Figure 4-1 - uploaded by Padraic P Levings
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Diagrammatic representation of the human and murine β-globin gene locus. The human β-globin gene locus: the embryonic ε-globin gene, the two fetal γ-globin genes, and the adult δ-and β-globin genes. The LCR is located upstream of the ε-globin gene and composed of at least 5 HS sites. The murine β-globin locus: the εγ and βh1 globin genes, which are co-expressed in the embryonic yolk sac and the βmaj and βmin globin genes, which are expressed during the fetal and adult stages of erythropoiesis. The overall organization of the LCR is highly conserved between the murine and the human β-globin locus. PCR fragments analyzed in the ChIP experiments are indicated as lines (horizontal bars) below the respective regions.
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Erythroid-specific, high level expression of the β-globin genes is regulated by the locus control region (LCR), composed of
multiple DNase I-hypersensitive sites and located far upstream of the genes. Recent studies have shown that LCR core elements
recruit RNA polymerase II (pol II). In the present study we demonstrate the following: 1) pol II and...
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... This potentially implies that TFIIH association to the basal promoter is linked to the recruitment of Pol II at basal promoter elements via TFIID/TFIIB-mediated recruitment. Pol II has been known to be recruited to HS sites on the LCR both in vitro and in vivo, which implies that transcription complexes first form at the LCR before they are eventually transferred to globin gene promoters (Vieria et al., 2004;Levings et al., 2002). Thus, a model has been proposed suggesting that the LCR serves as a reservoir for transcription complexes, which are transferred to the globin genes during transient contacts. ...
The human ꞵ-globin gene locus consists of five genes that encode subunits of hemoglobin. The locus control region (LCR) regulates these genes, acting as a super-enhancer to promote high-level of β-globin expression in erythroid cells. The LCR, like other super-enhancers, is known to recruit transcription complexes and generate non-coding enhancer RNA (eRNA). Past research has suggested that super-enhancers form phase-separated transcription initiation domains that concentrate RNA polymerase II (Pol II) and transfer Pol II to target gene promoters during transient looping interactions. The aim of this project is to examine the role of TFIIH, involved transcription initiation, in enhancer RNA production and Pol II transfer in the globin gene locus. The LCR was immobilized on magnetic beads and incubated with erythroid nuclear protein extracts. Proteins not bound were washed away and bound proteins were subjected to western blotting experiments. The data show that TFIIH is bound at the LCR and thus may be involved in eRNA production. Cut & Run experiments were performed to examine the localization of TFIIH in the globin gene locus in intact cells. In the future, the laboratory plans to deplete TFIIH and examine production of eRNA and Pol II recruitment at the LCR.
... They can physically interact with their target promoters by creating chromatin loops to amplify gene expression. They are bound by transcription factors and diverse co-factors (Noordermeer and de Laat, 2008;Vieira et al., 2013) and increase gene expression independently of their orientation or position (Banerji et al., 1981). Enhancers can even be on different chromosome and still activate gene expression by transvection in Drosophila (Geyer et al., 1990). ...
Gene expression is controlled by several factors including histone modifications, boundary elements (BEs) and their associated insulator proteins as well as 3D chromatin organization. To decipher the links between these key players, we used Drosophila Hox genes as a model. We compared cell types (cell lines or imaginal discs from L3 larva) harboring different transcriptional status for Hox genes: Wing discs, S2 and Kc167 cell lines are repressed for all Hox genes, whereas Genital discs, S3 and Sg4 cell lines express Abd-B. We confirmed that gene expression is associated with active histone modifications such as H3K4me3 and changes in 3D chromatin organization. Indeed, when Abd-B is active, it loops out of the repressed homeotic cluster that is covered by H3K27me3 repressive histone mark. Unexpecting, we found that the Abd-B dissociation was not equivalent between the active cell types and it used different BEs to isolate the active gene from the repressed surroundings namely Fab-7 for Sg4 cells, Fab-8 for S3 cells and both for Genital discs. Moreover, the differential usage of BEs correlates with the activation of distinct cis-regulatory regions that control Abd-B expression. This discrepancy was accompanied by a switch of Abd-B promoter expression between S3 and Sg4 cells. We then compared 3D chromatin organization and promoter expression in WT and Fab-7 deleted (Fab-7¹) Genital discs. We found that deletion of the Fab-7 BE led to increased Abd-B contacts over the repressed BX-C and overall decrease of Abd-B expression, but no changes in promoter expression were detected in cell population. We performed 5’ single cell RNA-seq in WT and Fab-7¹ imaginal discs and showed that BE deletion did not change the number of isoforms expressed but induced a shift of Abd-B promoter usage. In the mutant, promoter towards the 3’ tends to be more often detected in single cells. Together, our results showed that BE are tightly linked to the epigenetic landscape, the 3D chromatin organization and promoter expression.
... Subsequent reports demonstrated that LCR HS sites harbor promoter-like activity (15,16). While Tuan et al. proposed a facilitated tracking mechanism by which the enhancer is delivered to the globin gene promoters by a transcription mediated process (13), others hypothesized that LCR recruited transcription complexes are transferred to the globin gene promoters by looping mechanisms (17)(18)(19). Evidence for both mechanisms exist. ...
... In erythroid cells lacking the transcription factor NF-E2, adult -globin expression was reduced and Pol II accumulated at the LCR (17). Moreover, in vitro studies demonstrated that Pol II is transferred from an immobilized LCR to a -globin gene template in a process stimulated by NF-E2 (18). Deletion of the murine LCR reduced recruitment of Pol II at the adult globin gene promoter by about 50%; however, the remaining Pol II recruited to the globin gene was not transcriptionally competent (23). ...
... For example, Johnson et al. showed accumulation of Pol II at the LCR and reduced -globin expression in MEL cells lacking transcription factor NF-E2 (17). Moreover, we demonstrated that Pol II recruited to an immobilized LCR template is transferred to the -globin gene in a process depending on the -globin TATA-box and stimulated by NF-E2 (18). Finally, targeting a synthetic DNA-binding protein to the globin downstream promoter inhibited transcription elongation and accumulated Pol II at the LCR (56). ...
Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine β-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous β-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3′eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3′eRNA leads to a reduction in β-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult β-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.
... The LCR HSs are bound by a large number of ubiquitously expressed and tissue-restricted transcription factors, and were among the first enhancer elements shown to recruit Pol II and to transcribe non-coding RNA, now referred to as enhancer RNA (eRNA) [31][32][33][34]. It has been proposed that the LCR HSs constitute the primary sites for Pol II transcription complex recruitment and assembly and that elongation-competent Pol II complexes are transferred to strong basal promoter elements associated with the β-type globin gene promoters during transient looping interactions [32,33,[35][36][37]. ...
Genes under control of super-enhancers are expressed at extremely high levels and are frequently associated with nuclear speckles. Recent data suggest that the high concentration of unphosphorylated RNA polymerase II (Pol II) and Mediator recruited to super-enhancers create phase-separated condensates. Transcription initiates within or at the surface of these phase-separated droplets and the phosphorylation of Pol II, associated with transcription initiation and elongation, dissociates Pol II from these domains leading to engagement with nuclear speckles, which are enriched with RNA processing factors. The transitioning of Pol II from transcription initiation domains to RNA processing domains effectively co-ordinates transcription and processing of highly expressed RNAs which are then rapidly exported into the cytoplasm.
... We propose that PE3 also has a role in recruiting the transcriptional apparatus to Pax6 in differentiated β-TC3 pancreatic cells and may be an element important for general Pax6 gene activation in multiple tissues including the pancreas, consistent with its broad developmental expression pattern (Fig. 2). Recruitment of pre-initiation complex components to distal elements has been described at early stages of transcription at globin genes (47,48) and likely facilitates efficient transfer of factors ready for later differentiation. Using synthetic transcription factors, we confirmed that PE3 can transfer both activator and repressive signals over more than 45 kb to modulate Pax6 gene expression. ...
Complex diseases, such as diabetes, are influenced by comprehensive transcriptional networks. Genome-wide association studies have revealed that variants located in regulatory elements for pancreatic transcription factors are linked to diabetes, including those functionally linked to the paired box transcription factor, Pax6. Pax6 deletions in adult mice cause rapid onset of classic diabetes, but the full spectrum of pancreatic Pax6 regulators is unknown. Using a regulatory element discovery approach we identified two novel Pax6 pancreatic cis-regulatory elements in a poorly characterised regulatory desert. Both new elements, PE3 and PE4, are located 50 and 100 kb upstream, interact with different parts of the Pax6 promoter and nearby non-coding RNAs. They drive expression in the developing pancreas and brain, and code for multiple pancreas related transcription factor binding sites. PE3 binds CTCF and is marked by stem cell identity markers in embryonic stem cells, whilst a common variant located in the PE4 element affects binding of Pax4, a known pancreatic regulatory, altering Pax6 gene expression. To determine the ability of these elements to regulate gene expression synthetic transcriptional activators and repressors were targeted to PE3 and PE4, modulating Pax6 gene expression, as well as influencing neighbouring genes and lncRNAs, implicating the Pax6 locus in pancreas function and diabetes.
... Chez les eucaryotes, la transcription a lieu pendant tout le cycle cellulaire, alors que la réplication est restreinte à la phase S. La transcription est globalement active en phase S, mais elle a lieu au sein de territoires nucléaires distincts par rapport à la réplication (Wei et al. 1998, Helmrich et al. 2013). C'est le cas du gène codant pour la beta-globine, fortement exprimé dans les cellules érythroïdes (Vieira et al. 2004), ou du gène codant pour l'histone H4, présent en plusieurs copies sous l'influence de différentes séquences régulatrices (Holmes et al. 2005). Le contrôle du moment d'activation des origines suivant un programme de réplication très précis (Jackson 1995), permet aussi de limiter les conflits en séparant temporellement la transcription et la réplication (Gilbert et al. 2002). ...
L’activation d’oncogènes entraine une prolifération aberrante des cellules, un stress réplicatif et des cassures de l’ADN. Un lien a été établi entre l’instabilité génomique résultant des cassures et l’inhibition de checkpoints entrainant l’accumulation de mutations et finalement le cancer (Halazonetis et al. 2008). Cependant, les mécanismes liant ces différents évènements n’ont pas encore été caractérisés. Notre hypothèse est que la prolifération incontrôlée des cellules augmente les incidents dus aux conflits entre les polymérases responsables de la réplication et celles responsables de la transcription. Lors de la rencontre des deux polymérases, l’accumulation de surenroulements positifs de l’ADN induit un blocage des fourches de réplication. Ceci crée des zones de fragilité, notamment dues à l’exposition d’ADN simple brin, et pourrait être à l’origine des cassures observées chez les cellules tumorales. Pour valider cette hypothèse, les biologistes de l'équipe ont étudié plusieurs lignées de cellules HeLa dans lesquelles les conflits réplication-transcription sont augmentés et j'ai réalisé l'analyse bioinformatique des approches génomiques suivantes :-DRIP-seq pour la détection des R-loops, une structure double brin hybride ADN/ARN qui se forme lors de la transcription, exposant ainsi un brin d’ADN simple brin.-ChIP-seq de γ-H2AX, une marque d’histone indiquant les cassures de l’ADN.-ChIP-seq de phospho-RPA (S33), un substrat de la kinase ATR au niveau des fourches bloquées. Pour chaque expérience, nous avons utilisé une lignée contrôle et deux lignées dans lesquelles TOP1 et ASF/SF2 sont appauvries avec un shRNA inductible (shTOP1 et shASF). La Topoisomérase I (TOP1) est une enzyme qui relaxe les surenroulements de l’ADN. Le complexe ASF/SF2 est un facteur d’épissage responsable entre autres de l’assemblage des mRNP (ribonucleoprotein particles) au moment de la transcription, qui limitent la formation des R-loops. L’analyse bioinformatique de ces données, ainsi que d'autres données de la littérature, m'a permis d'identifier des régions à risque du génome, localisées en aval de gènes fortement transcrits et répliqués précocement en phase S par des fourches progressant en sens opposé à la transcription. J’ai également observé que les gènes impliqués dans le cancer sont surreprésentés dans ces régions à risque.
... Active transcription at enhancers was first observed over a decade ago in locus-specific molecular biology experiments (23)(24)(25). These observations were extended by the initial observation using ChIP-seq that Pol II is recruited to enhancers across the genome (22). ...
Transcriptional enhancers are DNA regulatory elements that are bound by transcription factors and act to positively regulate the expression of nearby or distally located target genes. Enhancers have many features that have been discovered using genomic analyses. Recent studies have shown that active enhancers recruit RNA polymerase II (Pol II) and are transcribed, producing enhancer RNAs (eRNAs). GRO-seq, a method for identifying the location and orientation of all actively transcribing RNA polymerases across the genome, is a powerful approach for monitoring nascent enhancer transcription. Furthermore, the unique pattern of enhancer transcription can be used to identify enhancers in the absence of any information about the underlying transcription factors. Here, we describe the computational approaches required to identify and analyze active enhancers using GRO-seq data, including data pre-processing, alignment, and transcript calling. In addition, we describe protocols and computational pipelines for mining GRO-seq data to identify active enhancers, as well as known transcription factor binding sites that are transcribed. Furthermore, we discuss approaches for integrating GRO-seq-based enhancer data with other genomic data, including target gene expression and function. Finally, we describe molecular biology assays that can be used to confirm and explore further the function of enhancers that have been identified using genomic assays. Together, these approaches should allow the user to identify and explore the features and biological functions of new cell type-specific enhancers.
... Although bacteria species lack temporal separation [2], eukaryotic replication and transcription occur within spatially and temporally separated domains [201]. Although the majority of transcriptional activity in eukaryotes occurs during G1 phase, there are transcriptionally active loci in S phase, and these loci seem to be spatially separated from replicating regions [202]. Transcription and replication of rRNA genes (rDNA) in mammalian cells are an excellent example of such coordination [203,204]. ...
All living organisms need to duplicate their genetic information while protecting it from unwanted mutations, which can lead to genetic disorders and cancer development. Inaccuracies during DNA replication are the major cause of genomic instability, as replication forks are prone to stalling and collapse, resulting in DNA damage. The presence of exogenous DNA damaging agents as well as endogenous difficult-to-replicate DNA regions containing DNA–protein complexes, repetitive DNA, secondary DNA structures, or transcribing RNA polymerases, increases the risk of genomic instability and thus threatens cell survival. Therefore, understanding the cellular mechanisms required to preserve the genetic information during S phase is of paramount importance. In this review, we will discuss our current understanding of how cells cope with these natural impediments in order to prevent DNA damage and genomic instability during DNA replication.
... Enhancers can directly recruit RNA pol II and other components of the PIC [48][49][50]. Genome-wide ChIP-seq experiments have confirmed RNA pol II presence at enhancers in a variety of tissues, often prior to activation of target genes [51][52][53], and it has been proposed that enhancer-bound RNA pol II might be directly transferred to target promoters to ...
... Prospects & Overviews .... activate their expression ( Fig. 1D and Ref. [50]). Treatment with a pause-release inhibitor of pTEFB (flavopiridol) decreases occupancy of RNA pol II at the PSA gene, but increases occupancy at the upstream enhancer [54]. ...
... Similarly, placement of an insulator element (a sequence capable of blocking enhancer activation) between an enhancer and its target promoter [55], or binding of a zinc-finger binding domain to the promoter (thus blocking RNA pol II elongation) [56], can also increase RNA pol II occupancy at an enhancer whilst decreasing promoter occupancy. RNA pol II transfer has also been observed between the b-globin LCR and b-globin gene in vitro [50]. Although RNA pol II transfer is consistent with the genome-wide presence of RNA pol II at distal enhancer elements, it so far lacks support as a general mechanism of enhancer function. ...
Enhancers can stimulate transcription by a number of different mechanisms which control different stages of the transcription cycle of their target genes, from recruitment of the transcription machinery to elongation by RNA polymerase. These mechanisms may not be mutually exclusive, as a single enhancer may act through different pathways by binding multiple transcription factors. Multiple enhancers may also work together to regulate transcription of a shared target gene. Most of the evidence supporting different enhancer mechanisms comes from the study of single genes, but new high-throughput experimental frameworks offer the opportunity to integrate and generalize disparate mechanisms identified at single genes. This effort is especially important if we are to fully understand how sequence variation within enhancers contributes to human disease.
... Whereas Pol II recruitment to promoters is commonly stimulated by enhancers, enhancers also recruit Pol II directly to the enhancer ( Johnson, Christensen, Zhao, & Bresnick, 2001; Johnson et al., 2003; Szutorisz, Dillon, & Tora, 2005). Consequences of Pol II recruitment to an enhancer include generation of enhancer-derived RNA transcripts (Lai & Shiekhattar, 2014), the exact function of which remains controversial, and Pol II transfer to the promoter, a poorly understood reaction ( Johnson et al., 2001; Vieira et al., 2004). In principle, transfer may involve processive Pol II tracking along the template (Ling, Ainol, et al., 2004) or DNA/chromatin looping in which the enhancer complex physically associates with promoter components (Cullen, Kladde, & Seyfred, 1993; Dekker, Rippe, Dekker, & Kleckner, 2002; Schleif, 1992; Willis & Seyfred, 1996). ...
