cell lineage commitment by Serpent, Lozenge, and U-shaped. In addition, we analyzed cis-regulation of ush expression in the lymph gland and identified similarities and differences between regulatory strategies used during embryonic and lymph gland hematopoiesis. The results of these studies provide information to analyze further the regulation of this conserved gene family and its role during hematopoietic lineage commitment.

Animals↗

PubMed2000

A notch-independent activity of suppressor of hairless is required for normal mechanoreceptor physiology.

Suppressor of Hairless [Su(H)]/Lag-1/RBP-Jkappa/CBF1 is the only known transducing transcription factor for Notch receptor signaling. Here, we show that Su(H) has three distinct functions in the development of external mechanosensory organs in Drosophila: Notch-dependent transcriptional activation and a novel auto-repression function, both of which direct cell fate decisions, and a novel auto-activation function required for normal socket cell differentiation. This third phase of activity, the first known Notch-independent activation function for Su(H) in development, depends on a cell type-specific autoregulatory enhancer that is active throughout adult life and is required for proper mechanoreception. These results establish a direct link between a broadly deployed cell signaling pathway and an essential physiological function of the nervous system.

Animals↗

PubMed2006

Transgenic analysis of Dlx regulation in fish tooth development reveals evolutionary retention of enhancer function despite organ loss.

It has been considered a "law" that a lost structure cannot reappear in evolution. The common explanation, that genes required for the development of the lost structure degrade by mutation, remains largely theoretical, however. Additionally, the extent to which this mechanism applies to systems of repeated parts, where individual modules are likely to exhibit few unique aspects of genetic control, is unclear. We investigated reversibility of evolution in one such system, the vertebrate dentition, using as a model loss of oral teeth in cypriniform fishes, which include the zebrafish. This evolutionary event, which occurred > 50 million years ago, has not been reversed despite subsequent diversification of feeding modes and retention of pharyngeal teeth. We asked whether the cis-regulatory region of a gene whose expression loss parallels cypriniform tooth loss, Dlx2b, retains the capacity for expression in oral teeth. We first created a zebrafish reporter transgenic line that recapitulates endogenous dlx2b expression. We then showed that this zebrafish construct drives reporter expression in oral teeth of the related characiform Astyanax mexicanus. This result, along with our finding that Dlx genes are required for normal tooth development, suggests that changes in trans-acting regulators of these genes were responsible for loss of cypriniform oral teeth. Preservation of oral enhancer function unused for > 50 million years could be the result of pleiotropic function in the pharyngeal dentition. If enhancers of other genes in the tooth developmental pathway are similarly preserved, teeth lost from specific regions may be relatively easy to reacquire in evolution.

Animals↗

PubMed2003

The functional analysis of insulator interactions in the Drosophila embryo.

Chromatin boundaries or insulators modulate enhancer-promoter interactions in complex genetic loci. However, the mechanism underlying insulator activity is not known. Previous studies showed that the activity of the Drosophila suHw insulator is abolished by the tandem arrangement (pairing) of the insulator elements, suggesting that interactions between insulators or like elements may be involved in their enhancer-blocking mechanism. To test whether such phenomenon reflects a general property of chromatin insulators, we tested the effect of pairing on enhancer-blocking activity of 11 homologous and heterologous insulator combinations using suHw, scs, or SF1 insulators. We found that, unlike the homologous pairing of suHw, the heterologous combinations of suHw with other insulators do not reduce their enhancer-blocking activity. Rather, paired insulators exhibit a higher level of enhancer-blocking activity than either single insulator alone, suggesting that they can function independently or additively. Furthermore, the analyses of two additional chromatin boundaries, scs and SF1, in homologous or heterologous pairing with other boundary elements, also showed no reduction but rather enhancement of insulator activity. We propose that diverse mechanisms may underlie insulator activity, and selective interactions among insulators could influence their function as well as the formations of independent chromatin domains.

Animals↗

PubMed2001

Treatment of spinal muscular atrophy by sodium butyrate.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, leading to muscular paralysis with muscular atrophy. No effective treatment of this disorder is presently available. Studies of the correlation between disease severity and the amount of survival motor neuron (SMN) protein have shown an inverse relationship. We report that sodium butyrate effectively increases the amount of exon 7-containing SMN protein in SMA lymphoid cell lines by changing the alternative splicing pattern of exon 7 in the SMN2 gene. In vivo, sodium butyrate treatment of SMA-like mice resulted in increased expression of SMN protein in motor neurons of the spinal cord and resulted in significant improvement of SMA clinical symptoms. Oral administration of sodium butyrate to intercrosses of heterozygous pregnant knockout-transgenic SMA-like mice decreased the birth rate of severe types of SMA-like mice, and SMA symptoms were ameliorated for all three types of SMA-like mice. These results suggest that sodium butyrate may be an effective drug for the treatment of human SMA patients.

Abnormalities, Multiple↗

PubMed1997

The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo.

The Drosophila gypsy retrotransposon disrupts gene activity by blocking the interactions of distal enhancers with target promoters. This enhancer-blocking activity is mediated by a 340 bp insulator DNA within gypsy. The insulator contains a cluster of binding sites for a zinc finger protein, suppressor of Hairy wing [su(Hw)]. Recent studies have shown that a second protein, mod(mdg4), is also important for normal insulator function. Mutations in mod(mdg4) exert paradoxical effects on different gypsy-induced phenotypes. For example, it enhances yellow2 but suppresses cut6. Here, we employ a stripe expression assay in transgenic embryos to investigate the role of mod(mdg4) in gypsy insulator activity. The insulator was inserted between defined enhancers and placed among divergently transcribed reporter genes (white and lacZ) containing distinct core promoter sequences. These assays indicate that mod(mdg4) is essential for the enhancer-blocking activity of the insulator DNA. Moreover, reductions in mod(mdg4)+ activity cause the insulator to function as a promoter-specific silencer that selectively represses white, but not lacZ. The repression of white does not affect the expression of the closely linked lacZ gene, suggesting that the insulator does not propagate changes in chromatin structure. These results provide an explanation for why mod(mdg4) exerts differential effects on different gypsy-induced mutations.

Animals↗

PubMed1991

LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function [corrected].

Lymphoid-specific cDNA clones were isolated that encode a nuclear protein with homology to the chromosomal nonhistone protein HMG-1 and to putative regulators of cell specialization, including the mammalian testis-determining factor SRY and fungal mating-type proteins. The gene represented by the isolated cDNA clones, termed LEF-1 (lymphoid enhancer-binding factor 1), is developmentally regulated and expressed in pre-B and T lymphocytes but not in later-stage B cells or nonlymphoid tissues. Both endogenous and recombinant LEF-1 were shown to bind to a functionally important site in the T-cell antigen receptor (TCR) alpha enhancer. Maximal TCR alpha enhancer activity was found to parallel the cell type-specific expression pattern of LEF-1. Moreover, forced expression of recombinant LEF-1 in late stage B cells increases TCR alpha enhancer function. Taken together, these data suggest that LEF-1 is a regulatory participant in lymphocyte gene expression and differentiation.

Amino Acid Sequence↗

PubMed2005

A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts.

Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of its organization. First, large regions of the genome, called gene deserts, are devoid of protein-coding sequences and have no obvious biological role. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) in all vertebrates. Most surprisingly, these structural features are strongly associated with genes that have essential functions during development. Among these, the vertebrate Iroquois (Irx) genes stand out on both fronts. Mammalian Irx genes are organized in two clusters (IrxA and IrxB) that span >1 Mb each with no other genes interspersed. Additionally, a large number of HCNRs exist within Irx clusters. We have systematically examined the enhancer activity of HCNRs from the IrxB cluster using transgenic Xenopus and zebrafish embryos. Most of these HCNRs are active in subdomains of endogenous Irx expression, and some are candidates to contain shared enhancers of neighboring genes, which could explain the evolutionary conservation of Irx clusters. Furthermore, HCNRs present in tetrapod IrxB but not in fish may be responsible for novel Irx expression domains that appeared after their divergence. Finally, we have performed a more detailed analysis on two IrxB ultraconserved non-coding regions (UCRs) duplicated in IrxA clusters in similar relative positions. These four regions share a core region highly conserved among all of them and drive expression in similar domains. However, inter-species conserved sequences surrounding the core, specific for each of these UCRs, are able to modulate their expression.

Animals↗

PubMed2005

Real-time monitoring of mesangial cell-macrophage cross-talk using SEAP in vitro and ex vivo.

BACKGROUND: Macrophage-mesangial cell interaction plays a crucial role in the pathogenesis of glomerulonephritis. We established a novel system for continuous, real-time monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo. METHODS: Rat mesangial cells were genetically engineered to produce secreted alkaline phosphatase (SEAP) under the control of the nuclear factor-kappaB (NF-kappaB) enhancer elements. The established sensor cells were exposed to macrophages or macrophage-derived factors, and the level of SEAP production was evaluated. RESULTS: In vitro, the established cells expressed and secreted SEAP when exposed to activated macrophages or to cytokines produced by macrophages. The kinetics of SEAP activity in culture media was closely correlated with the expression level of SEAP mRNA. The sensor cells also secreted SEAP in response to media conditioned by macrophage-accumulating, inflamed rat glomeruli. When the sensor cells were transferred adoptively into rat glomeruli subjected to acute anti-Thy 1 glomerulonephritis, the isolated glomeruli containing sensor cells secreted SEAP rapidly and progressively. CONCLUSION: These data suggested that the established system provides simple and useful tools for monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo. This approach would be useful for investigation of molecular mechanisms involved in mesangial cell-macrophage interaction and also for screening of therapeutic agents that efficiently interfere with the link between infiltrating leukocytes and resident glomerular cells.

Alkaline Phosphatase↗

PubMed1994

Lymphoid and non-lymphoid tumors in E kappa-myc transgenic rabbits.

We developed transgenic rabbits with a DNA construct containing the proto-oncogene c-myc conjugated to the Ig kappa-chain enhancer gene, E kappa. One of four transgenic rabbits was mated to a normal rabbit and we used the offspring to develop a colony of rabbits carrying the E kappa-myc transgene in their germline. Of a total of 19 E kappa-myc transgenic rabbits, eight developed tumors. The tumors were characterized histologically and four were diagnosed as lymphoma, and one each was diagnosed as embryonic carcinoma, hepatoma, ovarian carcinoma and basal cell carcinoma. By Southern analysis, we showed the four lymphomas were of B-lymphoid lineage and by nucleotide sequence analysis we found three of them most likely used VH1 in their VDJ gene rearrangements. Cells from the embryonic carcinoma, the hepatoma and two of the B-lymphomas were adapted to tissue culture. We discuss the possibility that tumors of non-lymphoid origin develop in the E kappa-myc transgenic rabbits because of the potential for NF-kappa B to activate the kappa-enhancer in cells other than B-lymphoid lineage cells.

Animals↗

PubMed2004

Conserved and acquired features of neurogenin1 regulation.

The telencephalon shows vast morphological variations among different vertebrate groups. The transcription factor neurogenin1 (ngn1) controls neurogenesis in the mouse pallium and is also expressed in the dorsal telencephalon of the evolutionary distant zebrafish. The upstream regions of the zebrafish and mammalian ngn1 loci harbour several stretches of conserved sequences. Here, we show that the upstream region of zebrafish ngn1 is capable of faithfully recapitulating endogenous expression in the zebrafish and mouse telencephalon. A single conserved regulatory region is essential for dorsal telencephalic expression in the zebrafish, and for expression in the dorsal pallium of the mouse. However, a second conserved region that is inactive in the fish telencephalon is necessary for expression in the lateral pallium of mouse embryos. This regulatory region, which drives expression in the zebrafish diencephalon and hindbrain, is dependent on Pax6 activity and binds recombinant Pax6 in vitro. Thus, the regulatory elements of ngn1 appear to be conserved among vertebrates, with certain differences being incorporated in the utilisation of these enhancers, for the acquisition of more advanced features in amniotes. Our data provide evidence for the co-option of regulatory regions as a mechanism of evolutionary diversification of expression patterns, and suggest that an alteration in Pax6 expression was crucial in neocortex evolution.

Amino Acid Sequence↗

PubMed2025

Repression of CADM1 transcription by HPV type 18 is mediated by three-dimensional rearrangement of promoter-enhancer interactions.

Upon infection, human papillomavirus (HPV) manipulates host cell gene expression to create an environment that is supportive of a productive and persistent infection. The virus-induced changes to the host cell's transcriptome are thought to contribute to carcinogenesis. Here, we show by RNA-sequencing that oncogenic HPV18 episome replication in primary human foreskin keratinocytes (HFKs) drives host transcriptional changes that are consistent between multiple HFK donors. We have previously shown that HPV18 recruits the host protein CTCF to viral episomes to control the differentiation-dependent viral transcriptional programme. Since CTCF is an important regulator of host cell transcription via coordination of epigenetic boundaries and long-range chromosomal interactions, we hypothesised that HPV18 may also manipulate CTCF to contribute to host transcription reprogramming. Analysis of CTCF binding in the host cell genome by ChIP-Seq revealed that while the total number of CTCF binding sites is not altered by the virus, there are a sub-set of CTCF binding sites that are either enriched or depleted of CTCF. Many of these altered sites are clustered within regulatory elements of differentially expressed genes, including the tumour suppressor gene cell adhesion molecule 1 (CADM1), which supresses epithelial cell growth and invasion. We show that HPV18 establishment results in reduced CTCF binding at the CADM1 promoter and upstream enhancer. Loss of CTCF binding is coincident with epigenetic repression of CADM1, in the absence of CpG hypermethylation, while adjacent genes including the transcriptional regulator ZBTB16 are activated. These data indicate that the CADM1 locus is subject to topological rearrangement following HPV18 establishment. We tested this hypothesis using 4C-Seq (circular chromosome confirmation capture-sequencing) and show that HPV18 establishment causes a loss of long-range chromosomal interactions between the CADM1 transcriptional start site and the upstream transcriptional enhancer. These data show that HPV18 manipulates host cell promoter-enhancer interactions to drive transcriptional reprogramming that may contribute to HPV-induced disease progression.

Humans↗

PubMed2004

Immunity regulatory DNAs share common organizational features in Drosophila.

Infection results in the rapid activation of immunity genes in the Drosophila fat body. Two classes of transcription factors have been implicated in this process: the REL-containing proteins, Dorsal, Dif, and Relish, and the GATA factor Serpent. Here we present evidence that REL-GATA synergy plays a pervasive role in the immune response. SELEX assays identified consensus binding sites that permitted the characterization of several immunity regulatory DNAs. The distribution of REL and GATA sites within these DNAs suggests that most or all fat-specific immunity genes contain a common organization of regulatory elements: closely linked REL and GATA binding sites positioned in the same orientation and located near the transcription start site. Aspects of this "regulatory code" are essential for the immune response. These results suggest that immunity regulatory DNAs contain constrained organizational features, which may be a general property of eukaryotic enhancers.

Animals↗

PubMed2003

Polycomb-group proteins are involved in silencing processes caused by a transgenic element from the murine imprinted H19/Igf2 region in Drosophila.

A subset of autosomal genes undergo genomic imprinting which results in expression from only the paternal or maternal chromosome. While this phenomenon is restricted to mammals and angiosperms, the underlying silencing mechanisms appear to be evolutionarily conserved. A biallelically unmethylated DNaseI hypersensitive region (A6-A4) between the imprinted Igf2 and H19 genes is conserved in humans and mice and functions as a tissue-specific maintenance element for the imprinted growth factor IGF2. In order to analyse A6-A4 for potentially conserved transcriptional maintenance properties, we have generated transgenic Drosophila harbouring the element in a reporter construct. These flies depicted silencing of the reporter genes lacZ and mini -white. The silenced state of the mini -white gene showed variegation and sensitivity to temperature changes. In addition, two members of the conserved Polycomb group, Enhancer of zeste and Posterior sex combs, were needed for repression. Polycomb group proteins are essential for gene silencing during development. Our results indicate that Polycomb group proteins may also be involved in the regulation of mammalian imprinted genes.

Animals↗

PubMed2006

PRE-mediated bypass of two Su(Hw) insulators targets PcG proteins to a downstream promoter.

Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. We show here that Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and that PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. On the other hand, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter.

Animals↗

PubMed2001

Loss of insulator activity by paired Su(Hw) chromatin insulators.

Chromatin insulators are regulatory elements that block the action of transcriptional enhancers when interposed between enhancer and promoter. The Drosophila Suppressor of Hairy wing [Su(Hw)] protein binds the Su(Hw) insulator and prevents enhancer-promoter interaction by a mechanism that is not understood. We show that when two copies of the Su(Hw) insulator element, instead of a single one, are inserted between enhancer and promoter, insulator activity is neutralized and the enhancer-promoter interaction may instead be facilitated. This paradoxical phenomenon could be explained by interactions between protein complexes bound at the insulators.

ATP-Binding Cassette Transporters↗

PubMed2000

TATA-binding protein mutants that increase transcription from enhancerless and repressed promoters in vivo.

Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery.

Animals↗