| 1. |
- Eberle, Andrea B., et al.
(författare)
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An Interaction between RRP6 and SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to Heterochromatin Maintenance in Drosophila melanogaster
- 2015
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Ingår i: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 11:9
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Tidskriftsartikel (refereegranskat)abstract
- RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin.
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| 2. |
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| 3. |
- Gañez-Zapater, Antoni, 1986-, et al.
(författare)
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The SWI/SNF subunit BRG1 affects alternative splicing by changing RNA binding factor interactions with nascent RNA
- 2022
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Ingår i: Molecular Genetics and Genomics. - : Springer Science and Business Media LLC. - 1617-4615 .- 1617-4623. ; 297:2, s. 463-484
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Tidskriftsartikel (refereegranskat)abstract
- BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes mainly associated with transcriptional initiation. They also have a role in alternative splicing, which has been shown for BRM-containing SWI/SNF complexes at a few genes. Here, we have identified a subset of genes which harbour alternative exons that are affected by SWI/SNF ATPases by expressing the ATPases BRG1 and BRM in C33A cells, a BRG1- and BRM-deficient cell line, and analysed the effect on splicing by RNA sequencing. BRG1- and BRM-affected sub-sets of genes favouring both exon inclusion and exon skipping, with only a minor overlap between the ATPase. Some of the changes in alternative splicing induced by BRG1 and BRM expression did not require the ATPase activity. The BRG1-ATPase independent included exons displayed an exon signature of a high GC content. By investigating three genes with exons affected by the BRG-ATPase-deficient variant, we show that these exons accumulated phosphorylated RNA pol II CTD, both serine 2 and serine 5 phosphorylation, without an enrichment of the RNA polymerase II. The ATPases were recruited to the alternative exons, together with both core and signature subunits of SWI/SNF complexes, and promoted the binding of RNA binding factors to chromatin and RNA at the alternative exons. The interaction with the nascent RNP, however, did not reflect the association to chromatin. The hnRNPL, hnRNPU and SAM68 proteins associated with chromatin in cells expressing BRG1 and BRM wild type, but the binding of hnRNPU to the nascent RNP was excluded. This suggests that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and influence their binding to the nascent pre-mRNA particle.
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| 4. |
- Gómez-Navarro, Natalia, et al.
(författare)
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Defects in the NC2 repressor affect both canonical and non-coding RNA polymerase II transcription initiation in yeast
- 2016
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Background: The formation of the pre-initiation complex in eukaryotic genes is a key step in transcription initiation. The TATA-binding protein (TBP) is a universal component of all pre-initiation complexes for all kinds of RNA polymerase II (RNA pol II) genes, including those with a TATA or a TATA-like element, both those that encode proteins and those that transcribe non-coding RNAs. Mot1 and the negative cofactor 2 (NC2) complex are regulators of TBP, and it has been shown that depletion of these factors in yeast leads to defects in the control of transcription initiation that alter cryptic transcription levels in selected yeast loci. Results: In order to cast light on the molecular functions of NC2, we performed genome-wide studies in conditional mutants in yeast NC2 essential subunits Ydr1 and Bur6. Our analyses show a generally increased level of cryptic transcription in all kinds of genes upon depletion of NC2 subunits, and that each kind of gene (canonical or ncRNAs, TATA or TATA-like) shows some differences in the cryptic transcription pattern for each NC2 mutant. Conclusions: We conclude that NC2 plays a general role in transcription initiation in RNA polymerase II genes that is related with its known TBP interchange function from free to promoter bound states. Therefore, loss of the NC2 function provokes increases in cryptic transcription throughout the yeast genome. Our results also suggest functional differences between NC2 subunits Ydr1 and Bur6.
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| 5. |
- Gutiérrez, Gabriel, et al.
(författare)
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Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
- 2017
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Ingår i: Epigenetics & Chromatin. - : Springer Science and Business Media LLC. - 1756-8935. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: TFIIS stimulates RNA cleavage by RNA polymerase II and promotes the resolution of backtracking events. TFIIS acts in the chromatin context, but its contribution to the chromatin landscape has not yet been investigated. Co-transcriptional chromatin alterations include subtle changes in nucleosome positioning, like those expected to be elicited by TFIIS, which are elusive to detect. The most popular method to map nucleosomes involves intensive chromatin digestion by micrococcal nuclease (MNase). Maps based on these exhaustively digested samples miss any MNase-sensitive nucleosomes caused by transcription. In contrast, partial digestion approaches preserve such nucleosomes, but introduce noise due to MNase sequence preferences. A systematic way of correcting this bias for massively parallel sequencing experiments is still missing.Results: To investigate the contribution of TFIIS to the chromatin landscape, we developed a refined nucleosome-mapping method in Saccharomyces cerevisiae. Based on partial MNase digestion and a sequence-bias correction derived from naked DNA cleavage, the refined method efficiently mapped nucleosomes in promoter regions rich in MNase-sensitive structures. The naked DNA correction was also important for mapping gene body nucleosomes, particularly in those genes whose core promoters contain a canonical TATA element. With this improved method, we analyzed the global nucleosomal changes caused by lack of TFIIS. We detected a general increase in nucleosomal fuzziness and more restricted changes in nucleosome occupancy, which concentrated in some gene categories. The TATA-containing genes were preferentially associated with decreased occupancy in gene bodies, whereas the TATA-like genes did so with increased fuzziness. The detected chromatin alterations correlated with functional defects in nascent transcription, as revealed by genomic run-on experiments.Conclusions: The combination of partial MNase digestion and naked DNA correction of the sequence bias is a precise nucleosomal mapping method that does not exclude MNase-sensitive nucleosomes. This method is useful for detecting subtle alterations in nucleosome positioning produced by lack of TFIIS. Their analysis revealed that TFIIS generally contributed to nucleosome positioning in both gene promoters and bodies. The independent effect of lack of TFIIS on nucleosome occupancy and fuzziness supports the existence of alternative chromatin dynamics during transcription elongation.
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| 6. |
- Jordán-Pla, Antonio, et al.
(författare)
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SWI/SNF regulates half of its targets without the need of ATP-driven nucleosome remodeling by Brahma
- 2018
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- Background: Brahma (BRM) is the only catalytic subunit of the SVVI/SNF chromatin-remodeling complex of Drosophila melanogaster. The function of SWI/SNF in transcription has long been attributed to its ability to remodel nucleosomes, which requires the ATPase activity of BRM. However, recent studies have provided evidence for a non-catalytic function of BRM in the transcriptional regulation of a few specific genes.Results: Here we have used RNA-seq and ChIP-seq to identify the BRM target genes in 52 cells, and we have used a catalytically inactive BRM mutant (K804R) that is unable to hydrolyze ATP to investigate the magnitude of the non-catalytic function of BRM in transcription regulation. We show that 49% of the BRM target genes in 52 cells are regulated through mechanisms that do not require BRM to have an ATPase activity. We also show that the catalytic and non-catalytic mechanisms of SVVI/SNF regulation operate on two subsets of genes that differ in promoter architecture and are linked to different biological processes.Conclusions: This study shows that the non-catalytic role of SWI/SNF in transcription regulation is far more prevalent than previously anticipated and that the genes that are regulated by SVVI/SNF through ATPase-dependent and ATPase-independent mechanisms have specialized roles in different cellular and developmental processes.
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| 7. |
- Planells, Jordi, et al.
(författare)
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The exosome degrades chromatin-associated RNAs genome-wide and maintains chromatin homeostasis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Chromatin-associated RNAs (caRNAs) modulate chromatin organization and function. The RNAexosome degrades different types of nuclear transcripts, but its role in chromatin has not beenaddressed. Here we have used Drosophila melanogaster S2 cells as a model system to identify therepertoire of caRNAs and establish the role of the exosome in their regulation. We have analyzed bothunique and repetitive sequences, and combining RNA-seq and ATAC-seq we show that thesimultaneous depletion of the exosome catalytic subunits RRP6 and DIS3 not only affects caRNAlevels but also changes the local chromatin accessibility at specific loci. We have identified a group ofexosome-sensitive genes that are involved in developmental regulation and are characterized by abalanced chromatin state in which Polycomb and Trithorax factors coexist. Our results reveal that RNAdegradation by the exosome is an important mechanism for the homeostasis of such balancedchromatin states. Given that eukaryotic genomes are repetitive to a large extent, we have also analyzed repetitive caRNAs (rep-caRNAs) and we show that the exosome is needed to control repcaRNAlevels and to maintain the degree of chromatin packaging in repetitive genomic regions. Thisrole is particularly relevant in the pericentromeric regions where the exosome is required to silenceLTR elements and maintain centromere organization.
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| 8. |
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| 9. |
- Yu, Simei, et al.
(författare)
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SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing
- 2018
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 46:16, s. 8557-8573
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Tidskriftsartikel (refereegranskat)abstract
- SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3′ end maturation by facilitating 3′ end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3′ end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.
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| 10. |
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