Nuclear Myosin VI Enhances RNA Polymerase II-Dependent Transcription Sarah Vreugde, Carmelo Ferrai, Annarita Miluzio, Ehud Hauben, Pier Carlo Marchisio, Massimo P. Crippa, Mario Bussi, Stefano Biffo  Molecular Cell  Volume 23, Issue 5, Pages 749-755 (September 2006) DOI: 10.1016/j.molcel.2006.07.005 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Myosin VI Is Present in the Nucleus and Associates with Nascent Transcripts and RNA Polymerase II (A) Immunolocalization of myosin VI in HeLa cells. Confocal images of cells, stained with Hoechst to visualize the nuclei (left column), with an affinity-purified anti-myosin VI antibody (middle column, top) and merged Hoechst/myosin VI (overlay, right column). The anti-myosin VI antibody stains both the nucleus and the cytoplasm. The nuclear myosin VI is distributed throughout the nucleoplasm in many discrete foci. Similar localization patterns are observed in cells transfected with a GFP-Myo VI fusion construct (middle row). Preadsorption of the anti-myosin VI antibody with the immunizing peptide abolishes the myosin VI-specific staining (bottom row). Scale bar, 20 μm. Asterisk on nontransfected cell. (B) Antibody to myosin VI recognizes a 150 kDa protein in the nucleus and cytoplasm. A protein immunoblot analysis with this antibody was performed on nuclear and cytoplasmic extracts from PC3 and HeLa cells. Lamin B, nuclear marker on 10% SDS-PAGE; LDH, cytosolic marker on 12% SDS-PAGE. (C) Nascent transcripts colocalize with myosin VI. Localization of myosin VI (green, left) relative to BrUTP (red, middle) in permeabilized HeLa cells. Most of the BrUTP foci colocalize with myosin VI (yellow in overlay, right). Scale bar, 10 μm. (D) Coimmunoprecipitation of myosin VI and RNAPII, using an antibody to myosin VI or to RNAPII to immunoprecipitate respective proteins from HeLa cells. Myosin VI and RNAPII coimmmunoprecipitated in both conditions, but not when a preimmune serum was used for immunoprecipitation. The migration of myosin VI and RNAPII in a total protein extract (input, 5%) is shown for comparison. (E) Coimmunoprecipitation of GFP-myosin VI and RNAPII. Anti-GFP antibodies were used to immunoprecipitate, respectively, GFP-myosin VI and GFP from transfected cells. Immunoblot analysis was conducted with antibodies to GFP or to RNAPII. The migration of RNAPII and GFP-myosin VI or GFP (Figure S1) in total protein extracts from transfected cells is shown for comparison (input, 5%). Coimmunoprecipitation is demonstrated by the presence of RNAPII-specific bands in GFP-myosin VI-immunoprecipitated complexes and absence of GFP-myosin VI and RNAPII-specific bands in the immunoprecipitation of GFP-transfected control cells. Molecular Cell 2006 23, 749-755DOI: (10.1016/j.molcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Transcriptional Arrest Induces a Redistribution of Nuclear Myosin VI and Impedes Its Interaction with RNAPII (A) HeLa cells were cultured in the presence or absence of actinomycin D or α-amanitin. After fixation, cells were stained with antibodies to myosin VI (top row) and to Ser2 or Ser5-phosphorylated fractions of RNAPII (middle row). Partial colocalization (yellow color in the overlay) of myosin VI and Ser2/Ser5-RNAPII can be observed in control cells (yellow in lower row), but upon transcriptional arrest, nuclear myosin VI is redistributed into rounded minimal domains (arrowhead) localized adjacent to Ser2/Ser5-RNAPII dots/speckles. Treatment with α-amanitin induces a redistribution of Ser2/Ser5-RNAPII in speckles and/or diminished levels of immunoreactivity, as described (Bregman et al., 1995; Szentirmay and Sawadogo, 2000). (B) Absence of coimmunoprecipitation of myosin VI and RNAPII in conditions of transcriptional arrest. RNAPII was detected in the immunocomplexes when an antibody to myosin VI was used for immunoprecipitation in nontreated cells (lane 2), but not when a preimmune serum was used for immunoprecipitation (lane 3). No coimmunoprecipitation of RNAPII and myosin VI was detected when antibodies to myosin VI (lane 4 and 5) or a preimmune serum (lane 6 and 7) were used to immunoprecipitate proteins from actinomycin D or α-amanitin-treated cells. The migration of myosin VI and RNAPII in a total protein extract (input, lane 1) is shown for comparison. Molecular Cell 2006 23, 749-755DOI: (10.1016/j.molcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Myosin VI Associates with Selected Gene Promoters and Intragenic Regions (A–E) ChIP was performed as described, and QT-PCR amplification was performed with primers defining, respectively, the uPA, LDLR, p27BBP/eIF6, and p21WAF1 promoter and intragenic regions. Results and error bars shown are the average and standard deviation of at least three independent PCRs from three independent ChIP experiments. Values are relative to immunoprecipitated input DNA. Immunoprecipitated genomic DNA is enriched in the promoter and/or intragenic regions of the uPA, LDLR, and p27/eIF6 genes, when antibodies to phosphorylated RNAPIIser2 and RNAPIIser5 fractions and to myosin VI are used. No enrichment is seen when unrelated antibodies are used (Ab) or when antibodies are omitted for immunoprecipitation (Mock). RNAPIIser2, but not RNAPIIser5, fractions are significantly enriched in intragenic regions as compared to promoter regions of all genes tested, as described (Komarnitsky et al., 2000). In these conditions, we were consistently unable to detect p21WAF1-specific DNA when antibodies to myosin VI were used for immunoprecipitation. Molecular Cell 2006 23, 749-755DOI: (10.1016/j.molcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Myosin VI Modulates RNAPII-Dependent Transcription (A) Western blot showing inhibition of myosin VI expression in PC3 cells transiently transfected with 5 nM of two different MyoVI-siRNA oligos, relative to cells transfected with equal amounts of negative control siRNAs, and with a pIRES-EGFP-myosin VI antisense sequence (pIRES-EGFP-MyoVI-AS), compared to pIRES-EGFP control vector. Blotting for actin as a control. (B) mRNA levels of uPA, LDLR, and p27/eIF6 in pIRES-EGFP-MyoVI-AS or different MyoVI-siRNA-transfected PC3 cells are significantly lower than mRNA levels of pIRES-EGFP or scrambled control siRNA-transfected cells arbitrarily set to 1. Error bars represent the standard deviation of three independent experiments. For all tests, p values are <0.01 (two-tailed t test). mRNA levels of the p21WAF1 gene are not significantly affected (p = 0.42; two-tailed t test) by transfection with pIRES-EGFP-MyoVI-AS vector. (C) In vitro run-off transcription is inhibited by antibodies to myosin VI. Transcription of a specific 400 nucleotide (nt) RNA was significantly inhibited (p < 0,01, two-tailed t test) in the presence of antibodies to myosin VI as compared to control samples (control) in a dose-dependent way and as compared to transcription in the presence of 4 μg of preadsorbed antibodies (pep ads anti-Myo VI) or denatured antibodies. Preadsorbing 4 μg of myosin VI antibodies with the immunizing peptide partially, but significantly, eliminated the inhibition (p = 0.006, two-tailed t test). This experiment was repeated three times, and the mean fractional transcription activities compared with the control are shown at the bottom, as quantified by a phosphorimager. Molecular Cell 2006 23, 749-755DOI: (10.1016/j.molcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions