Fig 1: 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 followed by IB. S4: SUMO4. (B) HA-tagged wild type CDK9 and 12 identified SUMOylation site reversing mutation constructs were co-overexpressed with Flag-tagged SUMO4 and Flag-tagged UBC9. The endogenous TRIM28 was knocked down with siRNAs. CDK9 and CDK9 mutants were IP with anti-HA-tag beads followed by IB. Asterisks represented the constructs whose SUMOylation bands disappeared upon TRIM28 knockdown. (C) Three angles of co-crystal structure of Cyclin T1 and CDK9 (PDB ID: 4EC8). Three SUMOylation sites Lys44, Lys56 and Lys68 were shown in ball-and-stick models. The two upper panels showed the ribbon models, while two lower panels showed the surface models. The inner six framed figures which numbered from I to VI represented the amplification views of Lys44, Lys56 and Lys68 sites.
Fig 2: 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 TZM-bl cell lines. The tag reads of the HIV-1 pseudotyped virus/minigenome 5’LTR integration sites were counted and normalized to the total mapped reads, and represented as relative tag density. The highest tag density was set as 100. Figures showed 2 kb range centered the 5’LTR integration sites. (C–D) ChIP assays with antibodies against CDK9 and Ser2 Pho-Pol II were performed in TZM-bl cell lines which were treated with siNC, siSUMO4 and siTRIM28, respectively. (E) Cyclin T1 or GFP was co-overexpressed with CDK9 in the absence or presence of SUMO4, UBC9 and TRIM28. Cyclin T1 and GFP were IP followed by IB. (F) Fold change of kinase activity when CDK9 was SUMOylated. Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t-test. *p<0.05, **p<0.01.
Fig 3: 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 samples were IB with specific antibodies targeting each proteins. (B) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 and different amount of Flag-tagged TRIM28. Target proteins were IB as in (A). (C) In vitro purified CDK9, SUMO4, SAE1, UBA2, UBC9 and TRIM28 were co-cultured in SUMO conjugation reaction buffer. Proteins including SUMOylated CDK9 were IB with antibodies against each targets. (D) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 or Flag-tagged TRIM28, and siNC. In the last group, CDK9 was co-overexpressed with SUMO4, UBC9 and siRNA against TRIM28. Target proteins were IB as in (A). (E) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9, Flag-tagged TRIM28 or two gradients of SENP3. Target proteins were IB as in (A).
Fig 4: 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 mutants. The luciferase activity was measured. Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t-test. *p<0.05, **p<0.01, ***p<0.001. (C–E) Endogenous TRIM28 in TZM-bl cells was knocked down by siRNA targeting 3’UTR of TRIM28 mRNA. Another three groups whose endogenous TRIM28 was knocked down were overexpressed with wild type TRIM28 construct or TRIM28 mutants without RING or PHD domain, respectively. ChIP assays with antibodies against H3K9me3, H3K9Acetyl and H3K27me3 were performed for each group. Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t-test. *p<0.05, **p<0.01.
Fig 5: 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 and GAPDH. (B) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9, Flag-tagged full length TRIM28 or Flag-tagged domain-truncated TRIM28 mutants. CDK9 was IP with anti-HA-tag beads, followed by IB with antibodies against HA-tag, Flag-tag and GAPDH. (C) GFP-tagged TRIM28 or TRIM28-dRING mutant was co-overexpressed with RFP-tagged CDK9 in HEK293T cells. The samples were fixed and dyed according to the immunofluorescence procedure, then visualized in Nikon A1 N-SIM. DAPI was used to dye DNA which was colored into blue. (D) Quantitation of co-localization of TRIM28 or TRIM28-dRING with CDK9. The percentage of co-localization was indicated by percentage of target protein voxels above threshold co-localized voxels. Both Pearson’s coefficient and thresholded Mander’s coefficient were used to evaluate co-localization. For Pearson’s coefficient, a value of 1 represents perfect co-localization, 0 no co-localization, and −1 perfect inverse co-localization. For thresholded Mander’s coefficient, a value of 1 represents perfect co-localization and 0 no co-localization.
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