Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes

Accumulating evidence shows the 26S proteasome is involved in the regulation of gene expression. We and others have demonstrated that proteasome components bind to sites of gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear, although prior observations suggest both proteolytic and non-proteolytic activities. Here, we define novel, non-proteolytic, roles for each of the three 19S heterodimers, represented by the 19S ATPases Sug1, S7, and S6a, in mammalian gene expression using the inflammatory gene CIITApIV. These 19S ATPases are recruited to induced CIITApIV promoters and also associate with CIITA coding regions. Additionally, these ATPases interact with elongation factor PTEFb complex members CDK9 and Hexim-1 and with Ser5 phosphorylated RNA Pol II. Both the generation of transcripts from CIITApIV and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by siRNA mediated knockdown of these 19S ATPases. Together, these results define novel roles for 19S ATPases in mammalian gene expression and indicate roles for these ATPases in promoting transcription processes

Knockdown of 19S ATPases does not activate the heat shock response.

<p>HeLa cells were transfected with HSE-Luciferase reporter, control siRNA, or S6a siRNA and were treated with MG132 six hrs prior to harvest. Cells were harvested following 48 hrs of incubation, lysed in cell lysis buffer, and analyzed by Luciferase assay. Luciferase readings obtained were normalized by Bradford assay. Data shown represents values obtained from three independent experiments. The negative control is a mixture of non-inducible reporter construct and constitutively expressing Renilla luciferase construct. The positive control is an inducible transcription factor-responsive construct expressing firefly luciferase, and a constitutively expressing Renilla luciferase construct.</p

Reduced expression of 19S ATPases via siRNA negatively impacts the generation of long transcripts from CIITA pIV.

<p>(A–B, D–E, G–H) Cells were transfected with siRNA, and mRNA was quantitated using CIITA mRNA primers and probes specific for transcripts from CIITA exon IV and exon VII. CIITA mRNA generated was normalized to GAPDH. Data shown represent the mean ± SEM of three biologically independent experiments. (C, F, I) Expression of Sug1, S7, and S6a was specifically decreased using ATPase specific siRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091200#pone.0091200.s001" target="_blank">Figure S1A, S1B, and S1C</a>). Blots shown are representative of three biologically independent experiments.</p

The 26S proteasome is composed of a 20S proteolytic core capped on one or both ends by 19S regulatory particle.

<p>The 20S core is a hollow cylindrical structure composed of two heptameric rings of α-subunits and two heptameric rings of β-subunits. The 19S regulatory particle is composed of a base and lid component. The lid component consists of nine non-ATPase subunits and the base is composed of six ATPases (S7, S4, S6a, S10b, Sug1 and S6b) and three non-ATPases (S1, S2, and S5b). Polyubiquitinated proteins are recognized, deubiquitinated, and unfolded by the 19S regulatory particle and the unfolded proteins are translocated to the 20S core where proteins are degraded into small peptides.</p

19S ATPases associate with the CIITA pIV proximal promoter.

<p>(A, C,E) ChIP assays were carried out in HeLa cells stimulated with IFN-γ for 0–2 hrs. Cell lysates were immunoprecipitated (IP’d) with control antibody or with antibody to endogenous 19S ATPase S6a, Sug1, or S7 and associated DNA was isolated and analyzed by real-time PCR using primers and probe spanning the CIITApIV proximal promoter. Real time PCR values were normalized to the total amount of DNA in the reaction (Input). IP values are represented as ATPase binding to CIITApIV promoter DNA relative to unstimulated samples. (B,D,F) ChIP signal at the inactive gene CD4. The control IgG values were 0.004±0.001. Values for control IgG and either Sug1 IP, S7 IP or S6a IP represent the mean ± SEM of three biologically independent experiments * p<0.05.</p

19S ATPases bind CIITA pIV within the coding region.

<p>(A–I) ChIP assays were carried out in HeLa cells stimulated with IFN-γ for 0–2 hrs. Cell lysates were IP’d with control antibody or with antibody to endogenous Sug1 (A and B), S7 (D and E), or S6a (G and H) and associated DNA was isolated and analyzed by real-time PCR using primers and probes spanning CIITApIV exon IV (A, C, E) and exon VII (B, D, F). Real time PCR IP values were normalized to the total amount of DNA (input); IP values are represented as ATPase binding to CIITApIV exon IV or exon VII DNA relative to unstimulated samples. (C,F,I) ChIP signal at the inactive gene CD4. The control IgG values were 0.005±0.001. Values for control and IP represent mean ± SEM of three biologically independent experiments. *p<0.05, **p<0.005. G. Mobility shift assay of Sug1 with a 90 nucleotide single stranded DNA on a native 8% polyacrylamide gel with a tris-borate magnesium running buffer; 0.7<b> </b>µM DNA, 0.85 µM sug1, and 500 µM ATP. DNA was visualized with SYBER Green II dye.</p

19S ATPases associate with elongation factors Hexim and CDK9.

<p>(A–C) HeLa cells were co-transfected with Myc tagged S6a, S7, or Sug1 and Flag tagged Hexim or HA tagged CDK9 as indicated. Cells were lysed and IP’d with Myc beads (lane 1) as a positive control, mouse isotype IgG (lane 2) as a negative control, flag beads (lane 3), and HA beads (lane 4). IP samples (top panel) and lysates (bottom panel) were IB’d for Myc, Flag, and HA as indicated. (D–E) HeLa cells were lysed and IP’d with either Hexim or CDK9 (lane 1) as a positive control, mouse isotype IgG (lane 2) as a negative control, or with S6a (lane 3), S7 (lane 4), and Sug1 (lane 5). IP samples (top panel) and lysates (bottom panel) were IB’d for Hexim or CDK9 as indicated. Results shown are indicative of data from three biologically independent experiments.</p

19S ATPases associate with Ser5 phosphorylated RNA pol II (Ser5p-RNA pol II).

<p>(A) HeLa cells were transfected with myc tagged S6a, S7 or Sug1 as indicated. Cells were lysed and IP’d with myc beads (first lane, top panels) as a positive control, with mouse isotype IgG (second lane, top panels) as a negative control, and with Ser5p-RNA Pol II antibody (third lane, top panels). IP’d samples (top panels) and lysates (middle and bottom panels) were IB for myc ATPases or for Ser5p-RNA pol II as indicated. (B) HeLa cells were lysed and IP’d with Ser5p-RNA Pol II (lane 1) as a positive control, mouse isotype IgG (lane 2) as a negative control, or with S6a (lane 3), S7 (lane 4), or Sug1 (lane 5). IP samples (top panel) and lysates (bottom panel) were IB’d Ser5p-RNA Pol II as indicated. Results shown are indicative of data from three biologically independent experiments.</p

Reduced expression of 19S ATPases decreases TBP recruitment to the CIITApIV proximal promoter.

<p>(A, C, E) ChIP assays were carried out in HeLa cells transfected with ATPase specific or with control siRNA and stimulated with IFN-γ for 0–2 hrs. Cell lysates were crosslinked, sonicated, lysed, and IP’d with either antibody against endogenous TBP or with control antibody (IgG). Associated DNA was analyzed via real-time PCR using primers and probe specific for the CIITApIV proximal promoter. Real time PCR IP values were normalized to total amount of reaction DNA (Input). The values for control IP and TBP IP are representative data *p<0.05, **p<0.005, ***p<0.0005 versus control siRNA. (B, D, F) ChIP signal at the inactive gene CD4. Sug1, S7, and S6a protein expression was effectively decreased using ATPase specific siRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091200#pone.0091200.s004" target="_blank">Figure S4A, S4B, and S4C</a>).</p

Effects of 19S ATPase knockdown on CIITApIV transcription are independent of effects on degradation.

<p>(A–B, D–E) HeLa cells were stimulated with IFN-γ as indicated and were harvested four hrs post treatment with 10 µM MG132 or 10 µM Lactacystin. mRNA was extracted and cDNA was generated using indicated reverse primers followed by amplification via real-time PCR. CIITA mRNA transcripts were obtained using primers and probes specific for CIITA exon IV and exon VII were normalized to GAPDH. (C) 18S rRNA transcripts for control and MG132 treated cells were obtained using primers and probe specific for 18S rRNA and were normalized to GAPDH. The 18 hr control sample was set to 100%. Data shown represents the mean ± SEM of three biologically independent experiments.</p