PMCID
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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See methods for a detailed description.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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The figure was generated by Biorender. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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B).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Depletion of CYTOR in stimulated primary CD4+ T cells using lentivirus encoding CYTOR shRNA.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Data were measured by RT-qPCR, normalized to GAPDH, and presented relative to cells expressing scrambled shRNA—set to 1.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Statistical significance is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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p≤0.05; n = 3. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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C).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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FACS analysis presenting effects of CYTOR knockdown (KD) on HIVGKO infection in primary CD4+ T cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Cells were stimulated and then transduced with HIVGKO before being analyzed by FACS for mKO2 and GFP expression. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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D).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Quantification of quadrate percentage from three independent experiments of FACS analysis for HIVGKO transduction in CD4+ primary T cells, where CYTOR is KD.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Statistical significance is based on the calculation of mean ±SD from three independent experiments (n = 3) using Two-way ANOVA. ***
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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p≤0.05.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Towards direct effects of CYTOR on HIV gene transcription, this would require its localization within the cell nucleus.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We therefore monitored the subcellular distribution of CYTOR lncRNA between the nucleus and cytoplasm in resting or activated conditions of primary CD4+ T cells by cell fractionation and subsequent RT-qPCR analysis (Fig 4A).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Levels of CYTOR RNA were compared to those of the abundant 7SK lncRNA, which is known to interact with inactive P-TEFb.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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RNA levels were normalized to the 7SL RNA, which does not bind to the HIV promotor and is commonly used as a specificity control for these experiments .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Our analysis showed that CYTOR is localized to the cytoplasm and the nucleus.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Notably, upon T cell stimulation, levels of nuclear CYTOR were elevated relative to the levels of nuclear 7SK, which were decreased (Fig 4A).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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To further extend our understanding of the mechanism by which CYTOR activates HIV gene expression, we tested whether CYTOR binds to the HIV promoter, thereby regulating HIV gene transcription.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We monitored CYTOR occupancy on the HIV promoter by employing Chromatin Isolation by RNA Purification (ChIRP) analysis in J-Lat 6.3 cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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In vitro-transcribed biotinylated CYTOR RNA was synthesized, purified, and then incubated with ChIP material isolated from unstimulated or P/I stimulated HIV J-Lat 6.3 cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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RNA-protein complexes were then specifically pulled down with streptavidin beads, and pulled down levels of CYTOR on the HIV promoter were monitored by RT-qPCR with specific primers that target the viral promoter (Fig 4B).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Our analysis showed that CYTOR binds to the HIV promoter even in unstimulated conditions.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Significantly, CYTOR occupancy on the viral promoter was further elevated following T-cell stimulation (Fig 4B).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR occupancy on gene promoters was also demonstrated for cellular genes that are known to be regulated by P-TEFb, such as NF-κB, IL21Ra, myc.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Of note, the binding of CYTOR to HIV downstream reverse transcriptase sequences was not observed, suggesting that the specificity of CYTOR within the HIV genome lies within the HIV promoter (S3 Fig). (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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A).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR is localized to the nucleus and its levels are elevated upon T-cell stimulation.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Resting or stimulated primary CD4+ T cells were subjected to cell fractionation, separating the samples into a nuclear fraction (grey bar) or cytoplasmic fraction (light blue bar).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Samples were then subjected to RT-qPCR and monitored for CYTOR or 7SK ncRNA levels.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Data were normalized to 7SL RNA in each of the cellular fractions and conditions.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Data are presented relative to cytoplasmic fraction in each condition—set to 1. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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B).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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ChIRP-qPCR analysis for CYTOR binding to the HIV promoter.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR-specific (black bar) or control lacZ (red bar) antisense biotinylated probes were incubated with lysates isolated from unstimulated or P/I stimulated J-Lat 6.3 cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Biotinylated RNA was pulled down with streptavidin beads, and following washing, associated DNA was eluted and analyzed by qPCR with primers for the HIV promoter.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Statistical significance was calculated between the two probes and between unstimulated and stimulated states.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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IgG served as a non-specific antibody for IP control (grey bar).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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The analysis is based on calculating mean ± SD from three independent experiments using two-way ANOVA. ***
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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p≤0.05. **
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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0.05≤p≤ 0.1; n.s—not significant. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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C, D) CYTOR affects the phosphorylation state of RNAPII CTD and histone landscape.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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ChIP qPCR analysis in control or CYTOR KD J-Lat 6.3 cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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ChIP material from cells was immune-precipitated (IP) with antibodies targeting RNAPII-Ser2P or RNAPII Ser5P (C); or for H3K4me3 and H3K27Ac histone activation marks (D).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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IP fraction was analyzed for enrichment of the indicated modifications on the HIV promoter by qPCR with specific primers.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Non-specific IgG served as a control (grey bar).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Percentage of input are means ±SD; n = 3; *** p≤0.05 calculated between scrambled and KD cells for each antibody.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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n.s—not significant.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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To further obtain insights into the mechanisms of action of CYTOR, we performed Chromatin immunoprecipitation (ChIP) qPCR from J-Lat 6.3 cells, where CYTOR expression was manipulated.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We monitored the levels of phosphorylated C-terminal domain (CTD) of RNA Polymerase II (RNAPII) at Ser2 (Ser2P) or Ser5 (Ser5P) residues on control or CYTOR KD expressing cells, using specific antibodies that target the CTD phosphorylation states of RNAPII (Fig 4C).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CDK9/P-TEFb phosphorylates Ser2 and marks RNAPII pause-release and elongation of transcription [50–52].
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CDK7/TFIIH phosphorylates Ser5P on the CTD and catalyzes transcription initiation and promoter clearance.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Our analysis demonstrated that in the context of CYTOR depletion, levels of Ser2P on the HIV promoter were decreased without affecting those of Ser5P, implying the involvement of P-TEFb in CYTOR-mediated HIV gene activation (Fig 4C).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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In addition, ChIP-qPCR was employed using antibodies that target the histone activation markers, H3K27Ac or H3K4me3.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Our analysis confirmed that CYTOR mediates its activation properties by modifying the histone landscape around the HIV.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Upon CYTOR depletion, levels of these histone activation markers were reduced (Fig 4D).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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These results further imply that CYTOR activates HIV gene expression via P-TEFb, affecting transcription elongation.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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To expand the above results on the mechanism by which CYTOR enhances HIV gene expression, we employed RNA-precipitation (RNA-IP; RIP) followed by RT-qPCR in J-Lat 6.3 cells under resting or stimulated conditions (Fig 5).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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As our above results indicate that CYTOR promotes the Ser2 phosphorylation on the CTD, which is mediated by CDK9, we monitored CYTOR association with P-TEFb.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Lysates isolated from nuclei from resting or stimulated J-Lat 6.3 cells were incubated with antibodies that target CDK9 or CYCLIN T1, and samples were IP followed by RT-qPCR to detect CYTOR RNA levels by using specific primers.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We show that upon T cell stimulation, the levels of CYCLIN T1 and CDK9 that were associated with CYTOR RNA increased.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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As expected, levels of 7SK that are associated with P-TEFb were reduced upon T cell stimulation.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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We also followed the association of P-TEFb with 7SL, which served as control.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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As expected, P-TEFb was not associated with 7SL in each of the tested conditions.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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These results indicate that CYTOR associates with P-TEFb in cells (Fig 5A). (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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A) RIP analysis demonstrates the association of CYTOR with P-TEFb.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Isolated ChIP material from resting or stimulated J-Lat 6.3 CD4+ T cells was subjected to immune precipitation with antibodies targeting CDK9 or CYCLIN T1 of P-TEFb, followed by RT-qPCR with primers for the relevant lncRNA (7SK or CYTOR).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Non-specific IgG served as a control for the IP step.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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7SL ncRNA served as a control for an RNA that does not associate with P-TEFb and, therefore, not precipitated with CDK9 or CYCLIN T1 antibodies.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Statistical significance is based on the calculation of mean ±SD from three independent experiments using two-way ANOVA. ***
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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p≤0.05. **
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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0.05≤p≤0.1; ns: not significant. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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B) CYTOR associates with P-TEFb in cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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RNA pull-down followed by western blotting where lysates from J-Lat 6.3 cells were incubated with an in-vitro transcribed biotinylated CYTOR anti-sense probe and reactions were pulled down with streptavidin beads.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Eluted RNP complexes were subjected to western blotting with indicated antibodies.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Non-specific IgG served as a control for non-relevant IgG. Scramble RNA served as RNA that does not associate with P-TEFb.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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7SK probe confirmed association with P-TEFb.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Input is 5% of the total cell lysate .
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Next, we performed RNA pull-down experiments combined with western blotting to detect P-TEFb subunits (CYCLIN T1/CDK9) that are associated with CYTOR.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Lysates from J-Lat 6.3 cells were incubated with an in-vitro transcribed biotinylated anti-sense CYTOR probe, and reactions were pulled down with streptavidin beads.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Eluted RNP complexes were then subjected to western blotting with antibodies that target CYCLIN T1 or CDK9, demonstrating the association of CYTOR lncRNA with P-TEFb within cells.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Non-specific IgG was used as a specificity control for the IP step, while a non-specific scramble RNA probe served as a control for RNA-protein association.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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In addition, a 7SK RNA probe confirmed the association with P-TEFb (Fig 5B).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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These results establish that CYTOR binds to the HIV promoter and suggest that its activation effects are mediated by association with P-TEFb.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Since CYTOR has been previously recognized as a regulator of cytoskeleton-regulating genes in fibroblasts , we assessed if it could also affect HIV gene expression by indirect mechanisms through regulation of its downstream targets.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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For this, we performed RNA-Seq analysis in stimulated primary CD4+ T cells, where CYTOR expression was depleted or over-expressed (n = 3).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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CYTOR modulation of expression did not affect the activation state of cells as monitored by staining with T cell activation markers (S4 Fig).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Analyzing changes in the cellular transcriptome of stimulated CD4+ T cells upon depletion of CYTOR revealed a modest change in the cell gene expression program (S2 Table).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Additional gene GO analysis identified significant enrichment scores in various cellular pathways, including those of gene expression, signal transduction as well as actin dynamics and T-cell activation (Fig 6A and 6B). (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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A).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Volcano plot of the expression pattern of genes from an RNA-Seq analysis upon CYTOR depletion following T cell stimulation.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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-Log10 P is shown on the y-axis, and Log2FC is on the x-axis.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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RNA was isolated from 3 biological replicates (n = 3).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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The fold of change cutoff is defined as FC ≥2.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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FDR of p≤0.05 was used as a cutoff for significance. (
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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B).
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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Gene Ontology analysis for enriched CYTOR gene targets.
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PMC11075828
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Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.
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For enrichment analysis, the DAVID program was employed to identify enriched pathways and terms associated with the selected genes. (
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