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. 2019 Jan 17;8:e42426. doi: 10.7554/eLife.42426

Figure 8. 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.

Figure 8.

Figure 8—figure supplement 1. The distribution and GO analysis of increased accessible regions upon TRIM28 depletion.

Figure 8—figure supplement 1.

(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, cellular component analysis and molecular function analysis were used to classify genes with increased accessible regions upon TRIM28 knockout in J-Lat 10.6 (C) and TRIM28 knockdown in TZM-bl (D).
Figure 8—figure supplement 2. The COG analysis of increased accessible regions and the chromatin accessibility variations on target genes.

Figure 8—figure supplement 2.

(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 accessibilities of the promoter of SEC16A within which the integrated pseudotyped HIV-1 located were annotated in both wild type and TRIM28 knockout J-Lat 10.6 cell lines. (D) The chromatin accessibilities of the promoter of housekeeping gene GAPDH were annotated in both wild type and TRIM28 knockout J-Lat 10.6 cell lines. (E) The chromatin accessibilities of the promoter of RALGDS within which the HIV-1 reporter provirus located were annotated in both wild type and TRIM28 knockdown TZM-bl cell lines. (F) The chromatin accessibilities of the promoter of housekeeping gene GAPDH were annotated in both wild type and TRIM28 knockdown TZM-bl cell lines.
Figure 8—figure supplement 3. Schematic of in vitro SUMOylation assay and CDK9 kinase assay.

Figure 8—figure supplement 3.

(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 and SUMO4; Group 3 (G3): CDK9, SUMO4 and E1 (SAE1 and UBA2); Group 4 (G4): CDK9, SUMO4, E1 and E2 (UBC9); Group 5 (G5): CDK9, SUMO4, E1, E2 and E3 (TRIM28). After in vitro SUMOylation, CDK9 substrate PDKtides and ATP were added and incubated for 120 min at room temperature. The ADP which was consumed during CDK9 kinase assay was converted to ATP and quantitated by luciferase assay.