The Non-specific Lethal(NSL) Histone Acetyltransferase Complex Transcriptionally Regulates Yin Yang 1-mediated Mammalian Cell Proliferation

The NSL(non-specific lethal)histone acetyltransferase(HAT)complex containing MOF(horses absent on the first)consists of 9 subunits and has acetylase activity at histone H4 lysine K5,K8 and K16 sites.Several subunits in the complex are shared with other epigenetic regulatory complexes,such as INO80 chromatin remodeling complex and MLL/SET histone methyltransferase,suggesting that there may be some interactions between these complexes.NSL complexes play a wide range of roles in cells and play a key role in regulating pathways related to tissue development and cell homeostasis.However,the distribution,regulation target genes and functions of NSL complex in human cell genome are still unclear.In this paper,we successfully constructed the key subunit NSL3 knockout(NSL3-KO)293T cell line in NSL complex by using CRISPR/cas9 technology.Taking NSL3 wild-type and knockout cell lines as experimental objects,we detected the catalytic subunit MOF,key subunit NSL3,histone H4K16 ac,H4K5ac,H4K8 ac,H3K4me1,H3K4me2,H3K4me3,H3K27 ac,H3K27me3 and pol II of NSL complex by Ch IP Seq.Through high-throughput biostatistics analysis,we screened more than100 candidate target genes for transcriptional modulation of NSL complex,including transcription factors such as YY1,KLF6,TAF15,MED30,XBP1 and FOXP2.The functional annotation results showed that these candidate target genes were mainly involved in cell proliferation,biological adhesion,movement and metabolism.YY1 is a member of the Gli Kruppel family of zinc finger protein transcription factors.It is widely expressed in mammalian cells and participates in the regulation of cell proliferation,differentiation and embryonic development.YY1 is frequently expressed abnormally in different tumors,suggesting that it is closely related to tumorigenesis and progression.It is worth mentioning that YY1 can act as a transcriptional inhibitor or activator in cells according to its regulatory target genes.We found that the Ch IP Seq recruitment peaks of MOF and NSL3 were colocated with H4K16 ac,H3K4me2 and H3K4me3 at the transcription initiation site(TSS)of YY1.In cell experiments,si RNA knockdown or overexpression of NSL3 can affect the expression level of YY1 m RNA and protein,which proves that YY1 is a potential target gene for transcriptional regulation of NSL complex.Furthermore,the double luciferase reporter gene experiment of YY1 and its downstream target gene CDC6 confirmed that NSL complex could affect the transcriptional activation of YY1 and CDC6.In the experiments of cell viability and clone formation,overexpression of NSL3 can improve the proliferation ability of He La and Hep G2 cells.In addition,knockdown of NSL3 by sh RNA can inhibit the proliferation of Hep G2 cells,which can be partially restored by overexpression of YY1,indicating that the transcriptional regulation of YY1 gene mediated by NSL complex is involved in the proliferation of Hep G2 cells.Through the analysis of Ch IP Seq high-throughput data,we found that H4K16 ac,H4K8ac and H4K5 ac had high correlation with the distribution of H3K4me2 / me3 related to transcriptional activation,while the distribution of H3K27me3 related to transcriptional inhibition had low correlation.However,in NSL3-KO cells,the distribution of H4K16 ac,H3K4me2 and H3K4me3 in the gene promoter region decreased,indicating that NSL complex may co regulate gene transcription through H4K16 ac,H3K4me2 and H3K4me3.Further,cluster analysis of the changes in the enrichment intensity ratio of H4K16 ac,H3K4me2 and H3K4me3 after NSL3 knockout showed that NSL3 could coordinate the distribution of H4K16 ac,H3K4me2 and H3K4me3 in some gene TSS regions.In addition,de novo motif analysis of MOF and NSL3 recruitment targets in the genome showed that the DNA binding motifs of NSL complex matched with transcription factors such as KLFs,ELF2 and YY1,suggesting that NSL complex may play a role as a transcriptional co regulatory factor in the process of gene transcription.We further performed EMSA experiments using DNA fragments containing related motifs upstream of YY1 gene promoter,and confirmed that NSL3(or NSL complex)can bind to the promoter region of YY1.In conclusion,through CRISPR/Cas9 mediated NSL3-KO cell line combined with Ch IP Seq,RNA Seq,cell proliferation ability experiment,double luciferase reporter gene experiment and EMSA,this paper identified the potential target genes and related signal transduction pathways of NSL complex transcriptional regulation,confirmed that YY1 is the regulatory target gene of NSL complex,and clarified that NSL complex participates in the proliferation of Hep G2 and He La cells through YY1.This study provides a new idea for the follow-up research and development of related tumor targeted therapeutic drugs.