mechanistically, our observations show that cytor directly binds to the hiv promoter and enhances the phosphorylation of the ser2 ctd of rnapii through association with p - tefb to activate viral gene expression ( figs 4 and 5 ). changes in histone activation marks around the viral promoter in cytor - depleted cells also imply that cytor activates the proviral gene expression ( fig 4 ). in addition to the direct effects of cytor on hiv gene expression, we also demonstrate that cytor controls global gene expression. cytor is recruited to other gene promoters that are regulated by p - tefb, like myc, nf - κb, and il2ra ( s3 fig ). among the identified enriched pathways that potentially are regulated by cytor are those that are involved in actin dynamics. consistently, reduced levels of cytor expression are associated with reduced polymerization of cortical actin in response to tcr engagement ( fig 6 ). in turn, elevated levels of cytor do not further increase actin polymerization in response to t cell stimulation and cannot induce morphological responses of t cells in the absence of stimulation ( s5 fig ). thus, cytor is an important regulator of tcr - induced actin polymerization in t cells. however, its normal endogenous expression levels are sufficient for a proper response. to test a mechanistic link between actin remodeling, cytor levels, and hiv gene expression, we inhibited actin dynamics with specific inhibitors ( fig 6i ). effects of inhibition of actin polymerization phenocopied the effect of cytor depletion on hiv gene expression, suggesting that cytor may affect hiv gene expression by the regulation of genes that control cellular actin dynamics ( fig 6i ). accordingly, we propose a model where cytor exerts its effects on global gene expression and promotes hiv gene expression by both direct and indirect effects ( fig 7 ). cytor directly binds the hiv promoter and recruits the elongation transcription machinery to enhance rnapii ctd phosphorylation and deposition of active histone markers around the hiv promoter, ultimately activating hiv gene expression. indirectly, cytor controls gene targets that regulate actin dynamics in the nucleus and at the plasma membrane to optimize the response to t cell activation, presumably via the regulation of cellular gene expression. 10. 1371 / journal. ppat. 1012172. g007fig 7 * * * a working model for cytor functions. * * * following t cell activation, levels of cytor are elevated in the nucleus. cytor is recruited to the hiv promoter and binds to p - tefb, leading to the activation of viral gene expression. cellular genes regulated by cytor include actin remodeling genes that promote actin polymerization and the indirect activation of hiv gene expression. like cytor, other lncrnas have been reported to occupy the hiv promoter and modulate its activity at either transcriptional or posttranscriptional levels [ 57 ]. most act as scaffolds that associate with other transcriptional activators or repressors to control hiv gene expression [ 35 – 39, 58 – 60 ]. in the case of cytor, its effects on gene expression occur by recruiting the transcription elongation machinery to activate gene expression, either from the viral promoter or other cellular promoters. it will be essential to identify other partners that are associated with cytor lncrna and control hiv promoter activity. as we also aim to dissect the role of cytor in gene expression control, specifically for hiv gene regulation, it will be essential to define how events within the nucleus are regulated by cytor and translated to the control of downstream effector functions of stimulated t cells. future studies will further identify the downstream targets of cytor that control actin dynamics upon t - cell activation. as additional pathways were identified by our rna - seq analysis in cytor - depleted cells, we visualize that future work will identify novel downstream targets of cytor and elucidate their mechanisms of function in regulating hiv gene expression and latency. these may open new ways for developing novel therapeutic tools that will be integrated or substitute current strategies to successfully eliminate the hiv reservoir. materials and methods analysis of actin dynamics in response to t cell activation actin remodeling in response to t cell receptor ( tcr ) engagement was monitored by forming circumferential f - actin rings as previously described [ 61, 62 ]. in brief, stimulatory coverslips were prepared by coating with a 0. 01 % poly - l - lysine ( pll; sigma ) solution for 10 minutes at room temperature, followed by wet - chamber incubation for 3 hours at 37°c with 7 μg / ml anti - cd3 antibod y ( 50 μl per coverslip, clone hit3a against cd3e; bd biosciences ) in phosphate - buffered saline ( pbs ). stimulatory coverslips were subsequently washed in pbs and stored at 4°c in pbs until use. 5x105 cells per anti - cd3 - coated coverslip, respectively ) were used to seed coverslips for 4 minutes to allow tcr - mediated actin ring formation. cells were subsequently fixed in 3 % paraformaldehyde for 15 minutes, permeabilized for 2 minutes in 0. 1 % tritonx-100, and blocked for 30 minutes in 1 % fetal calf serum ( fcs ) in pbs. f - actin was visualized with tetramethyl rhodamine iso thiocyanate ( trit c ) - conjugated phalloidin ( 1: 1, 000, 1 hour, room temperature; sigma ). samples were mounted on glass slides and analyzed by epifluorescence ( olympus ix81 s1f - 3, cellm software ) and confocal ( spinning - disc perkinelmer ultraview vox, velocity software ) microscopes. for quantification of phenotype frequencies, at least 100 transfected cells were counted. chromatin immunoprecipitation ( chip ) analysis control cells expressing scramble shrna or cells where cytor expression was depleted ( kd ) were cross - linked with 1 % formaldehyde for 10 minutes and then washed with pbs and reverse cross - linked with glycine ( 125mm; 5 minutes ). cells were then lysed for 10 minutes on ice in 130μl sonication buffer ( 20 mm tris ph - 7. 8, 2 mm edta, 0, 5 % sds, 0. 5 mm phenylmethylsulfonyl fluoride ( pmsf ), and 1 % protease inhibitor cocktail ), and the nuclear pellets were collected. dna was fragmented by sonication at the following settings: amplitude 20 % for 30 cycles at 10 seconds on / 10 seconds off. samples were centrifuged ( 15 minutes, 14, 000 rpm, 4°c ). the soluble chromatin fraction ( 25 μg ) was collected and immunoprecipitate d ( ip ) overnight at 4°c on a rotating wheel in ip buffer ( 0. 5 % trit on x - 100, 2 mm edta, 20 mm tris ph - 7. 8, 150 mm nacl and 10 % glycerol ) with 2. 5 μg of one of the indicated antibod ies. the next day, the ip material was incubated with 25 μl dynabeads protein g for two hours to ensure the binding of the antibod y to the magnetic beads. dna was eluted with freshly prepared elution solution ( 1 % sds and 0. 1 m nahco3 ) and heated at 65°c overnight to reverse - crosslink the samples. precipitated dna fragments were then extracted using a chip dna clean and concentrator kit ( zymo research ), and hiv dna levels were quantified by qpcr with the primers specifically located on the nfκb region at the hiv - ltr promoter. all signals were normalized relative to input dna. chip assays were also performed with an anti-rabbit or mouse igg as negative control. primers used for qpcr analysis primers on the hiv promoter: nfκb forward: 5 ’ - aggtttgacagccgccta - 3 ’ nfκb reverse: 5 ’ - agagacccagtacaggcaaaa - 3 ’ gapdh forward: 5 ’ - agccacatcgctcagacac - 3 ’ gapdh reverse: 5 ’ - gcccaaacgaccaaatcc - 3 ’ primers for cytor: forward: 5 ’ - aacttgccagcctccatc; reverse: 5 ’ - gagcttcctgtttcatctccc primers for 7sk: forward; 5 ‘ - gagggcgatctggctgcgacat reverse: 5 ‘ - acatggagcggtgagggaggaa source paper: pmc11075828