Differential Gene Expression and mRNA decay in Wild-Type and Myeloid-specific TTP Knock-out macrophages using RNA-Seq

Members of the tristetraprolin (TTP) family of RNA-binding proteins can bind to and promote the decay of specific transcripts containing AU-rich motifs. ZFP36 (TTP) is best known for regulating cytokine expression in myeloid cells; however, the mammalian paralogues ZFP36L1 and ZFP36L2 have not been viewed as important in controlling inflammation. To study potential functional overlaps of these three TTP family proteins in myeloid cells, we developed myeloid-specific knock-out (M-KO) mice of these genes, singly and together. M-Zfp36-KO mice exhibited a mild inflammatory syndrome late in life, while M-Zfp36l1-KO and M-Zfp36l2-KO mice had no apparent spontaneous phenotypes. Mice with simultaneous deficiency of all three TTP family members in myeloid cells developed a severe, spontaneous, inflammatory phenotype, with a median survival of 8 weeks. Macrophages derived from these mice contained many more stabilized transcripts than cells from M-Zfp36-KO mice, many encoding pro-inflammatory cytokines and chemokines. Our findings emphasize the importance of all three family members, acting in concert, in myeloid cell function. Overall design: We generated mRNA profiles of wild-type (WT) and M-TTP knock-out (KO) bone marrow-derived macrophages at 8 different timepoints in quadruplicate, using RNA-Seq. The first goal of this study was to compare transcriptome-wide gene expression in wild-type (WT) and myeloid-specific TTP (M-TTP) knock-out (KO) macrophages before and after lipopolysaccharide (LPS) treatment. The second goal of this study was to compare mRNA decay rates in WT and M-TTP KO macrophages after LPS and Actinomycin D (ActD) treatment.