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C2H2-zinc finger transcription factors bind RNA and function in diverse post-transcriptional regulatory processes.

ABSTRACT: C2H2-zinc finger transcription factors bind RNA and function in diverse post-transcriptional regulatory processes.

PROVIDER: PRJNA962822 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR24332600_1.fastq.gz	Fastqsanger.gz
	SRR24332600_2.fastq.gz	Fastqsanger.gz
	SRR24333600.fastq.gz	Fastqsanger.gz
	SRR24333700.fastq.gz	Fastqsanger.gz
	SRR24332601_1.fastq.gz	Fastqsanger.gz

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C2H2-zinc finger transcription factors bind RNA and function in diverse post-transcriptional regulatory processes [RNA-seq]

Project description:With over 700 members, the Cys2-His2 zinc finger proteins (C2H2-ZNFs) constitute the largest class of DNA-binding transcription factors (TFs), yet remain largely uncharacterized1. Although certain family members, including GTF3A, WT1 and CTCF, have been shown to bind both DNA and RNA2,3, the extent to which C2H2-ZNFs interact with – and regulate – RNA-associated processes is not known. Here, we utilized UV crosslinking and immunoprecipitation (CLIP) and found that 148 of 150 C2H2-ZNFs that we analyzed bound directly to RNA in human cells. By integrating data from new individual nucleotide resolution RNA-binding maps for 50 of these C2H2-ZNFs with data from chromatin immunoprecipitation sequencing (ChIP-seq), protein-protein interaction, and RNA-seq experiments4, we observe that the RNA-binding profiles of C2H2-ZNFs are generally distinct from their DNA-binding preferences and that they regulate a variety of post-transcriptional processes, including pre-mRNA splicing, cleavage and polyadenylation, and m6A modification of mRNA. Our results thus substantially expand the cellular repertoire of RBPs by systematically defining RNA targets and regulatory roles of C2H2-ZNFs, and they provide a valuable resource for elucidating crosstalk between transcriptional and post-transcriptional regulatory programs.

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C2H2-zinc finger transcription factors bind RNA and function in diverse post-transcriptional regulatory processes [iCLIP-seq]

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Project description:C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2 arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. We have determined the binding sites and motifs of 119 C2H2 zinc finger proteins and the expression pattern of 80 cell lines overexpressing C2H2 zinc finger proteins in order to study the role of C2H2 zinc finger proteins in gene regulation. We expressed GFP-tagged C2H2-ZF proteins in stable transgenic HEK293 cells. Total RNA was isolated using Trizol and sequencing libraries were constructed using TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold or TruSeq RNA Library Preparation Kit v2.

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Project description:The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. Using the bacterial 1-hybrid (B1H) system, we determined DNA-binding motifs for thousands of individual natural C2H2-ZFs, and correlated them with C2H2-ZF specificity residues. The data reported here includes results of protein-binding microarray (PBM) assays for 146 of these natural C2H2-ZFs, performed in order to validate B1H assays and to explore the DNA-binding specificity of C2H2-ZFs. Protein binding microarray (PBM) experiments were performed for a set of 185 variants of mouse Egr1 in which the third zinc finger was replaced by different C2H2-ZFs from different organisms. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006. Among the 185 variants examined, 146 variants yielded motifs in PBMs, which are included here.

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