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immunodeficiency

By Rick Gonzalez,2014-02-06 11:10
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immunodeficiency

Supplemental Data

    Supplemental Experimental Procedures

    Materials

    We purchased rabbit antibodies against acetyl lysine (Cell Signaling, Danvers, MA), CDK9, cyclin T1, HA (Roche, Indianapolis, IN), p300 (Abcam, Cambridge, MA), tubulin ;, and FLAG (M2) (each from Sigma-Aldrich, St. Louis, MO). TSA, NA, DRB, HMBA, and actinomycin D were from Sigma-Aldrich, TNF; from BioSource

    International (Camarillo, CA). Rabbit anti-Hexim1 antibody was a gift from Q. Zhou (UC Berkeley) and O. Bensaude (Ecole Normale Supérieure, Paris). HA-cyclin T1 plasmids were kindly provided by K. Jones (Salk Institute), cyclin T2 plasmids by D. Price (University of Iowa), FLAG-tagged CDK9 by A. Rice (Baylor College of Medicine), the 5xUAS construct by B. Spiegelman (Harvard Medical School), MYC-tagged p300 by M. Stallcup (USC), constructs encoding wild-type and C-terminally deleted GST-cyclin T1 proteins as well as wild-type Gal4-cyclin T1 plasmids by B. M. Peterlin (UCSF). The IL-8 reporter plasmid (Ott, M., Lovett, J.L., Mueller, L. and Verdin, E. (1998) Superinduction of IL-8 in T cells by HIV-1 Tat protein is mediated through NF-kappaB factors. J Immunol, 160, 2872-2880.) , HIV LTR luciferase reporter and the

    Tat expression vector (Kwon, H.S., Brent, M.M., Getachew, R., Jayakumar, P., Chen, L.F., Schnolzer, M., McBurney, M.W., Marmorstein, R., Greene, W.C. and Ott, M. (2008) Human immunodeficiency virus type 1 Tat protein inhibits the SIRT1 deacetylase

    , 158-167.) were previously and induces T cell hyperactivation. Cell Host Microbe, 3

    described by our laboratory. Cyclin T1 mutations were constructed with full-length HA-cyclin T1 using the Quick-change mutagenesis kit (Stratagene, La Jolla, CA). All mutant constructs were analyzed by dideoxynucleotide sequencing to ensure that the proper

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    mutations were present before further experiments. Mutant HA-cyclin T1 constructs were digested with BamH1 treated with Klenow enzyme (New England Biolabs, Beverly, MA), followed by digestion with ClaI. The fragment was cloned into the blunted Xba1 and regular ClaI site within the Gal4-cyclin T1 construct.

Antibody generation

    Chemically synthesized 11-mer K404-acetylated peptides were conjugated to keyhole limpet hemocyanin (Thermo Fisher Scientific, Rockford, IL), mixed with Freund’s

    adjuvant and injected four times into rabbits (Covance, Madison, WI). Antigen-specific immunoglobulin G fractions were purified using K404-acetylated peptide conjugated with Affigel 10 matrix (Biorad, Hercules, CA) according to the manufacturer’s

    instructions.

Real time RT-PCR

    Total RNA was isolated using the RNA STAT-60 kit (TEL-TEST INC, Friendswood, TX) from HeLa cells transfected by wild-type or 4R mutant cyclin T1 with or without treatment of TNF; (2 ng/ml) for 5 h. 2 g of total RNA was used to generate cDNA with

     kit (Invitrogen). Human IL-8 mRNA was the Superscript III reverse transcriptase

    quantified by QuantiTect gene expression assays (Qiagen, Valencia, CA) and SYBR Green I master mix (MCLab, South San Francisco, CA). To calculate IL-8 mRNA copy numbers, ten-fold serial dilutions of the IL-8 cDNA were prepared at concentrations of

    97 × 10 to 7 × 10 copies per reaction and used for real-time RT-PCR analysis along with RNA samples from HeLa cells. Relative copies of IL-8 RNA were determined based on

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    these standard curves. Real-time RT-PCR was performed in duplicate on three independent samples using ABI PRISM 7700 thermocycler (Applied Biosystems, Foster City, CA). P values (paired t-test) were used for statistical analysis. Mass spectrometry

    Synthetic peptides were purchased from Peptide Specialty Laboratories GmbH (Heidelberg, Germany). For MALDI-TOF MS nonacetylated and in vitro acetylated

    peptides were desalted and concentrated using 10-l C18 ZipTip pipette tips (Millipore,

    Billerica, MA). MALDI mass spectra were recorded in the positive ion reflector mode with delayed extraction on a Reflex II time-of-flight instrument (Bruker-Daltonic, Bremen, Germany) equipped with a SCOUT-26 inlet and a 337-nm nitrogen laser. Spectra were calibrated externally by a two-point linear fit with peptides of angiotensin I and the oxidized B-chain of bovine insulin.

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    Supplemental Figure 1. Treatment with HDAC inhibitors shifts cellular P-TEFb into the Hexim1-free (active) form. Glycerol gradient sedimentation of endogenous P-TEFb in HeLa cells, which were treated with trichostatin A (TSA; 400 nM) and nicotinamide (NA; 5 mM) for six hours or were left untreated.

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    Supplemental Figure 2. Dissociation of 7SK snRNA from P-TEFb in response to HDAC inhibitor treatment. (A) 293 cells were transfected with HA-cyclin T1 and subsequently treated with TSA/NA as indicated. Total cell extract (800 g protein) was

    subjected to immunoprecipitation using either protein-A agarose beads alone (mock) or together with ;-HA antibody (cyclin T1). Data are expressed as 7SK snRNA copy numbers per g of protein and are representative of three independent experiments. RNA was isolated from the immunoprecipitated material using the RNA STAT-60 kit (TEL-TEST INC) and subjected to reverse transcription with the Superscript III reverse

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     kit (Invitrogen). 7SK snRNA was measured by real-time RT-PCR with transcriptase

    SYBR Green I master mix (MCLab) and ABI PRISM 7900 thermocycler (Applied Biosystems). The primers used for 7SK snRNA analysis were: 5’-

    AGGACCGGTCTTCGGTCAA-3’ and 5’-TCATTTGGATGTGTCTGCAGTCT-3’. To

    calculate 7SK snRNA copy numbers, ten-fold serial dilutions of the 7SK snRNA cDNA

    102were prepared at concentrations of 9× 10 to 9× 10 copies per reaction and used for

    real-time RT-PCR analysis. (B) Real time RT-PCR of 7SK snRNA isolated from total cell extracts treated with TSA/NA. No change is observed.

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Supplemental Figure 3. In vitro kinase assays of P-TEFb containing wildtype or 4R

    mutant HA-cyclin T1. 293 cells were transfected with wild-type or 4R mutant HA-cyclin T1 and FLAG-CDK9-expressing constructs. Expression of overexpressed proteins was analyzed by western blotting. Immunoprecipitations were performed with HA antibodies. Immunoprecipitated material was subjected to in vitro kinase assays

    containing bead-bound P-TEFb, recombinant murine GST-CTD (Peterson, S.R., Dvir, A., Anderson, C.W., and Dynan, W.S. 1992. DNA binding provides a signal for phosphorylation of the RNA polymerase II heptapeptide repeats. Genes & Dev. 6: 426-

    438) and 10 l of magnesium/ATP cocktail (Millipore). Reactions were incubated at o30C for 20 min in a 50 l volume. As a positive control, 10 U recombinant

    CDK9/cyclin T1 expressed in insect cells was incubated with recombinant GST-CTD (Millipore). The complexes were resolved by SDS-PAGE and analyzed by western blotting with anti-RNA polymerase II-CTD repeat YSPTSPS (phospho S2) antibodies (Abcam, Cambridge, MA) or by coomassie staining. This experiment represents one of two independent experiments with identical results. The phosphorylation of GST-CTD was quantified using the ImageJ software available at http://rsb.info.nih.gov/ij.

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    Supplemental Figure 4. Dissociation of Hexim1 from P-TEFb by treatment with HMBA. Coimmunoprecipitation experiments of endogenous Hexim1 and CDK9 proteins with wild-type or 4R mutant HA-cyclin T1 in 293 cells treated with HMBA (10 mM) for indicated times. Wild-type and 4R mutant cyclin T1 proteins are dissociated from

     Hexim1 effectively.

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