Functions and Specificities of Tristetraprolin (TTP) Family Members

Members of the tristetraprolin (TTP) family of RNA-binding proteins bind to mRNAs that contain specific AU-rich element (ARE) binding sites and promote the decay of target mRNAs. The defining feature of all TTP family members is the presence of a tandem zinc finger (TZF) domain that binds to AREs in the 3’-untranslated regions (3’-UTR) of target mRNAs. Many family members also contain a CNOT1 binding domain that has been shown to bind to CNOT1, a large scaffolding protein of the CCR4-NOT complex. Mice expressing TTP protein with the CNOT1 binding domain deleted (CNBD mice), developed only a mild inflammatory phenotype, in stark contrast to the severe phenotype of TTP KO mice, or mice expressing TTP with a C116R point mutation in the tandem zinc finger domain. These data suggest that the CNOT1 binding domain is important for some of TTP’s physiological functions, but not as critical as the TZF domain for TTP’s function. Yet, it remains unclear whether the CNOT1 binding domain of TTP is important to regulate specific targets in specific tissues.Three TTP family proteins are conserved in mammals (TTP, ZFP36L1, and ZFP36L2), encoded by the mouse genes Zfp36, Zfp36l1, and Zfp36l2, respectively. TTP, ZFP36L1, and ZFP36L2 behave similarly biochemically in assays of RNA-binding, mRNA deadenylation, and decay. Yet, knock-out (KO) mice for each gene have very different phenotypes, suggesting that each TTP family member has specific physiological functions. ZFP36 (TTP) is known for regulating cytokine expression in myeloid cells, and its deficiency leads to a severe, spontaneous, inflammatory phenotype; however, ZFP36L1 and ZFP36L2 have not been viewed as important in controlling inflammation. It is unclear whether the biochemical activities of these proteins are interchangeable or independent, and/or whether effects on target transcripts are solely dependent on the cell-specific expression of each protein. It is also unknown whether synergistic interactions exist among TTP family members and whether they can compensate for one another when the expression levels are altered. In the major project described in this thesis, I studied potential functional overlaps of these proteins in myeloid cells, by developing 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, referred to as M-triple KO mice, developed a severe spontaneous inflammatory phenotype, with a median survival of 8 weeks. Histopathological evaluation showed severe arthritis of peripheral joints and dramatic myeloid hyperplasia in tissues and bone marrow, as well as soft tissue inflammatory cell invasion. MicroCT analysis of the front and hind paws indicated severe bone loss and joint destruction and ankylosis. RNA-Seq analysis of mRNA from triple KO macrophages treated with LPS, followed by actinomycin D to inhibit transcription and allow for measurement of mRNA decay rates, demonstrated abnormal stabilization of many more cytokine and chemokine mRNAs than were seen in similar studies of cells from myeloid-specific TTP KO mice. Cytokine immunoassays also demonstrated increased levels of pro-inflammatory cytokines in serum from triple KO mice and in medium from LPS-stimulated M-triple KO macrophages. These findings suggest that simultaneous deficiency of Zfp36, Zfp36l1, and Zfp36l2 in myeloid cells leads to the synergistic development of a lethal inflammatory syndrome due to excess accumulation of pro-inflammatory cytokines. Our findings emphasize the importance of all three family members, acting in concert, in myeloid cell function. As noted above, TTP has been shown to regulate cytokine mRNA stability, and loss of TTP leads to chronic excess levels of many pro-inflammatory cytokines. Many autoimmune diseases are characterized by chronic excess levels of the same cytokines that are increased in Zfp36-KO mice. Therefore, we speculated that increased expression of TTP could have a beneficial effect on inflammatory diseases. Mice with regulated overexpression of TTP are protected from many models of inflammatory diseases in mice. In a separate project, we and collaborators demonstrated that mice overexpressing TTP were protected from a two-stage carcinogenesis model. I used RNA-Seq to identify transcriptome changes, and found that many pro-inflammatory genes were down-regulated in the skin from mice overexpressing TTP, compared to WT, after exposure to 12-0 tetradeccanoylphorbol-13-accetate (TPA) and dimethylbenz[a]anthracene (DMBA) in an established two-stage model of skin carcinogenesis. In a third project described in this thesis, we hypothesized that the C-terminal portion of TTP, which contains the CNOT1 binding domain, is vital to recruit exonucleases and promote deadenylation of the target mRNA. To determine if deletion of the CNOT1 binding domain of TTP in mice has effects on transcript turnover in mice, I chose four tissues in which TTP is expressed (liver, spleen, colon, and adipose tissue), and performed transcriptome analysis and differential gene expression analysis in these tissues from WT, TTP KO, and CNBD mice. We found that potential TTP target transcripts were differentially regulated in tissues from mice expressing TTP protein lacking the CNOT1 binding domain. Some transcripts were up-regulated to similar levels in tissues from both TTP KO and TTP CNBD mice, while other transcripts were up-regulated at higher levels in tissues from TTP KO mice than in tissues from TTP CNBD mice. These data suggest that the CNOT1 binding domain is important, but not the only factor necessary, for the ability of TTP to regulate mRNA stability in tissues, such as liver, spleen, colon, and adipose tissue. The work described in this dissertation increases our understanding of the functions and specificity of TTP family members, and the therapeutic potential of TTP and its family members in the treatment of inflammatory diseases.