Transcriptome profiling of human HEK293F cell lines WT, KO-ZATT, and KO-ZATT cell line engineered to express FLAG- ZATT.WT, and FLAG-ZATT.D955N and RIP-seq analysis of ZATT interacting transcripts

Zinc-finger Associated with Tdp2 and Top2 (ZATT, aka ZNF451) is a multifunctional protein found in vertebrates, involved in critical nuclear processes such as the repair of Topoisomerase 2 (Top2) mediated DNA damage, DNA replication, and transcription. These activities have been linked to its intrinsic N-terminal E3 SUMO2/3 ligase activity. However, ZATT also contains an array of central C2H2 class Zinc finger motifs (Znf) and a C-terminal PIN/NYN family nuclease domain of unknown function. Here we report that in addition to its SUMO ligase activity, recombinant purified human ZATT harbors intrinsic ribonuclease (RNase) activity. To investigate the gene expression changes regulated by ZATT, we utilized a HEK293F knockout (KO) cell line for ZATT and generated two complemented cell lines expressing either FLAG-tagged wild-type ZATT (FLAG-ZATT.WT) or a nuclease-deficient mutant (FLAG-ZATT.D955N). Comparative RNA-seq analysis of these cell lines revealed differential gene expression driven by ZATT's nuclease-independent and nuclease-dependent activities. Furthermore, using RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq), we identified specific transcripts that bind directly to ZATT, providing insights into its functional interactions with RNA and its regulatory roles.