| Histone modifications are one of the earlier and most studied epigenetic mechanisms.In cells,different histone-modifying enzymes can modify specific amino acid sites on the histone N-terminal tail differently,including acetylation,methylation,ubiquitination and phosphorylation,thus changing the degree of density of chromatin structure and further affecting gene transcriptional activation,chromatin recombination and DNA repair.The human MOF(males absent on the first)is one member of the MYST histone acetyltransferase family,which is a direct homolog of the Drosophila MOF protein and is involved in a variety of important biological processes in cells.It has been reported that MOF,as a catalytic subunit,is involved in the composition of two histone acetyltransferase complexes,MSL(male-specific lethal)and NSL(non-specific lethal),both of which have the enzymatic activity of catalytic histone H4K16 ac,suggesting that the two complexes may have similarities in some functions.On the other hand,the different subunits involved in forming the two complexes result in the specificity in the catalytic substrate and biological functions.For example,in human cells,the MSL complex consists of four subunits,MOF and MSL1-3,and specifically acetylate H4K16,while the NSL complex consists of nine subunits including MOF and NSL1-3,with broader substrate specificity than the MSL complex and can simultaneously acetylate histone H4K5,K8 and K16.However,few studies report the similarities and differences regarding the biological functions of MSL and NSL complexes.To explore the biological functions of the two complexes in human cells,we investigated the effects of MOF-containing complex on epithelial-to-mesenchymal transition(EMT)progression and metastasis by CRISPR/Cas9 system,multi-omics high-throughput data analysis,and a series of biochemical experiments.First,CRISPR/Cas9-mediated MSL1(a key subunit of the MSL)-knockout(KO)and NSL3(a key subunit of the NSL)-KO HEK293 T cells were successfully established.Ch IP-Seq analysis indicated that the MSL may be mainly involved in cell growth and maintenance,while NSL may be mainly involved in phosphoinositide-mediated signaling pathways.Interestingly,MSL1-KO and NSL3-KO cells seem to prefer to alter cellular phenotype.Second,we confirmed that the it had very different effects on cell proliferation,cycle,cytoskeleton after MSL1-KO and NSL3-KO by using cell viability assays,colony formation assay,flow cytometry,and immunofluorescence staining assay.It is able to promote cell proliferation and clonogenic formation,enabling cell cycle arrest in G2 /M and S phase and cause multipolarized spindles by MSL1-KO.However,NSL3-KO inhibited cell proliferation and clonogenic formation,enabling cells mostly arrest in S phase,and lumen-like cells were observed in cell immunofluorescence staining.The above results suggest that the MSL and NSL complexes may be involved in the maintenance of cell proliferation,cell cycle,and cell division processes with different molecular mechanisms.Third,silencing of MSL1 promotes migration of HEK293 T or breast cancer cells(MCF-7 and MDA-MB-231)in transwell experiments.In contrast,silencing of NSL3 inhibits migration of HEK293 T or breast cancer cells(MCF-7 and MDA-MB-231).Silencing of MSL1 decreased the protein levels of the epithelial biomarker E-cadherin,and increased the protein levels of mesenchymal biomarkers such as N-cadherin,Vimentin and Snail.In contrast,the expression level of NSL3 was negatively correlated with the E-cadherin and positively with N-cadherin,Vimentin and Snail.The above results indicate that both MSL and NSL complexes are associated with epithelial-mesenchymal transformation,but the mechanisms are different.Finally,further studies have clarified that MSL1,but not NSL3,can specifcally bind to the E-box-containing Snail promoter region and thereby negatively regulate Snail transactivation by luciferase assay including E-cadherin,N-cadherin,Vimentin and Snail promoter plasmids.Also,silencing of MSL1 promotes MCF-7 cells xenograft growth in BALB/c-nude mice and the lung metastasis of B16F10 melanoma cells in mice.In summary,by using CRISPR/Cas9 gene editing and si RNA/sh RNA interference technology,combined with a series of biological experiments,we clarified that the MOF containing MSL and NSL HATs are involved in the EMT process in different ways.Under normal circumstances,the MSL HAT may act as a tumor suppressor,specifically binding to the E-box-containing Snail promoter and thereby inhibiting the Snail expression and EMT process.When the MSL HAT is functionally defective,it can induce EMT and a cell phenotype and promote tumor cell metastasis.However,in contrast to MSL HAT,the NSL HAT contributes to inhibit the proliferation and the EMT process in tumor cells.Our data provide new insights for elucidating the molecular mechanism of MOF-containing HATs in human cells. |
