IntroductionGastric cancer (GC) has a high incidence worldwide. GC accounts for a large portion of global mortality and has a low survival rate [1]. Therefore, identifying therapeutic targets is crucial for improving prognosis. Epigenetic alterations are frequently observed in GC, particularly DNA methylation within CpG islands of tumor suppressor genes such as CDKN2A and MLH1 [2,3,4]. The overexpression of epigenetic enzymes that act as transcriptional repressors, such as DNMT1/3 DNA methyltransferases and EZH2 histone methyltransferase (HMT), positively correlates with GC progression [5,6,7]. In contrast, SETD1A (KMT2F), a histone H3 lysine 4 HMT, is associated with transcriptional activation and promotes tumorigenesis, as well as malignant phenotypes in GC [8, 9].SETD1A is a member of the KMT2 H3K4 HMT family of mammals [10]. Each HMT KMT2 contains a catalytic SET domain. A comprehensive analysis defined the frequent mutation rates of H3K4 HMTs, including KMT2A/C/D in the GC microsatellite instability (MSI) subtype, and genes associated with the cell cycle and DNA repair pathways are upregulated in KMT2-mutant GCs [11]. KMT2C/D is subject to frequent mutational events in mucinous GC [12]. Notably, KMT2A promotes GC cell migration and invasion [13]. KMT2D promotes GC cell proliferation and suppresses apoptosis [14]. However, the importance of the catalytic activity of each enzyme associated with the SET domain in GC remains unclear.SETD1A is a well-characterized KMT2 HMT harboring catalytic and non-catalytic functions. The SET domain of SETD1A is essential for embryonic stem (ES) cell differentiation but not for proliferation and self-renewal in ES cells [15, 16]. The N-terminus of SETD1A, which consists of an RNA recognition motif (RRM) and a WDR82 binding motif, promotes the transcription of CpG-island-associated genes in ES cells [17]. In leukemia, the non-catalytic FLOS domain of SETD1A is necessary for cell survival and interacts with Cyclin K, a cofactor of CDK12/13 that phosphorylates RNAP2 [18]. Thus, SETD1A regulates RNAP2 for the active transcriptional elongation of the genes associated with DNA repair and mitochondrial respiration in leukemia cells [19]. However, the roles of SETD1A’s catalytic and non-catalytic domains in GC remain unclear. Understanding the functional roles of SETD1A may lead to the identification of novel therapeutic targets in GC. Here, we found that the non-catalytic domain of SETD1A is crucial for the cell cycle and the regulation of the expression of the general transcription factor TAF6 in GC. SETD1A cooperatively regulates TAF6 expression via the cell-cycle-related transcription factor E2F4. Collectively, our study demonstrates the essential role of SETD1A’s non-catalytic domain in promoting GC cell cycle progression.Materials and methodsCell lines and cultureThe 293T (American Type Culture Collection; ATCC) and Plat-A (gifted by Dr. Kitamura [20]) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. AGS (ATCC), SNU719 (Korean Cell Line Bank), and MKN45 (Japanese Cancer Research Resources Cell Bank) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS and 1% penicillin–streptomycin. AGS cells were supplemented with nonessential amino acid solution (Life Technologies). Cells were maintained in a humidified incubator at 37 °C and 5% CO2.Competitive growth assayDoxycycline (Dox)-inducible Cas9 (iCas9) cell lines were generated through the lentivirus infection of GC cells with the pCW-Cas9 vector (Addgene #50661), followed by the isolation of single-cell-derived clones that highly expressed Cas9 after Dox treatment. iCas9 cells were infected with sgRNA vectors (Tables S1 and S3). The percentage of GFP or tRFP657 was monitored three days after infection using a CytoFLEX Flow Cytometer (Beckman Coulter), and the cells were subsequently treated with Dox. The percentage of GFP or tRFP657 was monitored every 7 days after Dox treatment. sgEmpty and sgRPA3 were used as the negative and positive controls, respectively.cDNA rescue experimentpMSCV-SETD1A-ires-GFP and pLKO5.sgRNA.EFS.tRFP657.ires.Hygro vectors were constructed as described in previous studies [18, 21, 22]. SETD1A expression vectors were transduced into iCas9-AGS cells via retroviral infection. GFP-positive cells were sorted using a SH800 Cell Sorter (Sony), followed by transduction with sgRNA vectors. The competitive growth assay was performed as described in the previous section. tRFP657-expressing cells were treated with 1 mg/mL hygromycin for 7 days to establish cell lines that stably expressed sgRNA.CRISPR screeningWe designed four sgRNAs (Table S1) for each target using the CRISPick Tool (https://broad.io/crispick), along with 100 non-targeting controls, 10 sgRNAs as negative controls, and 25 sgRNAs targeting essential genes as positive controls. The pooled oligos were synthesized by Twist Bioscience and ligated into the BsmBI-digested sgRNA vector pLKO5.sgRNA.EFS.GFP (Addgene #57822), as described previously [22]. The pooled sgRNA vector was transduced into 1 × 106 iCas9-AGS cells via lentiviral infection. GFP-positive cells were sorted using an SH800 Cell Sorter on day three after infection. The sorted cells (5 × 105) were cultured with Dox and passaged every 3–4 days. Genomic DNA was extracted from 1 × 106 sorted cells on days 0 and 14 after Dox treatment using a NucleoSpin Tissue Kit (Takara Bio). sgRNA sequences were amplified, and the DNA library was prepared and sequenced on a NovaSeq 6000 (Illumina), as described previously [22]. Data were analyzed using PoolQ (3.3.2). We applied a threshold of Log2 FC