Minor splicing factor 65K/RNPC3 interacts with ANKRD11 and mediates HDAC3-regulated histone deacetylation and transcription

Pre-mRNA splicing is coupled with DNA- and other RNA-processing machineries in the nucleus to form multilayer networks for accurate regulation of gene expression. However, those established couplings are major spliceosome-related, whether the minor spliceosome is involved remains unclear. Here, we first identified a direct interaction between a minor-specific protein 65K/RNPC3 and the histone deacetylase HDAC3 cofactor ANKRD11 through affinity co-purification using Drosophila lysate. In vivo assays revealed that the head of both DmAnkrd11Δ/Δ and Dm65KΔ/Δ mutants exhibited increased histone acetylation at H3K9 and H4K5, consistent with the high level of ANKRD11 in the central nervous systems. We then found that the 65K-ANKRD11 interaction is conserved in human, in which the HsANKRD11 middle-stretched domain mediates Hs65K association with HDAC3. CUT&Tag assays demonstrated that HDAC3 and Hs65K synergistically bind on chromatin, and the knockdown of ANKRD11 simultaneously decreased their bindings at the same chromatin locations and also increased nearby acetylation of H3K9. Sequencing of mRNAs revealed that the deficiency of HsANKRD11 or Hs65K caused changes in gene expression correlated with their chromatin-binding changes of HDAC3 and Hs65K and the levels of H3K9ac. This study provides a novel strategy for the regulation of histone deacetylation and gene expression through a minor spliceosome-specific component. To investigate the cooperative function 65K/ANKRD11/HDAC3 complex in the regulation of histone acetylation and gene transcription, we established the Dm65K-deletion and DmAnkrd11-deletion Drosophila strain, RNA was from Proximal heads or adults from 100 flies (1 days old), then libraries was prepared for transcriptome sequencing. Comparative gene expression profiling analysis of RNA-seq data for WT, Dm65K-deletion and DmAnkrd11-deletion Drosophila strain. 293T cell lines in which each target gene has been knocked down by shRNA. Comparative gene expression profiling analysis of RNA-seq data for 293T cells and its KD derivatives (sh65K and shANKRD11). The CUT&Tag assay was performed as described (Kaya-Okur et al., 2019). CUT&Tag assay using 65K or HDAC3 antibody to find out binding sites in 293T cells or its KD derivatives (ctrl and shANKRD11). The ChIP assay was performed as described (Gorkin et al., 2020). Briefly, the HEK293T KD derivatives (ctrl, sh65K and shANKRD11) cells were cross-linked by 1% formaldehyde for 10 min and then the cell lysate was prepared by removing cytoplasm followed by sonication-based chromatin fragmentation. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Histone 3 lysine 9 acetylation DNAs were subjected to library preparation with the Scale ssDNA-seq Lib Prep Kit for Illumina V2 (#RK20228, Abclonal).