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

Dysregulated Recruitment of the Histone Methyltransferase EZH2 to the Class II Transactivator (CIITA) Promoter IV in Breast Cancer Cells

One mechanism frequently utilized by tumor cells to escape immune system recognition and elimination is suppression of cell surface expression of Major Histocompatibility Class II (MHC II) molecules. Expression of MHC II is regulated primarily at the level of transcription by the Class II Transactivator, CIITA, and decreased CIITA expression is observed in multiple tumor types. We investigate here contributions of epigenetic modifications to transcriptional silencing of CIITA in variants of the human breast cancer cell line MDA MB 435. Significant increases in histone H3 lysine 27 trimethylation upon IFN-γ stimulation correlate with reductions in transcription factor recruitment to the interferon-γ inducible CIITA promoter, CIITApIV, and with significantly increased CIITApIV occupancy by the histone methyltransferase enhancer of zeste homolog 2 (EZH2). Most compelling is evidence that decreased expression of EZH2 in MDA MB 435 variants results in significant increases in CIITA and HLA-DRA mRNA expression, even in the absence of interferon-γ stimulation, as well as increased cell surface expression of MHC II. Together, these data add mechanistic insight to prior observations of increased EZH2 expression and decreased CIITA expression in multiple tumor types

Investigating in-vitro functionality and in-vivo taste assessment of eco-friendly Tadalafil Pastilles

Tadalafil (TDL) has poor bioavailability due to the less aqueous solubility and bitter taste. Oral solid dosage forms, especially tablets, have a broad market worldwide. Constraints of tablets are a long process, pollution, high processing cost, and requiring more excipient. The research was performed to optimize an eco-friendly immediate-acting pastille of TDL to put forward an alternate formulation to a tablet using advanced data mining tools. Another objective is to assess the taste masking of TDL using the Brief Access Taste Aversion (BATA) model. The amount of PEG-4000, Polyox N-10, and Kyron T-314 were chosen as critical material attributes from failure mode effect analysis. Box-Behnken design (BBD) was utilized to optimize the pastilles and ascertained the significant impact of chosen variables on disintegration time and % CDR at 10 min. The control strategy and optimal region were located using an overlay plot. The pastilles were able to release the drug within 15 min due to faster disintegration. The formulated pastilles were of uniform size, shape, and mechanical strength. The bitter taste of TDL was masked and confirmed by the BATA model. The newer formulation may be helpful in the industry due to its eco-friendly, single-step, and economical process. It unlocks a new direction in the field of oral solid dosage form as an alternative to tablets

EZH2 knockdown significantly increases mRNA levels of CIITApIV and <i>HLA-DRA</i> but not CIITApIII in MDA-MB 435 variants.

<p>(A–B) MDA MB 435 variants were transfected with either EZH2 specific siRNA or with control siRNA and were stimulated as indicated with IFN-γ. Levels of <i>HLA-DRA</i> mRNA and CIITApIV mRNA (A) and levels of CIITApIII mRNA (B) were measured by Q-PCR and were normalized to levels of GAPDH mRNA. Q-PCR was performed in triplicate and results shown represent the mean ± SD of three independent experiments. ***P<0.0005, **P<0.005, *P<0.05.</p

Decreased levels of CIITA isoform III and isoform IV mRNA correlates with low levels of acetylation.

<p>(A–B) HeLa and MDA MB 435, 435-Brain 1, and 435-Lung 2 cells were treated as indicated with IFN-γ. Levels of CIITApIII mRNA (A) and CIITApIV mRNA (B) were measured by Q-PCR. Results shown represent the mean ± SD of four independent experiments. (C–D) Levels of acetylated H3 (C) and acetylated H4 (D) at CIITApIV in MDA MB 435 and HeLa cells. ChIP assays were carried out in cells stimulated as indicated with IFN-γ. Lysates were IP with control antibody, with antibody to acetylated H3, (C) or with antibody to acetylated H4 (D) and associated DNA was isolated and analyzed via Q-PCR using primers spanning the IRF-E-GAS box of CIITApIV. Values for control IPs and acetylated H3 (C) and acetylated H4 (D) IPs represent the mean ± SEM of three independent experiments. Control IP values for (C) and (D) were 0.6±0.5 *** P<0.0005, **P<0.005, *P<0.05.</p

IFN-γ stimulation results in specific and significantly increased binding of EZH2 to CIITApIV in MDA MB 435 cells.

<p>(A) EZH2 mRNA expression. MDA MB 435, 435-Brain 1, 435-Lung 2, and HeLa cells were stimulated as indicated with IFN-γ. Cell lysates were analyzed for levels of EZH2 mRNA as above. Q-PCR was performed in triplicate and results represent the mean ± SD of three independent experiments. (B–C) Levels of EZH2 at <i>HLA-DRA</i> (B) and CIITApIV (C) promoters post cytokine stimulation. Double crosslinking ChIP assays were carried out in cells stimulated as indicated with IFN-γ. Lysates were IP with control antibody or with antibody to EZH2 and associated DNA was isolated and analyzed via Q-PCR using primers spanning the W-X-Y box of <i>HLA-DRA</i> (B) and using primers spanning the IRF-E-GAS box of CIITApIV (C). Values for control IPs and for and EZH2 IPs represent mean ± SEM of three independent experiments. Control IP values for (A) and (B) were 2.1±0.5 ***P<0.0005. (D) DNA methylation analyses of CIITApIV. Sequence analysis of CIITApIV region 2 of MDA MB 435 cells and HeLa cells indicates no differences in CpG island methylation. Primers spanning region 2 of CIITApIV were used to amplify DNA and purified PCR product was sequenced and aligned with cells lines. Arrows indicate unmethylated CpG sites. Boxes indicate methylated CpG sites conserved between MDA MB 435 cells and HeLa cells.</p

Decreased recruitment of STAT-1 and IRF-1 to CIITApIV in metastatic breast cancer cells.

<p>(A–B) ChIP assays were carried out in MDA MB 435 variants and HeLa cells stimulated as indicated with IFN-γ. Lysates were IP with control antibody, with antibody to STAT-1 (A), or with antibody to IRF-1 (B), and associated DNA was analyzed as above. IP values shown are presented as increases in CIITApIV promoter DNA relative to unstimulated STAT-1 (A) and IRF-1 (B) IP samples. Values for control IPs, STAT-1 IP's, and IRF-1 IPs represent mean ± SEM of three independent experiments. Control IP values for (A) and (B) were 0.2±0.3.</p

Decreased levels of CIITApIV H3K18ac, H3K27ac and CBP correlate with significantly elevated levels of H3K27me3 in metastatic breast cancer cells.

<p>(A–E) Levels of acetylated H3K18, CBP, H3K9me3, H3K27ac, and H3K27me3 at CIITApIV. ChIP assays were carried out in MDA MB 435, 435-Brain 1, 435-Lung 2, and HeLa cells stimulated as indicated with IFN-γ. Lysates were IP with control antibody or antibody to indicated proteins and associated DNA was isolated and analyzed as above. Values shown represent mean ± SEM of three to five independent experiments. Control IP values for (A–E) were 1.3±0.7 ***P<0.0005, **P<0.005.</p

EZH2 knockdown decreases CIITApIV histone H3K27 trimethylation.

<p>(A–B) EZH2 knockdown is efficient and specific. MDA MB 435, 435-Brain 1, and 435-Lung 2 cells were transfected with either EZH2 specific siRNA or with control siRNA. 10% of lysates were subjected to immunoblot (IB) for EZH2 and tubulin. Results reported are representative data from three independent experiments. The remaining lysates were subjected to EZH2 mRNA isolation and quantitation by Q-PCR. Q-PCR was performed in triplicate and results shown represent the mean ± SD of three independent experiments. (C) Levels of trimethylated H3K27 in breast cancer cells. ChIP assays were carried out in MDA MB 435 variants transfected with either EZH2 siRNA or control siRNA and stimulated as indicated with IFN-γ. Lysates were IP with control antibody or with antibody to H3K27me3 and associated DNA was isolated and analyzed as above via Q-PCR using primers spanning the IRF-E-GAS box of CIITApIV. Values shown represent mean ± SEM of three independent experiments. Control IP values were 1.2±0.6 ***P<0.0005, **P<0.005.</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