American Society of Hematology

Figure 2.

$HBZ RNA inhibits interaction of TBP with LTR chromatin. (A) Schematic representation of ChIRP. Biotinylated probes (supplemental Table 2) were hybridized to target HBZ RNA, and chromatin complexes were purified using magnetic streptavidin beads. (B) Enrichment of HBZ RNA in ChIRP precipitates from CHOK1-LTRLuc cells transfected with pME18Sneo or pME18Sneo-TTG plasmids. Data are percentages of HBZ and GAPDH RNAs retrieved relatively to the input DNA from RNA fractions. (C) LTR chromatin was precipitated with HBZ-specific biotinylated probes. DNA was eluted and amplified by qPCR using GAPDH or HTLV U3-R LTR primers (LTR1; supplemental Table 1). (D) Retrieval of HBZ RNA-specific RNA by ChIRP. The RNA fraction of ChIRP precipitates from control Jurkat T cells and HTLV-1–infected lymphocytes (C8166 and MT4) was extracted and analyzed by RT-qPCR, using primers against GADPH and HBZ RNAs (HBZ2; supplemental Table 1). (E) DNA fraction from ChIRP of Jurkat, C8166, and MT4 was extracted and analyzed by qPCR using primers against HTLV LTR (LTR1) and a control locus (GADPH). (F) Schematic representation of ChIP. (G-H) C91PL lymphocytes transduced with pUCOE-SFFV-EGFP, HBZ, TTG, or SM lentiviruses were analyzed by ChIP using antibodies specific for RNAPII (G), TBP (H), CREB (I), and CREB phosphorylated at serine 133 (p-CREB; J). Data shown are percentages of LTR DNA immunoprecipitated from the input before ChIP. The values are averages and mean standard error of at least 3 independent experiments. (K) C91PL lymphocytes expressing EGFP, HBZ, or TTG (Figure 1) were analyzed by immunoblot using anti-TAX, anti-TBP, and anti-tubulin antibodies. Numbers indicate the mean quantification of band luminescence intensities of TAX and TBP relative to tubulin obtained from 2 independent experiments. Statistical significances were calculated using analysis of variance and the Tukey post hoc test. (L) Model of inhibition of 5′LTR-directed transcription by HBZ RNA.$

HBZ RNA inhibits interaction of TBP with LTR chromatin. (A) Schematic representation of ChIRP. Biotinylated probes (supplemental Table 2) were hybridized to target HBZ RNA, and chromatin complexes were purified using magnetic streptavidin beads. (B) Enrichment of HBZ RNA in ChIRP precipitates from CHOK1-LTRLuc cells transfected with pME18Sneo or pME18Sneo-TTG plasmids. Data are percentages of HBZ and GAPDH RNAs retrieved relatively to the input DNA from RNA fractions. (C) LTR chromatin was precipitated with HBZ-specific biotinylated probes. DNA was eluted and amplified by qPCR using GAPDH or HTLV U3-R LTR primers (LTR1; supplemental Table 1). (D) Retrieval of HBZ RNA-specific RNA by ChIRP. The RNA fraction of ChIRP precipitates from control Jurkat T cells and HTLV-1–infected lymphocytes (C8166 and MT4) was extracted and analyzed by RT-qPCR, using primers against GADPH and HBZ RNAs (HBZ2; supplemental Table 1). (E) DNA fraction from ChIRP of Jurkat, C8166, and MT4 was extracted and analyzed by qPCR using primers against HTLV LTR (LTR1) and a control locus (GADPH). (F) Schematic representation of ChIP. (G-H) C91PL lymphocytes transduced with pUCOE-SFFV-EGFP, HBZ, TTG, or SM lentiviruses were analyzed by ChIP using antibodies specific for RNAPII (G), TBP (H), CREB (I), and CREB phosphorylated at serine 133 (p-CREB; J). Data shown are percentages of LTR DNA immunoprecipitated from the input before ChIP. The values are averages and mean standard error of at least 3 independent experiments. (K) C91PL lymphocytes expressing EGFP, HBZ, or TTG (Figure 1) were analyzed by immunoblot using anti-TAX, anti-TBP, and anti-tubulin antibodies. Numbers indicate the mean quantification of band luminescence intensities of TAX and TBP relative to tubulin obtained from 2 independent experiments. Statistical significances were calculated using analysis of variance and the Tukey post hoc test. (L) Model of inhibition of 5′LTR-directed transcription by HBZ RNA.

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