RESUMEN
Repetitive sequences including transposable elements and transposon-derived fragments account for nearly half of the human genome. While transposition-competent transposable elements must be repressed to maintain genomic stability, mutated and fragmented transposable elements comprising the bulk of repetitive sequences can also contribute to regulation of host gene expression and broader genome organization. Here, we analyzed published ChIP-seq data sets to identify proteins broadly enriched on transposable elements in the human genome. We show 2 of the proteins identified, C2H2 zinc finger-containing proteins ZNF146 (also known as OZF) and ZNF507, are targeted to distinct sites within LINE-1 ORF2 at thousands of locations in the genome. ZNF146 binding sites are found at old and young LINE-1 elements. In contrast, ZNF507 preferentially binds at young LINE-1 sequences correlated to sequence changes in LINE-1 elements at ZNF507's binding site. To gain further insight into ZNF146 and ZNF507 function, we disrupt their expression in HEK293 cells using CRISPR/Cas9 and perform RNA sequencing, finding modest gene expression changes in cells where ZNF507 has been disrupted. We further identify a physical interaction between ZNF507 and PRMT5, suggesting ZNF507 may target arginine methylation activity to LINE-1 sequences.
Asunto(s)
Factores de Transcripción de Tipo Kruppel , Elementos de Nucleótido Esparcido Largo , Proteínas de Unión al ARN , Sitios de Unión , Elementos Transponibles de ADN , Genoma Humano/genética , Células HEK293 , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
DNA replication is essential for cell proliferation and is one of the cell cycle stages where DNA is more vulnerable. Replication stress is a prominent property of tumor cells and an emerging target for cancer therapy. Although it is not directly involved in nucleotide incorporation, Claspin is a protein with relevant functions in DNA replication. It harbors a DNA-binding domain that interacts preferentially with branched or forked DNA molecules. It also acts as a platform for the interaction of proteins related to DNA damage checkpoint activation, DNA repair, DNA replication origin firing, and fork progression. In order to find new proteins potentially involved in the regulation of DNA replication, we performed a two-hybrid screen to discover new Claspin-binding proteins. This system allowed us to identify the zinc-finger protein OZF (ZNF146) as a new Claspin-interacting protein. OZF is also present at replication forks and co-immunoprecipitates not only with Claspin but also with other replisome components. Interestingly, OZF depletion does not affect DNA replication in a normal cell cycle, but its depletion induces a reduction in the fork progression rate under replication stress conditions. Our results suggest that OZF is a Claspin-binding protein with a specific function in fork progression under replication stress.