thus, in addition to prmt3, other members of the prmt family should be explored as potential targets for developing effective antiviral drugs. in addition to its effects on viral gene expression, prmt3 ’ s regulation of host gene transcription has been implicated in various cellular processes, including tumorigenesis, oxaliplatin resistance in liver cancer, retinoic acid signaling, and hepatic lipogenesis18, 20, 28, 29, 59, 60. however, the precise mechanism ( s ) underlying these transcriptional effects of prmt3 remain unclear. in this study, the comparison of atac - seq and rna - seq data between wt and prmt3 ko cells reveals that prmt3 selectively regulates chromatin accessibility and transcription of a small subgroup of human genes, including some well - studied genes such as tgfb2, which is relevant to the potential pathogenic effects of prmt3 in tumorigenesis. the selectivity of prmt3 ’ s action is apparently achieved through forming a complex with tead4, which then co - localizes at specific gene promoter regions via binding to the tead4 - recognition motif. together, these findings have revealed the mechanistic basis for prmt3 ’ s control of the transcription of hiv - 1 and a selected group of host genes. although the prmt3 - created transcription hub containing tead4 and p - tefb is demonstrated in the current study as important for hiv - 1 to escape latency, it is presently unknown whether it also plays a role during the establishment of latency. it is conceivable that the loss of expression / function of any component of the hub could be responsible in this latter process. further studies are thus necessary to test this hypothesis and explore the possibility of targeting the prmt3 transcription hub to cure hiv / aids. methods dcas9 - targeted ltr proteome analysis one million nh1 cells with an integrated with hiv - 1 5 ’ ltr - driven luciferase reporter construct, which contains 424 bp of ltr. cells were fixed with 1 % formaldehyde for 15 min at room temperature and 0. 125 m glycine for 5 min to quench unreacted formaldehyde at room temperature. use 20 ml of cold pbs to wash cells twice. scrape cells with 2 ml cold pbs containing protease inhibitor and dithioth reitol ( dtt ). spin at 700 × g at 4 °c for 3 min to pellet cells. cells were lysed with lysis buffer [ 1 % sds, 10 mm edta, 50 mm tris, ph 8. 1 ] and incubated on ice for 20 min. cells were sheared with a covaris sonicator m220 until genomic dna was visualized to be at 200 – 1000 bp. the dna was cleared with a high - speed spin ( 15, 000 g for 10 min ) at 4 °c. the dna was diluted by chip dilution buffer [ 0. 01 % sds, 1 % trit on x - 100, 1. 2 mm edta, 16. 7 mm tris-hcl, ph 8. 1, 167 mm nacl ]. dcas9 - 3 × flag / sgrnas complex was added to the sheared dna and incubated at 4 °c overnight. the anti - flag m2 agarose resin ( sigma ) was added, followed by a 3 h incubation at 4 °c. the resin was spun down at 1500 × g at 4 °c for 1 min and washed with high salt wash buffer [ 0. 1 % sds, 1 % trit on x - 100, 2 mm edta, 20 mm tris-hcl, ph 8. 1, 300 mm nacl ] for once, low salt wash buffer [ 0. 1 % sds, 1 % trit on x - 100, 2 mm edta, 20 mm tris-hcl, ph 8. 1, 150 mm nacl ] for three times, and te buffer [ 10 mm tris-hcl, 1 mm edta, ph 8. 0 ] for twice, with each wash for 5 min rotated at 4 °c. the products concentrated by beads were used for subsequent experiments. mass spectrometry ( ms ) and analysis the sample of dcas9 - 3 × flag - sgltrs and dcas9 - 3 × flag - sggal4 ( control ) immnuoprecipitated proteins were prepared and analyzed by ms. in general, the eluted ltr binding proteins were reduced in 20 mm dtt at 95 °c for 5 min, and subsequently alkylated in 50 mm iodoacetamide for 30 min in the dark at room temperature. after alkylation, the samples were transferred to a 10 kd centrifugal spin filter ( millipore ) and sequentially washed with 200 μl of 8 m urea for three times and 200 μl of 50 mm ammonium bicarbonate for two times by centrifugation at 14, 000 × g. next, tryptic digestion was performed by adding trypsinat 1: 50 ( enzyme / substrate, m / m ) in 200 μl of 50 mm ammonium bicarbonate at 37 °c for 16 hours. peptides were recovered by transferring the filter to a collection tube and spinning at 14, 000 × g. to increase the yield of peptides, the filter was washed twice with 100 μl of 50 mm ammonium bicarbonate. peptides were desalted by stagetips. ms experiments were performed on a nanoscale easy - nlc 1200 uhplc system ( thermo fisher scientific ) connected to an orbitrap fusion lumos equipped with a nanoelectrospray source ( thermo fisher scientific ). mobile phase a contained 0. 1 % formic acid ( v / v ) in water; mobile phase b contained 0. 1 % formic acid in 80 % acetonitrile ( acn ). the peptides were dissolved in 0. 1 % formic acid ( fa ) with 2 % acetonitrile and separated on an rp - hplc analytical column ( 75 μm × 25 cm ) packed with 2 μm c18 beads ( thermo fisher scientific ) using a linear gra dien t ranging from 5 % to 22 % acn in 90 min and followed by a linear increase to 35 % b in 20 min at a flow rate of 300 nl / min. the orbitrap fusion lumos acquired data in a data - dependent manner, alternating between full - scan ms and ms2 scans. the spray voltage was set at 2. 2 kv, and the temperature of the ion transfer capillary was 300 °c. the ms spectra ( 350 – 1500 m / z ) were collected with 120, 000 resolutions, agc of 4 × 105, and 50 ms maximal injection time. selected ions were sequentially fragmented by hcd with 30 % normalized collision energy, specified isolated windows 1. 6 m / z, and 15, 000 resolutions. agc of 5 × 104 and 40 ms maximal injection time were used. dynamic exclusion was set to 40 s. unassigned ions or those with a charge of 2 + and > 7 + were rejected for ms / ms. raw data was processed using proteome discoverer ( pd, version 2. 4 ), and ms / ms spectra were searched against the reviewed swissprot human proteome database. only peptides with at least six amino acids in length were considered. the peptide and protein identifications were filtered by pd to control the false discovery rate ( fdr ) * * * generation of stable knockout cell line * * * sgrna sequence was ligate into plasmid pspcas9 ( bb ) −2a - puro ( px459 ), which was purchased from addgene ( # 48139 ). cells were transfected with the plasmid expressing sgprmt3 by using lipofect amine 3000 ( invitrogen ) according to the manufacturer ’ s protocol. after 48 h of transfection, cells that did not contain the plasmid were killed with puromycin for 2 days, then changed into dmem with no antibiot ics and plated into 96 - well plate for single clone selection. after verification with western blot, the genome was extracted and sent for sequencing. methods the cytotoxic effects of the a. incana dcm fraction were evaluated in a dose - dependent manner using the mtt assay on several cancer cell lines, with particular emphasis on hela cells. flow cytometry was used to assess cell cycle arrest and apoptosis, while rt - qpcr quantified changes in the expression of apoptotic markers ( bax, bcl - 2, and p53 ). chemical composition analysis was conducted using gas chromatography - mass spectrometry / flame ionization detection ( gc - ms / fid ) to identify the major bioactive compounds within the fraction. results the dcm fraction exhibited dose - dependent cytotoxicity in hela cells, with an ic50 value of 135. 6 µg / ml and a selectivity index ( si ) of 2. 72 relative to normal cells. flow cytometry analysis revealed g0 / g1 cell cycle arrest, significantly hindering progression through the s and g2 / m phases. moreover, there was a significant increase in both early and late apoptotic cell populations, correlating with the upregulation of pro - apoptotic genes ( bax and p53 ) and the downregulation of the anti - apoptotic gene bcl - 2. the chemical analysis identified 22 compounds in the unsaponifiable fraction, chiefly terpenoids such as phytol ( 65. 74 % ).