Volume 150, Issue 6, Pages (September 2012)

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Volume 150, Issue 6, Pages 1147-1157 (September 2012) Microprocessor, Setx, Xrn2, and Rrp6 Co-operate to Induce Premature Termination of Transcription by RNAPII  Alexandre Wagschal, Emilie Rousset, Poornima Basavarajaiah, Xavier Contreras, Alex Harwig, Sabine Laurent-Chabalier, Mirai Nakamura, Xin Chen, Ke Zhang, Oussama Meziane, Frédéric Boyer, Hugues Parrinello, Ben Berkhout, Christophe Terzian, Monsef Benkirane, Rosemary Kiernan  Cell  Volume 150, Issue 6, Pages 1147-1157 (September 2012) DOI: 10.1016/j.cell.2012.08.004 Copyright © 2012 Elsevier Inc. Terms and Conditions

Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Drosha and Dgcr8 Are Transcriptional Repressors of the Integrated HIV-1 Promoter (A and B) RNA isolated from HeLa LTR-Luc cells transfected with the indicated siRNAs was analyzed by reverse-transcription q-PCR and NRO using the primers indicated on the schematic above the graphs. Values were normalized to that of GAPDH in the same samples. The result for Scr-treated cells was attributed a value of 1. The knockdown of specific factors was validated by immunoblot (right). (C) Validation of RNAi knockdowns by immunoblot using the indicated antibodies. (D) ChIP assay was performed using the indicated antibodies and chromatin prepared from HeLa LTR-Luc cells transfected with control (Si Scr), Drosha, or Dgcr8 siRNAs as indicated. Locations of primers used are indicated on the schematic above the graphs. The amount of immunoprecipitated material for each PCR was normalized to the input DNA. (E) Native ChIP was performed using the indicated antibodies and chromatin from cells transfected with the indicated siRNAs. The promoter region was amplified by q-PCR. The amount of immunoprecipitated material was normalized to the input DNA. (F) ChIP was performed using anti-HP1g or a control IgG and chromatin prepared from HeLa LTR-Luc cells transfected with the indicated siRNAs. The promoter region was amplified by q-PCR. The amount of immunoprecipitated material was normalized to the input DNA. All graphs show mean ± SE from three independent experiments. See also Figure S1. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 HIV-1 TAR RNA Is Required for Drosha and Dgcr8-Mediated Transcriptional Repression of the HIV-1 Promoter (A) ChIP assay was performed as in Figure 1 using the indicated antibodies and chromatin prepared from HeLa LTR-Luc cells transfected with the indicated siRNAs. Results are defined as enrichment over that of a mock precipitation with an unrelated IgG antiserum. Locations of primers used to amplify promoter-proximal and coding region sequences are indicated on the schematic above the graph. (B) ChIP was performed using the indicated antibodies and chromatin prepared from HeLa LTR-Luc cells treated with Tat or mock treated as indicated. The presence of HIV-1 promoter region in immunoprecipitated materials was determined by q-PCR. Results are presented as fold enrichment over that of a mock precipitation using an unrelated IgG antiserum. (C) ChIP was performed using the indicated antibodies and chromatin prepared from HeLa LTR-Luc cells that had been pretreated with RNase or mock treated as indicated. The presence of HIV-1 promoter-proximal region in immunoprecipitated materials was determined by q-PCR. Results are presented as fold enrichment over that of a mock precipitation using an unrelated IgG antiserum. (D) ChIP assay was performed using the indicated antibodies and chromatin prepared from HeLa LTR-Luc (WT) and HeLa LTRΔTAR-Luc (ΔTAR) cells. The presence of HIV-1 promoter-proximal region in immunoprecipitated materials was determined by q-PCR. Results are presented as fold enrichment over that of a mock precipitation using an unrelated IgG antiserum. (E) Nuclei isolated from HeLa LTR-Luc (WT) or HeLa LTRΔTAR-Luc (ΔTAR) cells transfected with the indicated siRNAs were analyzed by NRO using the primers indicated. Values were normalized to that of GAPDH in the same samples. The result for scr-treated cells was attributed a value of 1. The knockdown of Drosha was validated by immunoblot (bottom). All graphs show mean ± SE from three independent experiments. See also Figure S2. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Drosha-Dependent Recruitment of Setx, Xrn2, and Rrp6 to the HIV-1 Promoter-Proximal Region (A) ChIP assay was performed using the indicated antibody and chromatin from Tat-treated or siRNA-treated HeLa LTR-Luc cells, as indicated. Locations of primers used to amplify the promoter-proximal and luc 3′ regions are indicated on the schematic above the graphs. Results are presented as fold enrichment over that of a mock precipitation using an unrelated IgG antiserum. Knockdown efficiency of the siRNA was assessed by immunoblotting using the indicated antibodies. (B–E) ChIP assay was performed using the indicated antibody and chromatin from siRNA-treated or Tat-treated HeLa LTR-Luc cells. Unless indicated otherwise, the promoter-proximal region was amplified by q-PCR. Results are presented as fold enrichment over that of a mock precipitation using an unrelated IgG antiserum or fold increase relative to the control sample (siScr), which was attributed a value of 1. Knockdown efficiencies of the siRNAs were assessed by immunoblotting using the indicated antibodies. (F) NRO performed using nuclei prepared from HeLa LTR-Luc cells transfected with the indicated siRNAs. Regions amplified by PCR are indicated above the graph. Values were normalized to the amount of GAPDH RNA in the same samples. The result for Scr-treated cells was attributed a value of 1. Knockdown efficiencies of the siRNAs were assessed by immunoblotting using the indicated antibodies. All graphs show mean ± SE from three independent experiments. See also Figure S3. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Small TAR-Derived RNAs Repress LTR Activity (A) HeLa LTR-Luc (WT) or HeLa LTRΔTAR-Luc (ΔTAR) cells transfected with control (scr) or Rrp6 siRNA, with or without TAR RNA (1 μg), as indicated, were harvested for luciferase assay (top) and immunoblotting using the antibodies indicated (bottom). For each condition, values were normalized to the control sample that was mock transfected with TAR RNA, which was assigned a value of 1. (B) Northern blot analysis of TAR and TAR-derived RNAs obtained from HIV-1-infected cells transfected with the indicated siRNAs. RNA decade marker run in parallel is shown at left. (C) HeLa LTR-Luc (WT) or HeLa LTRΔTAR-Luc (ΔTAR) cells transfected with control (scr) or Rrp6 siRNA and the indicated RNA oligonucleotides were harvested for luciferase assay (top) and immunoblotting using the antibodies indicated (bottom). For each condition, values were normalized to the control sample, which was assigned a value of 1. All graphs represent mean ± SE obtained from at least three independent experiments. See also Figure S4. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 ChIP-seq Analysis Reveals the Endogenous Loci that Are Targets for Drosha (A) Pie charts summarizing the relative abundance of LINES, SINES, LTR elements, and DNA transposons in the human genome (left) and those bound by Drosha (right). The observed and expected numbers of tags are significantly different (chi-square test, p < 10−15). See also Table S1. (B) Chr6:21,360,000–21,368,000 region containing a HERV-H endogenous retrovirus. Gray triangles represent the two LTRs. Pink, violet, and blue boxes represent genomic fragments whose translations have similarities with Gag, Pol, and Env retroviral proteins. ChIP-seq tags density difference between Drosha siRNA and control conditions along the sequence. Positive values indicate an excess of mapped tags in the Drosha condition. (C) Experiment was performed as in (B) except that RNAPII, RNAPII ser5, and RNAPII ser2 antibodies were used for ChIP-seq. (D) Analysis of genes targeted by Drosha. HeLa LTR-Luc cells transfected with the indicated siRNA were analyzed by ChIP assay using antibody against Drosha (top) or by NRO (bottom). Regions amplified by q-PCR using specific oligonucleotides are indicated. ChIP results are presented as fold enrichment over that of a mock precipitation using an unrelated IgG antiserum. For NRO, values were normalized to the amount of GAPDH RNA in the same samples. The result for Scr-treated cells was attributed a value of 1. All graphs show mean ± SE from at least three independent experiments. See also Table S2. (E) TR of RNAPII is modified by Drosha. ChIP-seq data using anti-RNAPII performed on chromatin from HeLa LTR-Luc cells transfected with the indicated siRNA were analyzed, and the TR of RNAPII was determined. (F) A proposed model for premature termination and transcriptional repression at the HIV-1 promoter. See text for details. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S1 Drosha and Dgcr8 Repress Transcription from the Integrated HIV-1 Promoter Independently of RNAi, Related to Figure 1 (A) Extracts of HeLa LTR-luc cells transfected with the indicated siRNAs were analyzed for Luciferase activity. Results are presented as fold activation relative to control (Scr) cells. (B) RNA isolated from HeLa LTR-Luc cells transfected with siRNA targeting Ago1, Ago2 or a control was analyzed by RT-q-PCR using the primers indicated on the schematic above the graph. Values were normalized to that of GAPDH in the same samples. The result for Scr-treated cells was attributed a value of 1. The knockdown of specific factors was validated by immunoblot (right). (C) Overexpression of Drosha siRNA-resistant mutant rescues HIV-1 LTR activity in Drosha knock-down cells. HeLa LTR-luc cells were transfected with siRNA directed against the 3′ UTR of Drosha, or a control, as indicated. Cells were re-transfected 24 hr later with a plasmid expressing Drosha lacking its 3′ UTR. Extracts were harvested 48 hr later and analyzed for Luciferase activity and western blot using the antibodies indicated. Results are presented as fold activation relative to control siRNA-transfected cells. (D) RNA isolated from HeLa cells infected with HIV-1 Tat(−) virus and transfected with the indicated siRNA was analyzed by NRO using primers in Gag. Values were normalized to that of GAPDH in the same samples. The result for Scr-treated cells was attributed a value of 1The knockdown of specific factors was validated by immunoblot (right). All graphs show mean ± SE from 3 independent experiments. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S2 Deletion of TAR Sequence Enhances Basal LTR Activity, Abolishes Tat-Mediated Transactivation but Does Not Diminish Activation by PMA, Related to Figure 2 HeLa LTR-Luc and HeLa LTRΔTAR-Luc cells were treated as indicated. Luciferase activity in cell extracts was measured 16 hr later. Fold activation is the ratio between values obtained for each sample and HeLa LTR-Luc mock-treated cells. The numbers indicated on the top of the bars are the ratios between the indicated samples. Graph shows mean ± SE from 3 independent experiments. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S3 Drosha Endonucleolytic Activity Is Required for Repression of the HIV-1 Promoter, Related to Figure 3 HeLa LTR-Luc cells were mock-transfected or transfected with increasing concentrations of a plasmid encoding a Flag-Drosha mutant lacking its endonucleolytic activity, and transduced with Tat, as indicated. Luciferase activity in cell extracts was measured 16 hr later. Fold activation is the ratio between values obtained for each sample and HeLa LTR-Luc mock-treated cells. Graph shows mean ± SE from 3 independent experiments. Extracts were immunoprobed with Flag or tubulin antibodies. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S4 Small TAR-Derived RNA Sequences Identified in HIV-1-Infected Cells, Related to Figure 4 Number of reads corresponding to TAR RNA sequence identified from HIV-1 infected cells analyzed by RNA-seq. Locations of the most abundant reads from TAR-5p and TAR-3p are shown on the schematic drawing of TAR at left. Sequences transfected into HeLa-LTR-luc cells (TAR1 nt 11-28 and TAR2 nt 40-58) are shown in red. Cell 2012 150, 1147-1157DOI: (10.1016/j.cell.2012.08.004) Copyright © 2012 Elsevier Inc. Terms and Conditions