Identification of PRDM5 as a new candidate oncogene in MM. (A) Schematic strategy for identification of coregulated chromatin regions. Genome browser snapshot of the coregulated chromatin region comprising the PRDM5 and NDNF locus. Displayed tracks represent the chromatin state annotation in MM patients and normal B cells, and an additional track of H3K27ac peaks in KMS-11 cell line (ENCODE Consortium, ENCSR715JBO). (B) PRDM5 and NDNF expression analyzed by RNA-seq (left and center panel), and correlated expression levels in MM patients (right panel). (C) Normalized levels of chromatin interaction frequencies from the indicated viewpoint (purple arrowhead) between PRDM5 and NDNF gene loci as analyzed by 4C-seq in the MM.1S MM cell line expressing both transcripts. Lowest contact frequencies are indicated in turquoise, and highest frequencies in red. PRDM5 and NDNF location are shown above the 4C-seq track; black bar represents area of increased interactions, while H3K27ac peaks in this cell line are shown below. (D) Validation of PRDM5 (upper panel) and NDNF (lower panel) knockdown after shRNA expression determined by RT-qPCR. Expression values are normalized to the same condition prior to addition of doxycycline. Statistical analysis compares the effect of each shRNA versus the scramble shRNA (shGFP). (E) Relative cell proliferation rate (%) of KMS-11 cell line normalized to the same condition prior to addition of doxycycline. The upper panel presents the results for PRDM5 knockdown cells and the lower panel for NDNF knockdown cells. Statistical analysis compares the effect of each shRNA versus the scramble shRNA (shGFP). (F) Effect of PRDM5 (upper panel) and NDNF (lower panel) knockdown in cell apoptosis, as determined by Annexin V flow cytometry analysis. (G) Heat map of gene expression levels (indicated as Z-scores) of differentially expressed genes in PRDM5 knockdown cells before and after 4 d of the addition of doxycycline (n = 2 for control and Dox groups; 1216 genes). (H) List of top 10 hallmark gene signatures, determined using MSigDB Collection for genes down-regulated (upper panel) or up-regulated (lower panel) in PRDM5 knockdown cells 4 d after the addition of doxycycline. (Dox) Doxycycline. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (ns) not significant.
