Fig 1: Integrator and NACK are required for the activated form of RNAPII phosphorylated at Ser5 (RNAPII-S5P) and other transcriptional co-factors on the Notch-dependent promoter. a Representative ChIP shows that the loss of NACK results in the decrease of Notch1, INTS1, INTS11, and RNAPII-S5P, required for transcriptional elongation, on the HES1 promoter. Bars represent standard deviation (N = 3). WB shows depletion of NACK b In HEK293T cells, NACK activates HES5 transcription in a NACK concentration-dependent manner. Bars represent standard deviation (N = 3). **p < 0.01 versus CTR sample. c Representative ChIP shows that in HEK293T cells expressing mutant Maml1 (Maml1(2S)) or mutant NACK (NACK(K)), NACK is unable to bind the NTC, thus HES5 is not activated due to the absence of Notch1 and INT complex (INTS1, INTS11) on the promoter. Bars represent standard deviation (N = 3). d Representative ChIP shows that the knockdown of INTS11 does not affect the occupancy of Notch1, Maml1, and NACK on the HES1 promoter. Bars represent standard deviation (N = 3). e Representative ChIP shows that the knockdown of INTS11 results in a decrease of RNAPII-S5P, but not unphosphorylated RNAPII, on the HES1 promoter. Bars represent standard deviation (N = 3). f Representative ChIP shows that the knockdown of INTS11 does not affect the occupancy of Notch1, Maml1, and NACK on the HES4 promoter. Bars represent standard deviation (N = 3). g Representative ChIP shows that the knockdown of INTS11 results in a decrease of RNAPII-S5P, but not unphosphorylated RNAPII, on the HES4 promoter. Bars represent standard deviation (N = 3). WB shows depletion of INTS11
Fig 2: Integrator together with NACK activates Notch-mediated transcription. a MS/MS analysis shows that subunits of the Integrator complex co-purify with the Notch Ternary Complex in the Notch-driven T-cell lymphoma cell line 4084 by affinity chromatography. Notch-independent lymphoma 6780 was used as a negative control. b Representative ChIP on the HES1, HES4, and CCND1 promoters in OE19 EAC. Bars represent standard deviation (N = 3). c Expression of Notch1 target genes in OE19 and OE33 upon INTS11 knockdown. Bars represent standard deviation (the mean from 3 biological replicates). **p < 0.01 versus CTR sample. d Expression of Notch1 target genes in OE19 and OE33 upon NACK knockdown. Bars represent standard deviation (the mean from 3 biological replicates). **p < 0.01 versus CTR sample
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