TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb
- Sun Yat-sen University, China
- Shenyang Medical College, China
- Sun Yat-sen University Cancer Center, China
- Guangzhou Eighth People’s Hospital, China
Figures
Figure 1 with 3 supplements
TRIM28 suppresses HIV-1 expression and contributes to HIV-1 latency.
(A) A siRNA library targeting 182 human genes was transfected into TZM-bl cell line, respectively. Three distinct siRNAs targeting each gene were transfected as a mixture. Forty-eight hours …
Figure 1—figure supplement 1
TRIM28 suppresses HIV-1 expression and is upregulated upon activation by PHA.
(A) TRIM28 in TZM-bl cells was knocked down by siRNAs targeting the coding sequence and 3’UTR of TRIM28 mRNA. The luciferase from clarified lysates was quantitated and normalized to siNC. Data …
Figure 1—figure supplement 2
Primary CD4+T cells populations’ identities.
(A–D) CD4+ T cells were stimulated with PHA for 2 days or left untreated. One part of PHA-activated CD4+ T cells was washed for removing PHA and cultured in RPMI1640 which contained low IL-2 for 1 …
Figure 1—figure supplement 3
TRIM28 contributes to HIV-1 latency and is enriched on HIV-1 LTR.
(A–D) J-lat 6.3, 8.4, 9.2 and 15.4 cell lines were treated as in Figure 1B. The reactivation efficiency for each group was analyzed as in Figure 1C. Data represents mean ±SEM in triplicates. …
Figure 2 with 1 supplement
Both RING and PHD domains E3 ligase activities are important for repressive epigenetic modifications.
(A) Schematic of wild-type TRIM28 and nine TRIM28 mutants. (B) Endogenous TRIM28 was knocked down by siRNA targeting 3’UTR in TZM-bl cells and re-expressed with wild type and different TRIM28 …
Figure 2—figure supplement 1
Positive controls for ChIP.
(A–D) Data represented positive controls of siTRIM28-related ChIP. Endogenous TRIM28 in TZM-bl cells was knocked down by siRNA targeting 3’UTR of TRIM28 mRNA. Another three groups whose endogenous …
Figure 3 with 1 supplement
TRIM28 SUMOylates many transcription factors and transferases.
(A) Schematic of global site-specific SUMO-MS. His-tagged SUMO mutants were co-overexpressed with UBC9 and TRIM28. The SUMOylated proteins were enriched by His-tag beads and separated by SDS-PAGE. …
Figure 3—figure supplement 1
STRING, MCODE and GO analysis of proteins SUMOylated by TRIM28.
(A) Twelve subclusters, which were extracted by MCODE analysis, were separately plotted. The STRING and MCODE analyses were performed with the following settings: a significance threshold below 10−7,…
Figure 4 with 1 supplement
SUMO4 suppresses HIV-1 expression and contributes to HIV-1 latency.
(A) SUMO4 in TZM-bl cells was knocked down by siRNAs targeting the coding sequence and 3’UTR of SUMO4 mRNA. The luciferase from clarified lysates was quantitated and normalized to siNC. Data …
Figure 4—figure supplement 1
SUMO4 is uregulated upon activation by PHA and is the major paralog used by CDK9 and TRIM28.
(A) The knockdown efficiency of different SUMO4 siRNAs was confirmed by qPCR. Data represents mean ±SEM in triplicates. p-Vvalues were calculated by Student’s t-test. ***p<0.001. (B) The expression …
Figure 5 with 1 supplement
CDK9 is SUMOylated by TRIM28.
(A) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, UBC9 or TRIM28. CDK9 was IP with anti-HA-tag beads, followed by IB with anti-HA and –Flag antibodies. TRIM28, UBC9 and GAPDH in total …
Figure 5—figure supplement 1
CDK9 is deSUMOylated by SENP3 and CDK9 SUMOylation occurs in primary CD4+T cells.
(A) siRNAs targeting six SENPs were transfected into TZM-bl cells. The luciferase from the clarified lysates of each group was quantitated and normalized to siNC. Data represents mean ±SEM in …
Figure 6 with 1 supplement
TRIM28 co-localizes with SUMO4 and CDK9.
(A) cSTORM image of endogenous TRIM28 and SUMO4 in HEK293T cells. The first row: the original whole nucleus; the second row: one of the amplified region of the nucleus; the third row: the 3D-cSTORM …
Figure 6—figure supplement 1
Amplified views of transformed co-localization.
(A–B) cSTORM-imaged protein molecules and complexes were transformed and displayed as in Figure 6C–D.Data represented amplified views of each transformed co-localization images. Green spots …
Figure 7 with 1 supplement
The RING domain of TRIM28 plays a key role in binding to and SUMOylating CDK9.
(A) HA-tagged CDK9 was co-overexpressed with Flag-tagged full length TRIM28 or domain-truncated TRIM28 mutants. Flag-tagged proteins were IP, followed by IB with antibodies against HA-tag, Flag-tag …
Figure 7—figure supplement 1
TRIM28 enriches CDK9 in the presence of RNase and SUMOylation status of TRIM28 mutants.
(A) Flag-tagged GFP and Flag-tagged TRIM28 were co-overexpressed with HA-tagged CDK9, respectively. Flag-tagged proteins were IP with anti-Flag beads. Flag-tagged proteins from another two similar …
Figure 8 with 3 supplements
CDK9 function is reduced when SUMOylated by TRIM28.
(A–B) TRIM28-defective (sgTRIM28) J-Lat 10.6 cell line was generated by CRISPR-CAS9 technique. ATAC-Seq was conducted with sgNT and sgTRIM28 J-Lat 10.6 cell lines, as well as siNC and siTRIM28 …
Figure 8—figure supplement 1
The distribution and GO analysis of increased accessible regions upon TRIM28 depletion.
(A–B) The distribution of increased accessible regions upon TRIM28 knockout in J-Lat 10.6 (A) and TRIM28 knockdown in TZM-bl (B). (C–D) GO analyses which included biological process analysis, …
Figure 8—figure supplement 2
The COG analysis of increased accessible regions and the chromatin accessibility variations on target genes.
(A–B) Clusters of Orthologous Groups of proteins (COGs) analysis of increased accessible regions upon TRIM28 knockout in J-Lat 10.6 (A) and TRIM28 knockdown in TZM-bl (B). (C) The chromatin …
Figure 8—figure supplement 3
Schematic of in vitro SUMOylation assay and CDK9 kinase assay.
(A) In vitro expressed and purified CDK9 was incubated with SUMO system components (SUMO4, E1, UBC9 and TRIM28) or left untreated. Five groups were set. Group 1 (G1): CDK9 only; Group 2 (G2): CDK9 …
Figure 9 with 1 supplement
The Lys44, Lys56 and Lys68 residues of CDK9 are SUMOylated with SUMO4.
(A) Different HA-tagged CDK9 reversing mutation constructs or wild type CDK9 were co-overexpressed with SUMO4, UBC9 and TRIM28, respectively. CDK9 and CDK9 mutants were IP with anti-HA-tag beads …
Figure 9—figure supplement 1
The Lys44, Lys56 and Lys68 residues of CDK9 are SUMOylated with SUMO4.
(A) Schematic of different CDK9 mutants. CDK9-K0R indicated that all lysines had been mutated to arginines. CDK9-KKR indicated that the third part of CDK9 had mutated all lysines to arginines. The …
Figure 10 with 4 supplements
TRIM28 depletion reactivates latent HIV-1 in cells from HIV-1-infected individuals.
(A) shRNAs targeting luciferase and TRIM28 were packaged into lentiviruses and infected CD4+ T cells from HIV-1-infected individuals. Unstimulated CD4 +T cells were used as negative control (NC). …
Figure 10—figure supplement 1
Cytotoxicity assay, cell viability assay and cell number counting used to evaluate the toxicity of targeting TRIM28.
(A) TRIM28 in Hela cells and HIV-1-infected CD4+ T cells was knocked down by siRNA targeting TRIM28. ShRNA and sgRNA lentiviruses targeting TRIM28 were used to knock down TRIM28 and knock out TRIM28 …
Figure 10—figure supplement 2
Cell proliferation assay used to evaluate the toxicity of targeting TRIM28.
(A–D) The experiment setup was conducted as in Figure 10—figure supplement 1. On Day 0, cells from each group were stained with CFSE. The percentage and mean fluorescence intensity (MFI) of …
Figure 10—figure supplement 3
TRIM28 depletion reactivates latent HIV-1 in cells from HIV-1-infected individuals.
(A) Schematic of experiments on primary CD4 +T cells from HIV-1-infected individuals. (B) The knockdown efficiency of shTRIM28 in HIV-1-infected CD4+ T cells. (C) The knockdown efficiency of …
Figure 10—figure supplement 4
SUMO4 depletion reactivates latent HIV-1 in cells from HIV-1-infected individuals.
(A) SiRNAs targeting NC and SUMO4 were nucleofected into CD4+ T cells from HIV-1-infected individuals. Unstimulated CD4+ T cells were used as Mock. Stimulation with αCD3/αCD28/IL-2 was used as …
Author response image 1
Attenuated Tat Protein Tat-R5M4 can reactivate more genetically-diversified HIV-1.
(Geng et al., 2016, Molecular Therapy, PMID: 27434587).
Author response image 2
Different LRAs reactivate genetically-diversified HIV-1 at different integration sites.
Videos
Video 1
3D-cSTORM movie of the 3D co-localization of TRIM28 with SUMO4.
Green spots indicate TRIM28. Red spots indicate SUMO4.
Video 2
3D-cSTORM movie of the 3D co-localization of TRIM28 with CDK9.
Green spots indicate TRIM28. Red spots indicate CDK9.
Video 3
Transformed 3D-cSTORM movie of the 3D co-localization of TRIM28 with SUMO4.
Green spots indicate TRIM28. Red spots indicate SUMO4.
Video 4
Transformed 3D-cSTORM movie of the 3D co-localization of TRIM28 with CDK9.
Green spots indicate TRIM28. Red spots indicate CDK9.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | E.coli DH5α: F-, φ 80dlacZ ΔM15, Δ(lacZYA -argF )U169, deoR , recA1 , endA1 , hsdR17 (rK-, mK+), phoA, supE44 , λ-, thi −1, gyrA96 , relA1 | Takara | Cat#9057 | |
| Strain, strain background (Escherichia coli) | E. coli HB101: F-, hsdS20(rB-, mB-), recA13, ara-14, proA2, lacY1, galK2, rpsL20 (str), xyl-5, mtl-1,supE44, leuB6, thi-1. | Takara | Cat#9051 | |
| Strain, strain background (Escherichia coli) | E.coli BL21: F-, ompT, hsdSB (rB-mB-), gal, dcm | Takara | Cat#9126 | |
| Strain, strain background (Escherichia coli) | E.coli Stbl3: F-, mcrB, mrr, hsdS20 (rB-, mB-), recA13, supE44, ara-14, galK2, lacY1, proA2, rpsL20 (StrR), xyl-5, λ- leu, mtl-1 | ThermoFisher | Cat#C7381201 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216; RRID: CVCL_0063 | female |
| Cell line (Homo sapiens) | HeLa | ATCC | CCL-2; RRID: CVCL_0030 | female |
| Cell line (Homo sapiens) | TZM-bl | NIH AIDS Reagent Program | Cat#8129 | female |
| Cell line (Homo sapiens) | J-Lat 6.3 | PMID: 12682019 | NIH AIDS Reagent Program Cat#9846 | Dr. Eric Verdin (The Buck Institute for Research on Aging, Novato, CA, USA) |
| Cell line (Homo sapiens) | J-Lat 8.4 | PMID: 12682019 | NIH AIDS Reagent Program Cat#9847 | Dr. Eric Verdin (The Buck Institute for Research on Aging, Novato, CA, USA) |
| Cell line (Homo sapiens) | J-Lat 9.2 | PMID: 12682019 | NIH AIDS Reagent Program Cat#9848 | Dr. Eric Verdin (The Buck Institute for Research on Aging, Novato, CA, USA) |
| Cell line (Homo sapiens) | J-Lat 10.6 | PMID: 12682019 | NIH AIDS Reagent Program Cat#9849 | Dr. Eric Verdin (The Buck Institute for Research on Aging, Novato, CA, USA) |
| Cell line (Homo sapiens) | J-Lat 15.4 | PMID: 12682019 | NIH AIDS Reagent Program Cat#9850 | Dr. Eric Verdin (The Buck Institute for Research on Aging, Novato, CA, USA) |
| Biological sample (Homo sapiens) | Blood samples from healthy individuals | Guangzhou Blood Center, Guangzhou | http://www.gzbc.org/ | |
| Biological sample (Homo sapiens) | Blood samples from HIV-1- infected individuals | Department of Infectious Diseases, Guangzhou 8th People’s Hospital, Guangzhou | http://gz8h.com.cn/ | |
| Antibody | Mouse Monoclonal anti-TRIM28 Antibody | Proteintech | Cat#66630–1-Ig; RRID: AB_2732886; Lot#10006062 | (1:1000) |
| Antibody | Rabbit Polyclonal anti-TRIM28 Antibody | Proteintech | Cat#15202–1-AP; RRID: AB_2209890; Lot#00051172 | (1:1000) |
| Antibody | Rabbit Polyclonal Anti-Histone H3 (tri methyl K4) Antibody | Abcam | Cat#ab8580; RRID: AB_306649; Lot#GR273043-3 | Use 2 µg for 25 µg of chromatin |
| Antibody | Rabbit Polyclonal Anti-Histone H3 (acetyl K9) Antibody | Abcam | Cat#ab4441; RRID: AB_2118292; Lot#GR270585-1 | Use 2 µg for 25 µg of chromatin |
| Antibody | Mouse Monoclonal Anti-Histone H3 (tri methyl K27) Antibody | Abcam | Cat#ab6002; Lot#GR275911-3 | Use 5 µg for 25 µg of chromatin |
| Antibody | Normal Rabbit Anti-IgG Antibody | CST | Cat#2729; RRID: AB_1031062 | Use 1 µg for 25 µg of chromatin |
| Antibody | Rabbit Polyclonal Anti-UBE2I Antibody | Abclonal | Cat#A2193; Lot#45473 | (1:1000) |
| Antibody | Rabbit Polyclonal Anti-UBA2 Antibody | Abclonal | Cat#A4363 | (1:1000) |
| Antibody | Rabbit Polyclonal Anti-SAE1 Antibody | Proteintech | Cat#10229–1-AP; RRID: AB_2182917; Lot#00040591 | (1:1000) |
| Antibody | Rabbit Monoclonal Anti-SUMO4 Antibody | Abcam | Cat#ab126606; RRID: AB_11128131; Lot#GR851138-12 | (1:1000) |
| Antibody | Rabbit Monoclonal Anti-CDK9 (C12F7) Antibody | CST | Cat#2316; Lot#6 | (1:1000) |
| Antibody | Rabbit Polyclonal Anti-SENP3 Antibody | Proteintech | Cat#17659–1-AP; RRID: AB_2301618; Lot#00025621 | (1:1000) |
| Antibody | Rabbit Polyclonal Anti-RNA polymerase II CTD repeat YSPTSPS (phosphor-Ser2) Antibody | Abcam | Cat#ab5095; RRID: AB_304749; Lot#GR278215-1 | Use 2 µg for 25 µg of chromatin |
| Antibody | Mouse Monoclonal Anti-Histone H3 (di methyl K9) Antibody | Abcam | Cat#ab1220; RRID: AB_449854 | Use 4 µg for 25 µg of chromatin |
| Antibody | Rabbit Polyclonal Anti-Histone H3 (tri methyl K9) Antibody | Abcam | Cat#ab8898; RRID: AB_306848 | Use 4 µg for 25 µg of chromatin |
| Antibody | Donkey Anti-Mouse IgG H and L (Alexa Fluor 647) Antibody | Abcam | Cat#ab150107; Lot#GR311164-3 | (1:200) |
| Antibody | Donkey Anti-Rabbit IgG H and L (Alexa Fluor 647) Antibody | Abcam | Cat#ab150075; Lot#GR3174006-4 | (1:200) |
| Antibody | Donkey Anti-Rabbit IgG (H + L), Highly Cross- Adsorbed, CF 568 Dye Conjugates, Single Label for STORM | Biotium | Cat#20803–500 μl; Lot#17C0626 | (1:200) |
| Antibody | Donkey Anti-Mouse IgG (H + L), Highly Cross- Adsorbed, CF 568 Dye Conjugates, Single Label for STORM | Biotium | Cat#20802–500 μl; Lot#17C1004 | (1:200) |
| Antibody | Rabbit Anti-DDDDK Tag Polyclonal Antibody, Unconjugated | MBL | Cat#PM020; RRID: AB_591224; Lot#026 | (1:1000) |
| Antibody | Mouse Monoclonal Anti-HA-Tag Antibody | MBL | Cat#M180-3; RRID: AB_10951811; Lot#008 | (1:10000) |
| Antibody | Mouse Monoclonal Anti-His-Tag Antibody | Proteintech | Cat#66005–1-Ig; RRID: AB_11232599; Lot#00083246 | (1:1000) |
| Antibody | Rabbit Polyclonal Anti-GAPDH Antibody | Proteintech | Cat#10494–1-AP; RRID: AB_2263076; Lot#00039889 | (1:10000) |
| Antibody | IRDye 680RD Goat anti-Mouse IgG (H + L), 0.5 mg Antibody | LI-COR Biosciences | Cat#926–68070; RRID: AB_10956588; Lot#C70613-15 | (1:10000) |
| Antibody | IRDye 800CW Goat Anti-Rabbit IgG, Conjugated Antibody | LI-COR Biosciences | Cat#926–32211; RRID: AB_621843; Lot#C70620-05 | (1:10000) |
| Antibody | PerCP-Cy 5.5 Mouse Anti-Human CD45RO | BD Biosciences | Cat#560607; RRID: AB_1727500; Lot#5338941 | (1:1000) |
| Antibody | APC/Cy7 anti- human CD45RA | BioLegend | Cat#304127; RRID: AB_10708419; Lot#B164612 | (1:1000) |
| Antibody | Anti-Human CD69 PE-Cy7 | ThermoFisher | Cat#25-0699-42; RRID: AB_1548714; Lot#E10154-1635 | (1:1000) |
| Antibody | Anti-Human CD62L PE-Cyanine7 | ThermoFisher | Cat#25-0629-42; RRID: AB_1257142; Lot#4291471 | (1:1000) |
| Antibody | Anti-Human CD4 FITC | ThermoFisher | Cat#11-0048-42; RRID: AB_1633390; Lot#E10526-1631 | (1:1000) |
| Antibody | PE-Cy5 Conjugated Amti-human CD25 (IL-2R) | ThermoFisher | Cat#15-0259-42; RRID: AB_1944361; Lot#E11289-102 | (1:1000) |
| Recombinant DNA reagent | VSV-G glycoprotein- expression vector | PMID: 9306402 | Addgene Plasmid #12259 | Dr. Didier Trono (School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) |
| Recombinant DNA reagent | Lentiviral packaging construct pCMVΔR8.2 | PMID: 9306402 | Addgene Plasmid #12263 | Dr. Didier Trono (School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) |
| Recombinant DNA reagent | Lentiviral construct vector pLKO.3G-RFP | This paper | N/A | Progenitor: pLKO.3G |
| Recombinant DNA reagent | Lentiviral construct vector lentiCRISPRv2 | PMID: 25075903 | Addgene Plasmid #52961 | Dr. Feng Zhang (Broad Institute of MIT and Harvard) |
| Recombinant DNA reagent | Plasmid: 10His- SUMO1-Q92R | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 10His -SUMO2-Q88R | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 10His- SUMO4-Q88R | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 3HA- CDK9-KKR | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 3HA- CDK9-RRK | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 3HA -CDK9-RKK | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 3HA- CDK9-KRR | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 3HA- CDK9-KRK | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 3HA- CDK9-RKR | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmid: 3HA- CDK9-K0R | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Recombinant DNA reagent | Plasmids: 3HA-CDK9-K0R-RXK (X represent mutation position) | This paper | Supplementary file 3 | Progenitor: pcDNA3.1(+) |
| Sequence- based reagent | siRNA Library | RiboBio | Supplementary file 1; http://www.ribobio.com/ | |
| Sequence- based reagent | ChIP-qPCR Primers | This paper | Supplementary file 2 | |
| Sequence- based reagent | siRNA targeting TRIM28 3’UTR:5’- GCTCTGTTCTCTGTCCTGT-3’ | RiboBio | http://www.ribobio.com/ | |
| Sequence- based reagent | shRNA targeting Luciferase:5’- ACCGCCTGAAGTCTCTGATTAA-3’ | PMID: 29863470 | N/A | |
| Sequence- based reagent | shRNA targeting TRIM28 CDS:5’- CCAGCCAACCAGCGGAAATGTGA-3’ | PMID: 18082607 | N/A | |
| Sequence- based reagent | sgRNA targeting Dummyguide (sgNT):5’- ACGGAGGCTAAGCGTCGCAA-3’ | PMID: 25075903 | N/A | Dr. Feng Zhang (Broad Institute of MIT and Harvard) |
| Sequence- based reagent | sgRNA targeting TRIM28 CDS:5’- CACCGATTGAGCTGGCAGTCTCGGC-3’ | PMID: 25075903 | N/A | Dr. Feng Zhang (Broad Institute of MIT and Harvard) |
| Sequence- based reagent | β-Actin qPCR Forward Primer:5’- GCATGGAGTCCTGTGGCA-3’ | PMID: 27291871 | N/A | |
| Sequence- based reagent | β-Actin qPCR Reverse Primer:5’- CAGGAGGAGCAATGATCTTGA-3’ | PMID: 27291871 | N/A | |
| Sequence- based reagent | TRIM28 qPCR Forward Primer:5’- CTACTCAAGTGCAGAGCCCC-3’ | This paper | N/A | |
| Sequence- based reagent | TRIM28 qPCR Reverse Primer:5’- GGGAAGACCTTGAAGACGGG-3’ | This paper | N/A | |
| Sequence- based reagent | HIVTotRNA Forward Primer:5’- CTGGCTAACTAGGGAACCCACTGCT-3’ | PMID: 27291871 | N/A | |
| Sequence- based reagent | HIVTotRNA Reverse Primer:5’- GCTTCAGCAAGCCGAGTCCTGCGTC-3’ | PMID: 27535056 | N/A | |
| Sequence- based reagent | 1 st round Nest PCR Forward Primer (E00):5’- TAGAAAGAGCAGAAGACAGTGGCAATGA-3’ | PMID: 27434587 | N/A | |
| Sequence- based reagent | 1 st round Nest PCR Reverse Primer (ES8B):5’- CACTTCTCCAATTGTCCCTCA-3’ | PMID: 27434587 | N/A | |
| Sequence- based reagent | 2nd round Nest PCR Forward Primer (E20):5’- GGGCCACACATGCCTGTGTACCCACAG-3’ | PMID: 27434587 | N/A | |
| Sequence- based reagent | 2nd round Nest PCR Reverse Primer (E115):5’- AGAAAAATTCCCCTCCACAATTAA-3’ | PMID: 27434587 | N/A | |
| Chemical compound, drug | (+)-JQ-1 | Selleckchem | Cat#S7110 | |
| Chemical compound, drug | Vorinostat (SAHA) | Selleckchem | Cat#S1047 | |
| Chemical compound, drug | Formaldehyde solution | Sigma-Aldrich | Cat#F8775-25ML | |
| Chemical compound, drug | TRIzol Reagent | ThermoFisher | Cat#15596018 | |
| Chemical compound, drug | 4',6-Diamidino-2- Phenylindole, Dihydrochloride (DAPI) | ThermoFisher | Cat#D1306 | |
| Chemical compound, drug | Cysteamine (MEA) | Sigma-Aldrich | Cat#30070–10G | |
| Chemical compound, drug | Glucose Oxidase from Aspergillus niger, Type VII, lyophilized powder, ≥100,000 units/g solid | Sigma-Aldrich | Cat#G2133-250KU | |
| Chemical compound, drug | Catalase from bovine liver , lyophilized powder, ≥10,000 units/mg protein | Sigma-Aldrich | Cat#C40-1G | |
| Chemical compound, drug | Sodium borohydride (NaBH4) | Sigma-Aldrich | Cat#213462–25G | |
| Chemical compound, drug | 16% Paraformaldehyde (formaldehyde) Aqueous Solution | Electron Microscopy Sciences | Cat#15710 | |
| Chemical compound, drug | 8% Glutaraldehyde Aqueous Solution | Electron Microscopy Sciences | Cat#16019 | |
| Chemical compound, drug | Normal Donkey Serum (NDS) | Jackson ImmunoResearch | Cat#017-000-121 | |
| Chemical compound, drug | Triton X-100 | Sigma-Aldrich | Cat#T8787-50ML | |
| Chemical compound, drug | Protease Inhibitor Cocktail (PIC) | Sigma-Aldrich | Cat#P8340-1ML | |
| Chemical compound, drug | N-Ethylmaleimide (NEM) | Selleckchem | Cat#S3692 | |
| Chemical compound, drug | EZview Red Anti-HA Affinity Gel | Sigma-Aldrich | Cat#E6779-1ML | |
| Chemical compound, drug | EZview Red Anti- FLAG M2 Affinity Gel | Sigma-Aldrich | Cat#F2426-1ML | |
| Chemical compound, drug | Anti-His-tag Agarose | Abcam | Cat#ab1231 | |
| Chemical compound, drug | Penicillin-Streptomycin, Liquid | ThermoFisher | Cat#15140122 | |
| Chemical compound, drug | L-Glutamine, 200 mM Solution | ThermoFisher | Cat#25030081 | |
| Chemical compound, drug | Fetal Bovine Serum (FBS) | ThermoFisher | Cat#10270–106 | |
| Chemical compound, drug | Phytohemagglutinin -M (PHA-M) | Sigma-Aldrich | Cat#11082132001 | |
| Peptide, recombinant protein | Recombinant Human TNF-α | PeproTech | Cat#300-01A | |
| Peptide, recombinant protein | Recombinant Human IL-2 | R&D Systems | Cat#202-IL-500 | |
| Peptide, recombinant protein | Recombinant Human SUMO Activating Enzyme E1 (SAE1/UBA2) | R&D Systems | Cat#E-315 | |
| Peptide, recombinant protein | Recombinant Human UBE2I/Ubc9 | R&D Systems | Cat#E2-645-100 | |
| Peptide, recombinant protein | Recombinant Human CDK9 | Abcam | Cat#ab85603 | |
| Peptide, recombinant protein | Recombinant Human SUMO4 | This paper | N/A | |
| Peptide, recombinant protein | Recombinant Human TRIM28 | Abcam | Cat#ab131899 | |
| Commercial assay or kit | SUMO Conjugation Reaction Buffer Kit | R&D Systems | Cat#SK-15 | |
| Commercial assay or kit | Human Lymphocyte Separation Kit | TBDsciences | Cat#LTS1077 | |
| Commercial assay or kit | BD IMag Human CD4 + T Lymphocyte Enrichment Set-DM | BD Biosciences | Cat#557939 | |
| Commercial assay or kit | Luciferase Assay System | Promega | Cat#E4550 | |
| Commercial assay or kit | SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) | CST | Cat#9003S | |
| Commercial assay or kit | TruePrep DNA Library Prep Kit V2 for Illumina | Vazyme | Cat#TD501 | |
| Commercial assay or kit | HIV-1 p24 ELISA Kit | Abcam | Cat#ab218268 | |
| Commercial assay or kit | ProteoSilver Plus Silver Stain Kit | Sigma-Aldrich | Cat#PROTSIL2 -1KT | |
| Commercial assay or kit | CDK9/CyclinK Kinase Enzyme System | Promega | Cat#V4104 | |
| Commercial assay or kit | ADP-GloTM Kinase Assay | Promega | Cat#V6903 | |
| Commercial assay or kit | Cell Counting Kit-8 | Dojindo | Cat#CK04; Lot#KT793 | |
| Commercial assay or kit | Zombie Violet Fixable Viability Kit | BioLegend | Cat#423113; Lot#B256957 | |
| Commercial assay or kit | CellTrace CFSE Cell Proliferation Kit - For Flow Cytometry | ThermoFisher | Cat#C34554 | |
| Software, algorithm | Prism 5 | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | MEGA 7 | MEGA | https://www.megasoftware.net/ | |
| Software, algorithm | Cytoscape (3.6.1) | Cytoscape Consortium | RRID:SCR_015784 | |
| Software, algorithm | STRING | Cytoscape Consortium | RRID:SCR_005223 | |
| Software, algorithm | MCODE | Cytoscape Consortium | RRID:SCR_015828 | |
| Software, algorithm | BD LSRFortessa cell analyzer | BD Biosciences | http://www.bdbiosciences.com/in/instruments/lsr/index.jsp | |
| Software, algorithm | FlowJo V10 | Tree Star | https://www.flowjo.com/ | |
| Software, algorithm | Odyssey CLX Imager | LI-COR Biosciences | https://www.licor.com/bio/products/imaging_systems/odyssey/ | |
| Software, algorithm | Image Studio Lite Ver 4.0 | LI-COR Biosciences | https://www.licor.com/bio/products/software/image_studio_lite/ | |
| Software, algorithm | CFX Manager | BIO-RAD | http://www.bio-rad.com/ | |
| Software, algorithm | GloMax 96 Microplate Luminometer Software (version 1.9.3) | Promega | https://www.promega.com/resources/software-firmware/detection-instruments-software/promega-branded-instruments/glomax-96-microplate-luminometer/ | |
| Software, algorithm | SkanIt SW for Microplate Readers | ThermoFisher | https://www.thermofisher.com/order/catalog/product/5187139?SID=srch-srp-5187139 | |
| Software, algorithm | NIS-Elements Advanced Research microscope imaging software | Nikon | https://www.nikoninstruments.com/Products/Software | |
| Software, algorithm | PyMOL | Schrödinger | RRID:SCR_000305 | |
| Software, algorithm | FastQC | Babraham Institute | RRID:SCR_014583 | |
| Software, algorithm | Hisat2 | PMID: 25751142 | RRID:SCR_015530 | |
| Software, algorithm | DEGseq | Bioconductor | RRID:SCR_008480 | |
| Software , algorithm | gplots | R Foundation | https://www.rdocumentation.org/packages/gplots/versions/3.0.1 | |
| Software, algorithm | Bowtie2 | PMID: 22388286 | RRID:SCR_016368 | |
| Software, algorithm | Samtools | PMID: 19505943 | RRID:SCR_002105 | |
| Software, algorithm | igvtools | Broad Institute | https://software.broadinstitute.org/software/igv/igvtools | |
| Software, algorithm | Imaris (Version 9.2) | BITPLANE | RRID:SCR_007370 |
Additional files
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Supplementary file 1
SiRNA library used to screen HIV-1 suppression and latency contributors.
SiRNA library, which targeted several cellular pathways within the nucleus including chromatin binding, epigenetic modification, chromatin remodeling, ubiquitination, SUMOylation, and chromosome organization, was transfected into TZM-bl cells respectively. Both library and negative control siRNA were synthesized from RiboBio (Guangzhou, China).
- https://doi.org/10.7554/eLife.42426.034
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Supplementary file 2
ChIP primers used to explore the enrichment of target proteins on HIV-1.
Eight ChIP-qPCR primers targeting integrated HIV-1 reporter provirus in TZM-bl cell line were designed. G5: Cellular DNA and viral 5’LTR junction; A: Nucleosome 0 assembly site; B: Nucleosome free region; C: Nucleosome one assembly site; V5: Viral 5’LTR and gag leader sequence junction; L: Luciferase region; V3: Viral poly purine tract and 3’LTR junction; G3: Viral 3’LTR and cellular DNA junction. For ChIP-qPCR conducted in J-Lat 10.6, G5’ represented cellular DNA and viral 5’LTR junction; E represented envelop; G3’ represented viral 3’LTR and cellular DNA junction; A, B, C, V5 and V3 represented as in TZM-bl cell lines.
- https://doi.org/10.7554/eLife.42426.035
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Supplementary file 3
SUMO mutants used in SUMO-MS and CDK9 mutants used to identify SUMOylation sites.
The sequences of SUMO1-Q92R, SUMO2-Q88R and SUMO4-Q88R mutants, which mimicked yeast SUMO Smt3 to enable efficient identification of SUMO-acceptor lysines by MS, were represented below. Table also listed the major CDK9 mutants used in reversing mutation assay to identify SUMOylation sites on CDK9. All the sequences were verified by Sanger Sequencing to insure the accuracy.
- https://doi.org/10.7554/eLife.42426.036
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Supplementary file 4
SUMOylated proteins at significance threshold below 10−7.
Table showed 1,329 SUMOylated proteins identified in global site-specific SUMO-MS at significance threshold below 10−7.
- https://doi.org/10.7554/eLife.42426.037
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Supplementary file 5
Subclusters clustered by MCODE analysis.
Twelve highly interconnected functional subclusters were extracted from STRING network by MCODE analysis. Interconnectivity scores ranged from 14 to 96. Genes from each cluster were listed.
- https://doi.org/10.7554/eLife.42426.038
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Supplementary file 6
Go analysis of SUMOylated proteins.
Biological process analysis, molecular function analysis, cellular component analysis and protein class analysis were conducted for the identified SUMOylated proteins. Table showed gene numbers and percentages of each group.
- https://doi.org/10.7554/eLife.42426.039
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Supplementary file 7
SUMOylated proteins at significance threshold below 10−8.
Table showed 715 SUMOylated proteins identified in global site-specific SUMO-MS at significance threshold below 10−8.
- https://doi.org/10.7554/eLife.42426.040
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Transparent reporting form
- https://doi.org/10.7554/eLife.42426.041
