1. Volume 11, Issue 4, Pages 1055-1066 (April 2003)
Methylation of SPT5 Regulates Its Interaction with RNA Polymerase II and Transcriptional Elongation Properties 
Youn Tae Kwak, Jun Guo, Shashi Prajapati, Kyu-Jin Park, Rama M. Surabhi, Brady Miller, Peter Gehrig, Richard B. Gaynor 
Molecular Cell 
Volume 11, Issue 4, Pages (April 2003)
DOI: /S (03)
Copyright © 2003 Cell Press Terms and Conditions

2. Figure 1
Analysis of SPT5-Associated Proteins and MALDI Mass Spectrum of Trypsin-Digested Wild-Type SPT5 Protein
(A) After transfection of 293 cells with an expression vector encoding Flag-SPT5 or the parental expression vector alone, extracts (SPT5-Flag and control) were prepared and subjected to affinity chromatography using beads coupled with Flag antibody. Eluates were analyzed by SDS-polyacrylamide gel electrophoresis and silver staining. The positions of SPT5, PRMT5, and SPT4 were determined by Western blot analysis with specific antibodies directed against these proteins.
(B) Affinity-purified Flag-SPT5 and associated proteins were digested with trypsin and precipitated with TCA, and the peptides were analyzed by an LCQ-DECA ion-trap mass spectrometer and compared against a human database. The peptide sequences that were identified are included in the table.
(C) The molecular mass of a tryptic peptide extending between residues 669 and 700 in SPT5 detected with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is shown. The arginine residues found to be dimethylated are shown in bold and are underlined. The MALDI mass spectrum of the SPT5 peptide extending from residue 669 to 700 is shown with signals consistent with either three dimethylarginines (DMA) (m/z ; calculated m/z: ) or two dimethylarginines and one monomethylarginine (MMA) (m/z ; calculated m/z: ). The signals at m/z and can be assigned to fragment ions resulting from the gas phase loss of dimethylamine from an asymmetric methylarginine (aDMA; calculated m/z: ) and of methylamine from symmetric methylarginine (sDMA; calculated m/z: ), respectively. The m/z values of these postsource fragment ions were then calculated.
Molecular Cell  , DOI: ( /S (03) )
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3. Figure 2 Association of the SPT5 Protein with PRMT1 and PRMT5
(A) PRMT1, PRMT3, PRMT4, and PRMT5 proteins were immunoprecipitated from 293 cell extracts following transfection of expression vectors encoding each of these Flag-tagged proteins. These proteins were used in in vitro methylation assays with a GST-SPT5 fusion protein extending from residues 519 to 755 (top panel). Western blot analysis of these epitope-tagged PRMT proteins was performed with a monoclonal antibody (1:2000) directed against the Flag epitope.
(B) Wild-type or point mutants in either PRMT1 (G80R) (lanes 1 and 2, top panel) or PRMT5 (R368A) (lanes 3 and 4, top panel) were transfected into 293 cells, immunoprecipitated with Flag antibody (1:300), and analyzed in in vitro methylation assays. The expression of the PRMT proteins was determined by Western blot analysis with antibody directed against the Flag epitope (lower panel).
(C) In vivo methylation of endogenous SPT5 by wild-type and mutant PRMT1 and PRMT5 proteins in the presence of S-adenosyl-L-[methyl-3H]methionine was analyzed in nontransfected 293 cells (lane 1) or in cells transfected with expression vectors encoding either wild-type (lanes 2 and 3) or mutant (lanes 4 and 5) PRMT1 and PRMT5 (top panel). Quantitation of SPT5 methylation is shown under the specific lanes. The expression of endogenous SPT5 and the Flag-tagged PRMT proteins are indicated (lower panels).
(D) The association of endogenous SPT5 protein with PRMT1, PRMT3, PRMT4, and PRMT5 was analyzed following immunoprecipitation of HeLa extracts (500 μg) with antibodies directed against either the indicated PRMT proteins (1:200) or control immunoglobulin and Western blot analysis (1:1000) was performed as indicated (top panel). The expression of SPT5, PRMT1, PRMT3, PRMT4, and PRMT5 was analyzed by Western blot (middle and lower panels).
(E) Immunoprecipitation of HeLa lysates with SPT5 antibody (1:200) or control immunoglobulin was performed followed by Western blot analysis with the indicated PRMT antibodies (1:1000).
(F) A schematic of the SPT5 protein is shown. An analysis of the domains in SPT5 that interact with either PRMT5 or PRMT1 was performed by transfecting expression vectors encoding either Myc-tagged PRMT5 or Myc-tagged PRMT1 and the indicated Flag-tagged SPT5 constructs into 293 cells (left panel). Extracts were prepared and immunoprecipitated with Flag antibodies (1:300), and Western blot analysis was performed with the anti-Myc antibody to detect the Myc-tagged PRMT5 (left top panel) or PRMT1 (right top panel). The bottom panels indicate a control immunoprecipitation with anti-HA (1:200), and a Western blot of the extracts transfected with the Flag-SPT5 constructs, Myc-PRMT5, and Myc-PRMT1 is shown (right panel).
Molecular Cell  , DOI: ( /S (03) )
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4. Figure 3 SPT5 Is Methylated In Vitro by the PRMT1 and PRMT5 Proteins
(A) Affinity-purified PRMT5 (top panel) and PRMT1 (middle panel) proteins (10 ng) isolated from 293 cell extracts were used in in vitro methylation assays with wild-type or the indicated GST-SPT5 mutants (10 μg) as well as MBP as substrates in the presence of S-adenosyl-L-[methyl-3H]methionine (lanes 1–8). The arrows indicate the positions of the methylated GST-SPT5 proteins. A Coomassie stain of the GST-SPT5 substrates is shown in the lower panel, and the arrows indicate the position of each of these substrates. (B) Either wild-type or mutants of specific arginine mutants using either truncated GST-SPT5 proteins (aa 519–755) or (C) full-length GST-SPT5 proteins were used in in vitro methylation assays with the affinity-purified PRMT5 and PRMT1 proteins (top and middle panels). A Coomassie stain of each substrate is also shown (lower panel). (D) The position of potential arginine methylation sites in the SPT5 protein are shown with arginine residues 681, 696, and 698 that were found to be methylated by either PRMT1 or PRMT5 indicated in bold. Arginine residues 747 and 749 in the RQR sequence were also mutated. Asterisks and filled circles over specific arginine residues indicate the site of PRMT1 and PRMT5 methylation, respectively. (E) A comparison of the regions of SPT5 in proximity to the C-terminal KOW domains with potential sites of PRMT-directed arginine methylation in several different species is indicated.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

5. Figure 4
TNFα Treatment Leads to Enhanced SPT5 Association and Decreased PRMT1 and PRMT5 Association with Cytokine-Inducible Promoters
(A and B) HeLa cells were treated with TNFα (10 ng/ml), and cells were harvested at 0, 15, 30, and 60 min. ChIP assays were performed using the indicated antibodies followed by PCR and ethidium bromide staining on both the promoter elements and open reading frames of the (A) IκBα and (B) IL-8 genes. Immunoprecipitation with a control immunoglobulin and the PCR analysis of the input DNAs in the absence of immunoprecipitation are also indicated. PCR reactions using 10%, 1%, 0.1%, and 0.01% of input DNAs were loaded to determine the linear range of the signal.
(C) Quantitative analysis of these ChIP assays was performed by calculating the amount of the immunoprecipitated IκBα and IL-8 promoter DNAs bound to the indicated factors as a percentage of these input DNAs at various times following TNFα treatment.
(D) The levels of IκBα and IL-8 transcripts were monitored using RT-PCR with GAPDH used as a control. Quantitation of each experimental set is indicated under the specific lanes.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

6. Figure 5
In Vitro Transcription Analysis of Wild-Type and SPT5 Methylation Mutants
(A) Immunodepletion of SPT5 from HeLa nuclear extract was performed using either IgG- (lane 1) or SPT5-specific antisera (lane 2) followed by Western blot analysis with antibodies (1:1000) directed against SPT5, CDK9, RNA polymerase II (8WG16), CDK7, and TFIIB.
(B) Affinity-purified wild-type and the different SPT5/SPT4 mutant proteins were analyzed by SDS-polyacrylamide gel electrophoresis (left panel), and Western blot analysis of the affinity-purified wild-type and mutant Flag-SPT5 proteins and the associated Myc-tagged SPT4 is shown (lanes 1–5, right panel).
(C) In vitro transcription analysis was performed in the presence or absence of DRB with the pTF3 adenoviral template containing a G-less cassette using untreated HeLa nuclear extract (lanes 1 and 2), the SPT5-immunodepleted HeLa nuclear extract assayed either alone (lanes 3 and 4) or following the addition of SPT4, and the wild-type or mutant affinity-purified SPT5 proteins (20 ng) (lanes 5–16).
(D) In vitro transcription analysis was performed with the HIV-1 LTR wild-type (top panel) or the HIV-1 LTR loop mutant (lower panel) templates using HeLa nuclear extract (lanes 1 and 2), the SPT5-immunodepleted HeLa nuclear extract alone (lanes 3 and 4), or the SPT5-immunodepleted HeLa nuclear extract in the presence of affinity-purified SPT4 and wild-type or mutant SPT5 proteins (20 ng) (lanes 5–16). Tat (25 ng) was added to the odd-numbered lanes, and GST (25 ng) was added to the even-numbered lanes in the in vitro transcription assays. Following the in vitro transcription analysis for both the pTF3 and HIV-1 templates containing G-less cassettes, the labeled RNAs were digested with RNase T1 and gel electrophoresis, and autoradiography were performed. Quantitation of three different in vitro transcription assays performed under similar conditions is also shown in (C) and D).
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

7. Figure 6 PRMT1 and PRMT5 Repress Tat-Activated HIV-1 Gene Expression
(A) Either wild-type or (B) a TAR loop mutant HIV-1 LTR luciferase reporter construct (0.1 μg) was transfected into HeLa cells with different amounts (0.1–3.0 μg) of CMV expression vectors encoding PRMT1 or PRMT5 in the presence and absence of an expression vector encoding Tat (0.1 μg). The luciferase activity was normalized for differences in transfection efficiency using an RSV-β-galactosidase reporter construct. (C) A wild-type HIV-1 LTR luciferase reporter and a CMV expression vector encoding Tat were transfected into HeLa cells in the presence of wild-type or mutant PRMT1 and PRMT5 (0.1–3.0 μg), and luciferase activity was determined. (D) A thymidine kinase luciferase vector (0.1 μg) was cotransfected into HeLa cells with various quantities of expression vectors encoding wild-type or point mutants of PRMT1 and PRMT5 (0.1–3.0 μg), and luciferase activity was determined. (E) An HIV-1 LTR luciferase reporter construct was transfected into HeLa cells with or without a CMV expression vector encoding Tat. At 24 hr posttransfection, adenosine dialdehyde was added at the indicated concentrations, and luciferase activity was measured. (F) A similar experiment was performed as in (E), but expression vectors either coding PRMT1 or PRMT5 were also transfected, and luciferase activity was determined. (G) A thymidine kinase luciferase vector (0.1 μg) was transfected into HeLa cells in either the presence or absence of adenosine dialdehyde (10 μM), and the luciferase activity was determined. (H) An HIV-1 proviral construct was transfected into 293T cells, and 1 day later adenosine dialdehyde was added. After 24 hours, the culture supernatants were collected and assayed for p24 antigen by ELISA. The plotted data were averaged from three independent experiments ± standard deviation.
Molecular Cell  , DOI: ( /S (03) )
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8. Figure 7
SPT5 Methylation Mutants Exhibit Increased Association with RNA Polymerase II
(A) The in vivo methylation of endogenous (lane 1 and 2) or transfected (lane 3 and 4) SPT5 was determined following treatment of HeLa cells with adenosine dialdehyde (10 μM) for 12 hr, labeling with S-adenosyl-L-[methyl-3H] methionine, and autoradiography (top panel). Expression of the SPT5 proteins was determined by Western blot analysis (lower panel).
(B) HeLa cells were treated with adenosine dialdehyde (10 μM) for 12 hr and then treated with TNFα for the indicated times. ChIP assays were performed using the indicated antibodies to determine the association of the SPT5, PRMT1, PRMT5, RNA polymerase II, and p65 proteins to the IL-8 promoter followed by PCR analysis and ethidium bromide staining.
(C) HeLa cells were transfected with wild-type Flag-SPT5 (left panel) or the SPT5 mutant (R681/696/698K), and following TNFα treatment (10 ng/ml), ChIP assays were performed with the IL-8 promoter as described. Flag-tagged SPT5 proteins were immunoprecipitated with antibody directed against the Flag epitope in the CHIP assays, and their expression was determined by Western blot analysis with Flag antibody.
(D) 293 cells were either untransfected (lanes 1 and 2) or transfected with an expression vector encoding Flag-tagged SPT5 and treated with 25 μM of adenosine dialdehyde for 48 hr. Immunoprecipation of these extracts was then performed with either polyclonal anti-SPT5 (lanes 1 and 2) or anti-Flag (lanes 3 and 4) followed by Western blot analysis with 8WG16 antibody. Western blot analysis of these extracts with 8WG16 to detect RNA polymerase II and either anti-SPT5 (lanes 1 and 2) or anti-Flag antibody was performed.
(E and F) Flag-tagged SPT5 cDNAs encoding either wild-type or mutants were cotransfected with Myc-SPT4 into 293 cells. Following immunoprecipitation with anti-Flag monoclonal antibody (1:300), the cell lysates were subjected to Western blot analysis with either (E) 8WG16, H-5, or H-14 monoclonal antibodies (1:1000) directed against the RNA polymerase II CTD or anti-Myc (1:1000) to detect SPT4 or (F) either anti-CDK9 (1:1000) or anti-cyclin T1 (1:1000). Quantitation of the binding of RNA polymerase II, CDK9, and cyclin T1 is shown under the specific lanes (top panels). Western blot analysis was performed with specific antibodies directed against RNA polymerase II, CDK9, cyclin T1, Myc-SPT4, or Flag-SPT5 (lower panels).
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions