Fig 2: DYRK1A is associated with enhancer regions in T98G cells. (A) Venn diagram analysis of ChIP-seq data of DYRK1A, p300 and CBP in cycling T98G cells. Raw ChIP-seq data were obtained from GEO (GSE63712 and GSE21026) (12,20). (B) Profiles of CBP (black), p300 (red) centered on DYRK1A (yellow) peak. (C) Analysis of DYRK1A, p300 and CBP ChIP-seq data in cycling T98G cells. Occupancy levels of DYRK1A, p300 and CBP are shown. Profiles are centered on DYRK1A-occupied peaks (±10 kb) and sorted in descending order of DYRK1A occupancy. (D) Analysis of DYRK1A, p300 and CBP ChIP-seq data as in (C), but centered at TSS of the nearest gene and extends (±10 kb) on each side. (E) Genome browser track example showing CBP, p300 and DYRK1A ChIP-seq signal in T98G cells. Bottom track shows DYRK1A ChIP-seq signal in HeLa cells. 16 and 42 are arbitrary units depicting enrichment in genome tracks. (F) ChIP analysis shows enrichment of DYRK1A, CBP and H3K27acetylation at enhancer sites upstream to DNAJC12, WNT11, and DYNC1H1 in HEK293 cells. Data represent the mean ± SD (n = 3 biological replicates). Student's t test were done to compare samples. P value = * (P < 0.05). (G) GO term analysis of genes located near or downstream to DYRK1A occupied peaks.

Fig 3: Knockdown of DYRK1A, p300 and CBP in THP1 cells affects cell proliferation. THP1 cells were infected with shRNA lentivirus targeting DYRK1A, p300 and CBP, and sorted to isolate GFP expressing cells. Sorted cells were seeded in 6cm plates in triplicates. Cell proliferation was measured by counting number of cells using haemocytometer every 48 hours for six days. (A) Cell numbers are plotted. (B) Area under curve (AUC) analysis of data in (A). P values between control and experimental group were calculated using a two-tailed Student's t test. Data represent the mean ± SD (n = 3 biological replicates). P value = * (P < 0.05).

Fig 4: Reduction in DYRK1A leads to a weak reduction in expression of p300 and CBP. (A) RT-qPCR analysis of CBP and p300 mRNA levels after knockdown of DYRK1A with shRNA in 293 cells. GAPDH mRNA was used to normalize RNA in RT-qPCR samples. Data represent the mean ± SD (n = 3 biological replicates). (B) Western blot analysis of p300, CBP and DYRK1A proteins levels in whole cell extracts, after knockdown of DYRK1A with shRNA in 293 cells (n = 3 biological replicates). Actin was used as loading control in all western samples. Right graph shows quantification of western signal as the mean ±SD (n = 3 biological replicates). Signals were quantitated using Image J software. (C and E) RT-qPCR analysis of DYRK1A mRNA levels after knockdown of CBP or p300 with shRNAs in 293 cells. GAPDH mRNA was used to normalize RNA in RT-qPCR samples. Data represent the mean ± SD (n = 3 biological replicates). (D and F) Western blot analysis of DYRK1A protein levels in whole cell extracts, after knockdown of CBP or p300 with shRNAs in 293 cells. Actin was used as a loading control in all western samples. (n = 3 biological replicates). Graph on the right shows quantitation as in figure B. Student's t test were done to compare samples. P value = * (P < 0.05).

Fig 5: Overexpression of DYRK1A leads to hyper-phosphorylation of CBP and p300. (A) Whole cell lysates from 293T cells co-transfected with Myc-p300 and Flag-DYRK1A-WT or Flag-DYRK1A-KD were treated with ? phosphatase, and analysed by western blot. Arrows indicate the slower migrating p300 signal in vector vs DYRK1A overexpression. (B) Whole cell lysates from 293T cells co-transfected with HA-CBP and Flag-DYRK1A-WT or Flag-DYRK1A-KD were treated with ? phosphatase, and analysed by western blot. Arrows indicate the slower migrating CBP signal in vector vs DYRK1A overexpression.