Page 131 - 2018_12-Haematologica-web

BET inhibition modulates miRNAs in DLBCL
A
B
C
D
Figure 3. OTX015 reduces binding of BRD4 to PRMT5 and diminishes recruitment of PRMT5 to the microRNA-96-5p promoter. (A) Analysis of publicly available chromatin immunoprecipitation (ChIP) sequencing data of diffuse large B-cell lym- phoma (DLBCL) cells treated with the BET inhibitor JQ1 showed that BRD4 binds to the 5’ regulatory region of PRMT5 and that BET inhibitor treatment reduces BRD4 recruitment to PRMT5. (B,C) ChIP was performed for DLBCL cells treated with dimethyl sulfoxide (DMSO) or 500 nM OTX015 for 48 h. Anti-BRD4, anti-PRMT5 and anti-IgG (nega- tive control) antibodies were used for immunopre- cipitations. (B) Chromatin pulled down with anti- BRD4 and anti-IgG in DMSO- and OTX015-treated SU-DHL-2 cells was amplified with primers specific for the 5’ regulatory region of PRMT5 identified by the analysis in (A). (C) Chromatin pulled down with anti-PRMT5 and anti-IgG antibodies in DMSO- and OTX015-treated HBL-1 cells was amplified with primers specific for the promoter of miR-96-5p. Amplification of the same immunoprecipitated chromatin samples was performed with primers specific for the chromosome 4 human alpha satel- lite sequence as an additional negative control (representative results from one of two biological replicates are shown). The graphs show the mean fold-difference between DMSO- and OTX015-treat- ed cells after normalization to input and IgG back- ground subtraction. ChIP-quantitative polymerase chain reaction experiments were repeated twice in triplicate. (D) Proposed model for the upregulation of miR-96-5p expression following treatment of DLBCL cells with OTX015. Upper panel; previous work by others has shown that PRMT5 is overex- pressed in lymphoma cells in which it mediates transcriptional repression of miR-96 and that over- expression of miR-96 negatively regulates PRMT5 translation.39,49 Lower panel; here we showed that BRD4 binds to the upstream regulatory region of PRMT5. Treatment of DLBCL cells with a BET inhibitor reduced BRD4 occupancy at the PRMT5 locus and also reduced the expression of PRMT5 mRNA and protein. Additionally, the BET inhibitor diminished the occupancy of PRMT5 at the miR- 96 promoter and increased miR-96 expression. **P<0.01. Error bars denote the standard error.
haematologica | 2018; 103(12)
2055