Littérature scientifique sur le sujet « Distal regulatory element »

The apo(a) [apolipoprotein(a)] gene is responsible for variations in plasma lipoprotein(a), high levels of which are a risk factor for atherosclerosis and myocardial infarction. The apo(a) promoter stimulates the expression of reporter genes in HepG2 cells, but not in HeLa cells. In the present study, we demonstrate that the 1.4 kb apo(a) promoter comprises two composite regulatory regions: a distal negative regulatory module (positions −1432 to −716) and a proximal tissue-specific module (−716 to −616). The distal negative regulatory module contains two strong negative regulatory regions [polymorphic PNR (pentanucleotide repeat region) and NREβ (negative regulatory element β)], which sandwich the postive regulatory region PREβ (positive regulatory element β). The PNR was shown to bind to transcription factors in a tissue-specific manner, whereas the ubiquitous transcription factors hepatocyte nuclear factor 3α and GATA binding protein 4 bound to NREβ to repress gene transcription. The proximal tissue-specific module contains two regulatory elements: an activating region (PREα) that activates transcription in HepG2 cells, and NREα, which is responsible for repressing the apo(a) gene in HeLa cells. NREα binds to a HeLa-specific repressor. These multiple regulatory elements might work co-operatively to finely regulate apo(a) gene expression. Although the tissue-specific module is required for apo(a) gene activation and repression in a tissue-specific manner, the combinatorial interplay of the distal and proximal regulators might define the complex pathway(s) of apo(a) gene regulation.

No fewer than six different positive regulatory elements concentrated within 130 base pairs constitute the rat albumin promoter, which drives highly tissue specific transcription in rat hepatoma cells in culture. Inactivation of each element led to a decrease in transcriptional efficiency: from upstream to downstream, 3- to 4-fold for distal elements III and II, 15-fold for distal element I, and 50-fold for the CCAAT box and the proximal element (PE). Three of these elements, distal elements III and II and, more crucially, the PE, were found to be involved in the tissue-specific character of transcription, with an additional negative regulation possibly superimposed at the level of the PE. Finally, our mapping of these regulatory elements in vivo entirely coincided with footprint data obtained in vitro, thereby allowing the tentative assignment of specific factors to the effects observed in vivo.

No fewer than six different positive regulatory elements concentrated within 130 base pairs constitute the rat albumin promoter, which drives highly tissue specific transcription in rat hepatoma cells in culture. Inactivation of each element led to a decrease in transcriptional efficiency: from upstream to downstream, 3- to 4-fold for distal elements III and II, 15-fold for distal element I, and 50-fold for the CCAAT box and the proximal element (PE). Three of these elements, distal elements III and II and, more crucially, the PE, were found to be involved in the tissue-specific character of transcription, with an additional negative regulation possibly superimposed at the level of the PE. Finally, our mapping of these regulatory elements in vivo entirely coincided with footprint data obtained in vitro, thereby allowing the tentative assignment of specific factors to the effects observed in vivo.

Abstract Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a BAC transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not NK cells, macrophages and dendritic cells. This distal regulatory element is a Runx3 binding site in Th1 cells, and is needed for RNA polymerase II recruitment to IFNG, but not for histone acetylation of the IFNG locus. These results support a model whereby IFNG utilizes distinct regulatory elements to achieve cell-type expression.

The 3D conformation of the chromatin creates complex networks of noncoding regulatory regions (distal elements) and promoters impacting gene regulation. Despite the importance of the role of noncoding regions in complex diseases, little is known about their interplay within regulatory hubs and implication in multigenic diseases such as schizophrenia. Here we show that cis-regulatory hubs (CRHs) in neurons highlight functional interactions between distal elements and promoters, providing a model to explain epigenetic mechanisms involved in complex diseases. CRHs represent a new 3D model, where distal elements interact to create a complex network of active genes. In a disease context, CRHs highlighted strong enrichments in schizophrenia-associated genes, schizophrenia-associated SNPs, and schizophrenia heritability compared with equivalent structures. Finally, CRHs exhibit larger proportions of genes differentially expressed in schizophrenia compared with promoter-distal element pairs or TADs. CRHs thus capture causal regulatory processes improving the understanding of complex disease etiology such as schizophrenia. These multiple lines of genetic and statistical evidence support CRHs as 3D models to study dysregulation of gene expression in complex diseases more generally.

Regulation of hairy and enhancer of split homologue-1 (HES-1) by estradiol and all-trans retinoic acid affects proliferation of human breast cancer cells. Here, we identify and characterize cis-regulatory elements involved in HES-1 regulation. In the distal 5′ promoter of the HES-1 gene, we found a retinoic acid response element and in the distal 3′ region, an estrogen receptor α(ER)α binding site. The ERα binding site, composed of an estrogen response element (ERE) and an ERE half-site, is important for both ERα binding and transcriptional regulation. Chromatin immunoprecipitation assays revealed that ERα is recruited to the ERE and associates with the HES-1 promoter. We also show recruitment of nuclear receptor co-regulators to the ERE in response to estradiol, followed by a decrease in histone acetylation and RNA polymerase II docking in the HES-1 promoter region. Our findings are consistent with a novel type of repressive estrogen response element in the distal 3′ region of the HES-1 gene.

We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair β-myosin heavy chain (βMyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized βMyHC proximal promoter elements contribute to NWB regulation.

Abstract The transcription factor PU.1 (also known as Spi-1) plays a critical role in the development of the myeloid lineages, and myeloid cells derived from PU.1−/− animals are blocked at the earliest stage of myeloid differentiation. Expression of the PU.1 gene is tightly regulated during normal hematopoietic development, and dysregulation of PU.1 expression can lead to erythroleukemia. However, relatively little is known about how the PU.1 gene is regulated in vivo. Here it is shown that myeloid cell type–specific expression of PU.1 in stable cell lines and transgenic animals is conferred by a 91-kilobase (kb) murine genomic DNA fragment that consists of the entire PU.1 gene (20 kb) plus approximately 35 kb of upstream and downstream sequences, respectively. To further map the important transcriptional regulatory elements, deoxyribonuclease I hypersensitive site mapping studies revealed at least 3 clusters in the PU.1 gene. A 3.5-kb fragment containing one of these deoxyribonuclease I hypersensitive sites, located −14 kb 5′ of the transcriptional start site, conferred myeloid cell type–specific expression in stably transfected cell lines, suggesting that within this region is an element important for myeloid specific expression of PU.1. Further analysis of this myeloid-specific regulatory element will provide insight into the regulation of this key transcriptional regulator and may be useful as a tool for targeting expression to the myeloid lineage.

In order to search for additional regulatory elements in the human CYP17 (steroid 17α-hydroxylase) gene and to compare it with potential regulatory elements in bovine CYP17 genes, 3.5 kb of 5′ flanking region of CYP17 was cloned and analyzed. The newly acquired sequence was shown to be a highly defective copy of the human endogenous retrovirus HERV-K family. This retroviral sequence was itself interrupted by a novel element, a low copy number repeat occurring about 20 times in the human genome, including a known copy in the human catechol-O-methyltransferase gene. A reanalysis of the entire 5′ flanking region of human CYP17 indicates that only the 300 bp immediately distal to the promoter is of unique sequence; other regulatory sequences, including any that are similar to the upstream region of the bovine genes, are unlikely to occur within 5.5 kb of the promoter.Key words: Human CYP17 gene, endogenous retrovirus, low-copy-number repeats.

The MyoD gene can orchestrate the expression of the skeletal muscle differentiation program. We have identified the regions of the gene necessary to reproduce transcription specific to skeletal myoblasts and myotubes. A proximal regulatory region (PRR) contains a conserved TATA box, a CCAAT box, and a GC-rich region that includes a consensus SP1 binding site. The PRR is sufficient for high levels of skeletal muscle-specific activity in avian muscle cells. In murine cells the PRR alone has only low levels of activity and requires an additional distal regulatory region to achieve high levels of muscle-specific activity. The distal regulatory region differs from a conventional enhancer in that chromosomal integration appears necessary for productive interactions with the PRR. While the Moloney leukemia virus long terminal repeat can enhance transcription from the MyoD PRR in both transient and stable assays, the simian virus 40 enhancer cannot, suggesting that specific enhancer-promoter interactions are necessary for PRR function.

Sox2 codifica per un fattore trascrizionale necessario per la pluripotenza delle cellule staminali embrionali. Mutazioni eterozigoti in Sox2 nell’uomo causano difetti nello sviluppo dell’occhio (anoftalmia, microftalmia) e dell’ippocampo, con insorgenza di patologie come epilessia, problemi nel controllo motorio e difetti di apprendimento. Tramite “knock-out” condizionale di Sox2 in topo, abbiamo osservato l’importanza di Sox2 per lo sviluppo del cervello e per il “self-renewal” delle staminali neurali. Di recente è emerso che elementi regolatori possono trovarsi molto lontano dai geni che controllano lungo il cromosoma. Mutazioni in tali elementi talvolta causano patologie dovute a deregolazione del gene associato. In collaborazione con la Dr. C.-L. Wei (California), abbiamo comparato le interazioni “long-range” nella cromatina di cellule di precursori neurali (NPCs) di topi “wild-type” (wt) e Sox2-deleti, usando la tecnica ChIA-PET: su 7000 interazioni mappate in NPCs wt, 2700 erano perse in NPCs Sox2-delete. Tra queste 2700 interazioni, molte coinvolgevano geni legati allo sviluppo neurale e sequenze identificate come enhancer telencefalici per la presenza di siti di legame per p300. Abbiamo poi determinato la mappa genomica dei siti di legame per SOX2 nella cromatina di NPCs wt (in collaborazione con il Dr. F. Guillemot; Londra). Circa metà delle interazioni “long-range” SOX2-dipendenti presentava un picco di ChIP-seq per SOX2, suggerendo un ruolo diretto di SOX2 nel loro mantenimento. Il mio progetto di tesi intende definire se sequenze distali, associate in modo SOX2-dipendente a geni neurali (candidati “target” per SOX2), sono elementi di regolazione trascrizionale attivi durante lo sviluppo embrionale del cervello e se la loro attività è regolata da SOX2. Abbiamo selezionato 13 putativi elementi regolatori distali (DREs), tra le interazioni ChIA-PET perse nelle NPCs Sox2-delete, per caratterizzarli funzionalmente in esperimenti di transgenesi in zebrafish. Ho condotto questi esperimenti in vivo nel laboratorio della Dr. P. Bovolenta a Madrid, supportata da una “EMBO short-term fellowship”. Abbiamo clonato le 13 DREs in un plasmide (ZED), a monte di un promotore minimo e del gene GFP. Il plasmide è stato iniettato in embrioni allo stadio di 1 cellula e il DNA si è integrato nel genoma di pesce. Gli embrioni sono stati osservati durante lo sviluppo per analizzare se, e dove, la sequenza testata guidava l’espressione di GFP. La GFP era espressa riproducibilmente in 12 DREs su 13 nel cervello in via di sviluppo e/o in regioni neurali più posteriori, sovrapponendosi al pattern di espressione del gene associato. Ciò indica che i DREs da soli guidano l’espressione del gene reporter. Ho quindi selezionato embrioni GFP+ transienti (F0) di 8 DREs per ottenere linee transgeniche stabili F1. Per testare se l’attività enhancer dei DREs è regolata da SOX2, ho usato un approccio “perdita di funzione”. Ho iniettato un oligonucleotide antisenso (morfolino), specificamente diretto contro l’mRNA di Sox2, in embrioni F2 di zebrafish allo stadio di 1 cellula. In 2 linee stabili su 8, l’espressione telencefalo-specifica di GFP era ridotta a precoci stadi di sviluppo. Abbiamo anche clonato alcuni DREs in vettori luciferasi per esperimenti di transfezione in colture cellulari. Uno dei DREs mostrava un aumento di attività luciferasica in cotransfezione con vettori di espressione per Sox2 e Mash1, suggerendo un meccanismo di regolazione in cui SOX2 opera insieme al cofattore MASH1. Possiamo concludere che alcuni DREs testati, selezionati tra le interazioni “long-range” di ChIA-PET perse nelle NPCs Sox2-delete, agiscono come elementi regolatori in esperimenti in vivo e sono direttamente regolati da SOX2.
Sox2 encodes a transcription factor required for embryonic stem cell pluripotency. Heterozygous Sox2 mutations in humans cause defects in the development of eyes (anophthalmia, microphthalmia) and hippocampus, with neurological pathology including epilepsy, motor control problems and learning disabilities. Using a Sox2 conditional knock-out in mouse, we discovered that Sox2 is important for brain development and for neural stem cell maintenance. Recently, it was found that transcriptional regulatory elements are not always localized in proximity of the gene they control, but often they lie very far from it on the linear chromosome map. Mutations in these elements can cause pathology, due to the deregulation of the associated gene. In collaboration with Dr. C.-L. Wei’s lab (California), we compared long-range DNA interactions in chromatin of wild-type mouse neural stem/precursor cells (NPCs) and Sox2-deleted cells, using the ChIA-PET technique: out of a total of 7000 long-range interactions mapped in wild-type NPCs, 2700 were lost in Sox2-deleted cells. Many of the lost interactions involved genes important for neural development and sequences already identified as forebrain enhancers by p300 binding in mouse developing telencephalon. In parallel, we determined the genome-wide map of SOX2 binding sites in chromatin of wild-type NPCs, by ChIP-seq (in collaboration with Dr. F. Guillemot; London). At least half of the SOX2-dependent long-range interactions contain a SOX2 ChIP-seq peak, suggesting that SOX2 has a direct role in their maintenance. My project seeks to define if distal sequences, associated in a SOX2-dependent way to neural genes (candidates to be putative SOX2 targets), represent transcriptional regulatory elements active during embryonic brain development and if their activity is regulated by SOX2. We selected 13 putative distal regulatory elements (DREs), among the ChIA-PET interactions lost in Sox2-deleted cells, to functionally characterize them in transgenic experiments in zebrafish. I did the transgenesis experiments in Dr. P. Bovolenta’s lab in Madrid, supported by an EMBO short-term fellowship. We cloned the 13 DREs upstream of a minimal promoter and a GFP gene (in a “ZED” plasmid). The plasmid is injected in 1-cell stage embryos and the DNA is integrated into the fish genome. After injection, the embryos are observed during development to analyze if, and where, the tested sequences drive GFP expression. I found that 12 out of 13 DREs give rise to reproducible GFP expression in the developing forebrain and/or in more posterior neural regions, matching the expression pattern of the associated gene. This indicates that the selected DREs alone are able to guide reporter gene expression. I collected the transient GFP+ embryos (F0) of 8 DREs to obtain F1 stable transgenic lines. To test if the enhancer activity of DREs is regulated by SOX2, I used a loss of function experiment. I injected a morpholino antisense oligonucleotide, specifically directed against the Sox2 mRNA, in F2 zebrafish embryos at 1-cell stage. Two, out of 8, stable lines showed a reduced GFP expression specifically in forebrain in early developmental stages. We have also cloned some of the selected DREs in a luciferase vector to test them by transfection in cultured cells. One of the DREs showed a significant increase in luciferase activity if co-transfected with Sox2 and Mash1 expressing vectors, suggesting a regulatory mechanism operated by SOX2 on this element in presence of the cofactor MASH1. We can conclude that some of the tested DREs, involved in ChIA-PET interactions lost in Sox2-deleted cells, work as regulatory elements in in vivo experiments and are directly regulated by SOX2.

Cellular development and function necessitate precise patterns of gene expression. Control of gene expression is in part orchestrated by a class of remote regulatory elements, termed enhancers, which are brought into contact with promoters via DNA looping. Enhancers typically contain clusters of transcription factor binding sites, and TF recruitment to them is thought to play a key role in transcriptional control. In this thesis I have addressed two issues regarding gene regulation by enhancers. First, with recent genome-wide enhancer mapping, it is becoming increasingly apparent that genes are commonly regulated by multiple enhancers in the same cell type. How a gene’s regulatory information is encoded across multiple enhancers, however, is still not fully understood. Second, numerous recent studies have found that enhancers are enriched for expression-modulating and disease-associated genetic variants. However, understanding and predicting the effects of enhancer variants remains a major challenge. I focussed on a human lymphoblastoid cell line (LCL), GM12878, for which ChIP-Seq data are available for 52 different TFs from the ENCODE project. Significantly, Promoter Capture Hi-C data for the same LCL are available, making it possible to link enhancers to target genes globally. In the first part of the thesis, I investigated how gene regulatory information is encoded across enhancers. Specifically, I asked whether a gene tends to use multiple enhancers to bring the same or distinct regulatory information. I found that there was a general trend towards a “shadow” enhancer architecture, whereby similar combinations of TFs were recruited to multiple enhancers. However, numerous examples of “integrating” enhancers were observed, where the same gene showed large variation in TF binding across enhancers. Distinct groups of TFs were associated with these contrasting models of TF enhancer binding. To investigate the functional effects of variation at enhancers, I additionally took advantage of a panel of LCLs derived from 359 individuals, which have been genotyped by the 1000 Genomes Project, and for which RNA-Seq data are publically available. I used TF binding models to computationally predict variants impacting TF binding, and tested the association of these variants with the expression of the target genes they contact based on Promoter Capture Hi-C. Compared to the standard eQTL calling approach, this offers increased sensitivity as only variants physically contacting the promoter and predicted to impact TF binding are tested. Using this approach, I discovered a set of predicted TF-binding affinity variants at distal regions that associate with gene expression. Interestingly, a large proportion of these binding variants fall at the promoters of other genes. This finding suggests that some promoters may be able to act in an enhancer-like manner via long-range interactions, consistent with very recent findings from alternative approaches.

The sequence-specific DNA binding function of many proteins is recognized as one of the central mechanisms of regulating transcription and DNA replication and repair. The ability of these proteins to select a short (usually 10 to 20 basepair) sequence out of the entire genome with which to form a stable complex is a prime example of molecular recognition. Atomic resolution structural studies using NMR and X-ray crystallography have emerged as essential techniques in understanding the basis of specificity and stability in these systems. While NMR studies of small DNA-binding domains of proteins have become almost routine (see Kaptein, 1993 for a review) relatively few NMR studies of protein-DNA complexes have been reported. These include the lac repressor headpiece complex (Chuprina et al., 1993). the Antennapedia homeodomain complex (Billetere et al., 1993), the GATA-1 complex (Omichinski et al., 1993). and the Myb DNA binding domain complex (Ogata et al., 1993); all of these complexes are smaller than 20 kDa. In most cases, size limitations have meant that only the DNA binding domain of the protein in complex with a single binding element have been studied. In vivo, however, most DNA binding proteins are much larger than these domains and often function as oligomers. The decrease in quality and increase in complexity of spectra as the molecular weight of the sample increases, limits the number of systems amenable to study using NMR and influences the decision to focus on single domains of multidomain proteins. However, since many DNA-binding proteins are regulated by the binding of ligands, other proteins or phosphorylation, often at sites distal from the DNA-binding domain, it is preferable to study as much of the intact protein as possible in order to characterize allosteric and regulatory mechanisms (Pabo and Sauer, 1992). E. coli trp repressor is a 25 kDa homodimer that regulates operons involved in tryptophan biosynthesis. The dimer is one of the smallest intact proteins that binds sequence specifically to DNA and whose affinity is modulated by an effector (L-tryptophan).