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Transcriptome-wide identification of RNA binding protein binding sites using seCLIP-seq

ABSTRACT: Transcriptome-wide identification of RNA binding protein binding sites using seCLIP-seq

PROVIDER: PRJNA749175 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15222627.fastq.gz	Fastqsanger.gz
	SRR15222628.fastq.gz	Fastqsanger.gz
	SRR15222629.fastq.gz	Fastqsanger.gz
	SRR15222630.fastq.gz	Fastqsanger.gz

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Project description:Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SUUR protein is found at late-replicating intercalary heterochromatin sites and in pericentric heterochromatin. We performed a genome-wide mapping of SUUR target genes in the non-polytenic Drosophila Kc cells by using DamID. This approach is based on the ability of a chromatin protein fused to Escherichia coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled (using Cy3 or Cy5) and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000). We generated both an N- and a C-terminal fusion of the full-length SuUR open reading frame with Dam (Dam-SUUR and SUUR-Dam, respectively). For each SUUR fusion protein we performed four independent replicates. We used for this study a cDNA array developed by the GeneCore facility in EMBL (Heidelberg, Germany), covering the DGC1 and DGC2 cDNA libraries from the Berkeley Drosophila Genome Project, which represents more than 70% of the coding Drosophila genome. We found that SUUR preferentially binds to genes that are transcriptionally silent and late replicated. We compared the SUUR binding profile to the binding profile of three PcG proteins, which are known to bind to many intercalary heterochromatin sites, and found that there is a significant overlap with Pc and esc, but less with Sce. A significant overlap is also detected with two markers of pericentric heterochromatin, the heterochromatin proteins HP1 and SU(VAR)3-9. Finally, we demonstrated that SUUR binding profile negatively correlates with DNA polytenization level in salivary gland polytene chromosomes. Taken together, these results suggest that SUUR modulates the level of underreplication by direct binding to intercalary and pericentric heterochromatin.

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Transcriptome-wide identification of RNA binding protein binding sites using seCLIP-seq

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