The human males absent on the first (MOF)-containing non-specific lethal (NSL) histone acetyltransferase (HAT) complex acetylates histone H4 at lysine K5, K8, and K16. This complex shares several subunits with other epigenetic regulatory enzymes, which highlights the complexity of its intracellular function. However, the effect of the NSL HAT complex on the genome and target genes in human cells is still unclear. By using a CRISPR/Cas9-mediated NSL3-knockout 293T cell line and chromatin immunoprecipitation-sequencing (ChIP-Seq) approaches, we identified more than 100 genes as NSL HAT transcriptional targets, including several transcription factors, such as Yin Yang 1 (YY1) which are mainly involved in cell proliferation, biological adhesion, and metabolic processes. We found here that the ChIP-Seq peaks of MOF and NSL3 co-localized with H4K16ac, H3K4me2, and H3K4me3 at the transcriptional start site of YY1. In addition, both the mRNA and protein expression levels of YY1 were regulated by silencing or overexpressing NSL HAT. Interestingly, the expression levels of cell division cycle 6, a downstream target gene of YY1, were regulated by MOF or NSL3. In addition, the suppressed clonogenic ability of HepG2 cells caused by siNSL3 was reversed by overexpressing YY1, suggesting the involvement of YY1 in NSL HAT functioning. Additionally, de novo motif analysis of MOF and NSL3 targets indicated that the NSL HAT complex may recognize the specific DNA-binding sites in the promoter region of target genes in order to regulate their transcription.

Transactivation of p21 (cyclin-dependent kinase inhibitor 1A, CDKN1A) is closely related to the recruitment of transcription cofactors at the p53 responsive elements (p53REs) in its promoter region. Human chromatin remodeling enzyme INO80 can be recruited to the p53REs of p21 promoter and negatively regulates p21. As one of the key subunits of the INO80 complex, YY1 has also been confirmed to bind to the p53RE sites of p21 promoter. Importantly, YY1 was recently reported to be bound and stabilized by BCCIP (BRCA2 and CDKN1A-interacting protein). Therefore, we hypothesized that the YY1/BCCIP complex plays an important role in regulating the transactivation of p21. Here we present evidence that the YY1/BCCIP complex coordinatively regulates p53RE-mediated p21 transactivation. We first confirmed the cross-interaction between YY1, BCCIP, and p53, suggesting an intrinsic link between three proteins in the regulation of p21 transcription. In dual luciferase assays, YY1 inhibited p53RE-mediated luciferase activity, whereas BCCIP revealed the opposite effect. More interestingly, the region 146–270 amino acids of YY1, which bound to BCCIP, increased p53-mediated luciferase activity, indicating the complexity of the YY1/BCCIP complex in co-regulating p21 transcription. Further in-depth research confirmed the co-occupancy of YY1/BCCIP with p53 at the p53RE-proximal region of p21. Lentiviral-mediated knockdown of BCCIP inhibited the recruitment of p53 and YY1 at the p53RE proximal region of p21; however, this phenomenon was reversed by expressing exogenous YY1, suggesting the collaborative regulation of YY1/BCCIP complex in p53RE-mediated p21 transcription. These data provide new insights into the transcriptional regulation of p21 by the YY1/BCCIP complex.

Yin Yang 1 (YY1) is a well-known transcription factor that controls the expression of many genes and plays an important role in the occurrence and development of various cancers. We previously found that the human males absent on the first (MOF)-containing histone acetyltransferase (HAT) complex may be involved in regulating YY1 transcriptional activity; however, the precise interaction between MOF-HAT and YY1, as well as whether the acetylation activity of MOF impacts the function of YY1, has not been reported. Here, we present evidence that the MOF-containing male-specific lethal (MSL) HAT complex regulates YY1 stability and transcriptional activity in an acetylation-dependent manner. First, the MOF/MSL HAT complex was bound to and acetylated YY1, and this acetylation further promoted the ubiquitin–proteasome degradation pathway of YY1. The MOF-mediated degradation of YY1 was mainly related to the 146–270 amino acid residues of YY1. Further research clarified that acetylation-mediated ubiquitin degradation of YY1 mainly occurred through lysine 183. A mutation at the YY1K183 site was sufficient to alter the expression level of p53-mediated downstream target genes, such as CDKN1A (encoding p21), and it also suppressed the transactivation of YY1 on CDC6. Furthermore, a YY1K183R mutant and MOF remarkably antagonized the clone-forming ability of HCT116 and SW480 cells facilitated by YY1, suggesting that the acetylation–ubiquitin mode of YY1 plays an important role in tumor cell proliferation. These data may provide new strategies for the development of therapeutic drugs for tumors with high expression of YY1.

Both OGT1 (O-linked β-N-acetylglucosamine (O-GlcNAc) transferase isoform 1) and NSL3 (nonspecific lethal protein 3) are crucial components of the MOF (males absent on the first)/NSL histone acetyltransferase complex. We previously described how global histone H4 acetylation levels were modulated by OGT1/O-GlcNAcylation-mediated NSL3 stability. However, the specific modification site of NSL3 and its molecular mechanism of protein stability remain unknown. Here, we present evidence from biochemical experiments arguing that O-GlcNAcylation of NSL3 at Thr755 is tightly associated with holoenzyme activity of the MOF/NSL complex. Using in vitro O-GlcNAc-transferase assays combined with mass spectrometry, we suppose that the residue Thr755 on NSL3 C-terminus is the major site O-GlcNAc-modified by OGT1. Importantly, O-GlcNAcylation of this site is involved in the regulation of the ubiquitin-degradation of NSL3, because this site mutation (T755A) promotes the ubiquitin-mediated degradation of NSL3. Further in-depth research found that ubiquitin conjugating enzyme E2 S (UBE2S) accelerated the degradation of NSL3 via direct binding to it. Interestingly, OGT1 and UBE2S competitively bind to NSL3, suggesting the coordination of OGT1–UBE2S in regulating NSL3 stability. Furthermore, O-GlcNAcylation of NSL3 Thr755 site regulates the histone H4 acetylation levels at lysine 5, 8, and 16, suggesting that the O-GlcNAcylation of NSL3 at Thr755 is required for maintaining the integrity and holoenzyme activity of the MOF/NSL complex. In colony formation assays, we found that the integrity of the complex impacts the proliferation of the lung carcinoma type II epithelium-like A549 cells. Taken together, our results provide new insight into the elucidation of the molecular mechanism of the MOF/NSL complex.