931 research outputs found

    Alternative polyadenylation couples to transcription initiation: Insights from ELAV-mediated 3' UTR extension

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    Transcription initiation and mRNA maturation were long considered co-occurring but separately regulated events of gene control. In the past decade, gene promoters, the platforms of transcription initiation, have been assigned additional functions such as the regulation of splicing and 3' end processing. In a recent study, Oktaba and Zhang and al. reveal that neural 3' UTR extension is dependent on promoter sequences. In Drosophila neurons, promoter regions of a subset of genes recruit the RNA-binding protein ELAV, which is required for subsequent ELAV-mediated alternative polyadenylation. Intriguingly, RNA Polymerase II pausing at promoters seems to facilitate ELAV recruitment. How transcription initiation and alternative polyadenylation, processes separated by an entire gene length, are functionally linked, remains unsolved. In this article, I summarize recent findings and discuss possible mechanisms

    Targeting determinants of dosage compensation in Drosophila

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    The dosage compensation complex (DCC) in Drosophila melanogaster is responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. We have used chromatin immunoprecipitation to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and we have demonstrated that they possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, we provide evidence that this range of recruitment potential is due to differences in affinity of the complex to these sites. We were also able to establish that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, we have uncovered a number of short sequence motifs, which in combination may contribute to DCC recruitment. Our findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high-and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Our bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs

    Abnormal Dosage Compensation of Reporter Genes Driven by the Drosophila Glass Multiple Reporter (GMR) Enhancer-Promoter

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    In Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3′ end. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex

    The Negative Elongation Factor Complex – a poorly understood multi-faceted transcript-processing complex

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    With the advent of whole genome screens, promoter-proximal pausing of RNA polymerase II (RNAPII) has been shown to play a much more significant role in eukaryotic systems than previously thought. This type of transcription inhibition is dependent on the binding of the Negative Elongation Factor (NELF) complex which is composed of four sub-units, presenting unique domains and consequently fulfilling different functions. Numerous questions still surround the mechanism by which NELF is recruited, stabilised and dissociated from the RNAPII complex. Furthermore, not much is known about which other transcription stages the NELF complex is involved in and through which sub-units it carries out such functions. Based on the current knowledge of the role of NELF in transcription pausing, it is hypothesised that different interaction partners are required to direct context-specific functions of the NELF complex. This review covers some of the known roles and contexts in which NELF acts in an attempt to identify key questions for future NELF-dependent transcriptional research

    Genome-wide analysis of dMi-2 binding sites

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    ATP-dependent chromatin remodelers regulate gene expression. The actions of chromatin remodelers on the nucleosome removal and assembly, the histone variants exchange and the modifications of the nucleosome array modify the accessibility of the transcriptional machinery to DNA. Transcription is also influenced by the chromatin context. Indeed, the presence of transcription factors, nucleosome-depleted regions and histone modifications, facilitate the recruitment of specific histone modifying enzymes, chromatin modifying enzymes and chromatin remodelers. Thus, several chromatin features influence the transcription outcome. The ATP-dependent chromatin remodeler dMi-2 is typically associated with transcription repression, but its implication in active transcription has also been reported. The dMi-2 binding sites on polytene chromosomes suggest that dMi-2 binds mainly in open chromatin regions. However, the resolution of polytene staining is approximate and does not give any information about the chromatin context surrounding dMi-2. Thus, the genome-wide dMi-2 binding sites have been identified by ChIP-sequencing and correlated with existing data of histone modifications, RNA polymerase II, nucleosome-depleted regions, transcription, transcription factors and chromatin states. All in all, dMi-2 is located in open chromatin regions and in vicinity of developmental genes. Although dMi-2 mainly represses the expression of its associated genes, it binds close to features linked to active transcription and it is enriched in promoters and in potential regulatory regions. Upon heat shock, the inducible hsp70 gene is actively transcribed, and dMi-2 is important for its expression. To investigate the factors influencing the recruitment of dMi-2 in a context of active transcription, the dMi-2 genome-wide binding sites in un-induced and heat shock conditions have been identified by ChIP-sequencing. dMi-2 is selectively enriched on 7 hsp genes. The chromatin features associated to the hsp70 promoter or a nucleosome-depleted region does not suffice to recruit dMi-2. Moreover, a strong transcription is not sufficient to recruit dMi-2, even though its recruitment on the heat shock genes is transcription dependent. Notably, dMi-2 distribution encompasses the gene body and extent beyond the polyadenylation site of the heat shock genes. Thus, the results suggest that dMi-2 follow the transcriptional activity

    GAGA Factor Maintains Nucleosome-Free Regions and Has a Role in RNA Polymerase II Recruitment to Promoters

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    Previous studies have shown that GAGA Factor (GAF) is enriched on promoters with paused RNA Polymerase II (Pol II), but its genome-wide function and mechanism of action remain largely uncharacterized. We assayed the levels of transcriptionally-engaged polymerase using global run-on sequencing (GRO-seq) in control and GAF-RNAi Drosophila S2 cells and found promoter-proximal polymerase was significantly reduced on a large subset of paused promoters where GAF occupancy was reduced by knock down. These promoters show a dramatic increase in nucleosome occupancy upon GAF depletion. These results, in conjunction with previous studies showing that GAF directly interacts with nucleosome remodelers, strongly support a model where GAF directs nucleosome displacement at the promoter and thereby allows the entry Pol II to the promoter and pause sites. This action of GAF on nucleosomes is at least partially independent of paused Pol II because intergenic GAF binding sites with little or no Pol II also show GAF-dependent nucleosome displacement. In addition, the insulator factor BEAF, the BEAF-interacting protein Chriz, and the transcription factor M1BP are strikingly enriched on those GAF-associated genes where pausing is unaffected by knock down, suggesting insulators or the alternative promoter-associated factor M1BP protect a subset of GAF-bound paused genes from GAF knock-down effects. Thus, GAF binding at promoters can lead to the local displacement of nucleosomes, but this activity can be restricted or compensated for when insulator protein or M1BP complexes also reside at GAF bound promoters

    Chromatin organization and transcriptional control of gene expression in Drosophila

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    It is increasingly clear that the packaging of DNA in nucleosome arrays serves not only to constrain the genome within the nucleus, but also to encode information concerning the activity state of the gene. Packaging limits the accessibility of many regulatory DNA sequence elements and is functionally significant in the control of transcription, replication, repair and recombination. Here, we review studies of the heat-shock genes, illustrating the formation of a specific nucleosome array at an activatable promoter, and describe present information on the roles of DNA-binding factors and energy-dependent chromatin remodeling machines in facilitating assembly of an appropriate structure. Epigenetic maintenance of the activity state within large domains appears to be a key mechanism in regulating homeotic genes during development; recent advances indicate that chromatin structural organization is a critical parameter. The ability to utilize genetic, biochemical and cytological approaches makes Drosophila an ideal organism for studies of the role of chromatin structure in the regulation of gene expression. Keywords: Cellular memory; Chromatin remodeling complexes; Gene silencing; Heat shock genes; PcG and trxG protein

    An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila engrailed gene

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    Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila engrailed gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila
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