Effect of overexpression of cleavage/polyadenylation factor CFIm25 on monocyte-macrophage differentiation
Macrophages play a crucial role in our body's development and tissue repair and protect against microbial attacks via cytokine secretion or phagocytosis. A previous study in our lab revealed a crucial role for regulation of mRNA 3' end cleavage and polyadenylation (C/P) in monocyte to macrophage differentiation. Among the various C/P factors involved, the subunit CFIm25 showed a striking increase at an early time point upon treatment of monocytes with the differentiating agent Phorbol Myristate Acetate (PMA), suggesting that CFIm25 may promote this process. To study its role in macrophage differentiation, CFIm25 was overexpressed in the HL-60 and THP-1 monocytic cells, followed by PMA-mediated differentiation. Both cell lines showed a significant increase in macrophagic characteristics and a decrease in monocytic features. Cell cycle was slowed down, accompanied by a greater decrease in the proliferation markers PCNA and cyclin D1 coupled with increased 3'UTR lengthening of cyclin D1 mRNA. Since alternative polyadenylation (APA) could be greatly affected by manipulating CFIm25, we performed mRNA 3' end-focused sequencing to reveal changes in the global APA landscape during differentiation and after CFIm25 overexpression. Among the genes whose 3'UTRs were shortened, TAB2 and TBL1XR1 which are known positive regulators of the NF-?ß signaling pathway, underwent shortening followed by increased protein expression compared to the control. Examination of other components of the NF-?ß pathway showed an increase at earlier time in NF-?ß-p50 and phosphorylated NF-?ß-p65 and in the NF-?ß, targets p21, Bcl-XL, ICAM1 and TNF-a upon CFIm25 overexpression There was also an increase in the shorter isoforms of NF-?B1 mRNA that encodes the NF-?ß p50 precursor. Depletion of CFIm25 suppressed the NF-?ß pathway, as evident by lengthening of TAB2, TBL1XR1, and NF-?B1 mRNAs accompanied by decreased expression of TAB2, TBLXR1, and the NF-?ß proteins. In conclusion, our study supports a model in which CFIm25 accelerates the monocyte to macrophage transition by promoting APA events which lead to activation of the NF-?ß pathway. Overall design: To determine the effects of CFIm25 on global PAS usage during macrophage differentiation, we used the QuantSeq 3' mRNA-sequencing method which can provide information on differential APA events. PolyA+-selected RNA samples from HL-60 cells overexpressing CFIm25 and control cells after 6 h of PMA treatment were sequenced and analyzed to identify changes in isoform usage.