Browsing by Author "Blackshear, Perry J"

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.

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.

The tristetraprolin (TTP) protein family of RNA-binding proteins contains three widely expressed mammalian protein members: TTP (ZFP36), ZFP36L1, and ZFP36L2, all of which can regulate gene expression by binding to specific AU-rich sequences located in the 3'-untranslated regions (3’-UTR) of mRNAs and accelerating their decay. Unique among the three, ZFP36L2 plays a pivotal role in maintaining hematopoietic stem cells (HSC) during development. ZFP36L2-deficient mice exhibit severely impaired definitive hematopoiesis and die approximately two weeks after birth due to severe anemia, thrombocytopenia, and internal hemorrhage. Recent single-cell RNA sequencing (scRNA-seq) studies have demonstrated widespread Zfp36l2 expression in HSC and the hematopoietic system during both development and adulthood.

Despite the recognized importance of ZFP36L2 in maintaining HSC, there are still numerous aspects of its role that are not fully understood, hindering our understanding of hematopoietic regulation. While prior studies have provided valuable insights into the physiological function of ZFP36L2, its molecular mechanisms in HSC development and hematopoietic system maintenance remain poorly defined. To decipher its involvement in hematopoiesis, it is crucial to identify the mRNA targets and pathways regulated by ZFP36L2 and determine whether this regulation is intrinsic to HSC. Moreover, the premature death of ZFP36L2-deficient mice makes it unclear to what extent this protein governs adult HSC function. Lastly, considering the pivotal position of HSC in the hematopoietic hierarchy, it is important to investigate how ZFP36L2's activity in HSC affects the subsequent differentiation of hematopoietic lineages. Clarifying this relationship could yield valuable insights into the post-transcriptional mechanisms that govern HSC biology and potentially lead to the identification of new therapeutic targets for hematological disorders.

To address the knowledge gap, we employed a detailed analysis combining flow cytometry and scRNA-seq to examine HSC and hematopoietic progenitor cells (HSPC) at several critical developmental stages. Our studies revealed that the absence of ZFP36L2 resulted in significant reductions in both HSC and immature progenitors during mouse development, primarily due to HSC-autonomous dysregulation. In addition, scRNA-seq analysis of HSC and progenitors revealed that ZFP36L2 deficiency caused abnormal upregulation of transcripts related to cell cycle regulation and lymphoid specification, leading to aberrant cell cycle progression and premature lymphoid lineage commitment. This ultimately resulted in cellular damage and HSC exhaustion at birth. These findings demonstrate that ZFP36L2 is essential for maintaining the homeostasis of HSC, and emphasizes the significance of restraining lineage commitment and excessive self-renewal during HSC development.

In a related study, we investigated possible functional overlap of ZFP36L2 and TTP. We developed mice (L2KO/TTP∆ARE) that lacked Zfp36l2 but modestly overexpressed TTP throughout the body. L2KO/TTP∆ARE mice not only survived but also exhibited normal peripheral blood counts, except for residual moderate thrombocytopenia. We took advantage of this rescued ZFP36L2-deficient model and investigated the role of ZFP36L2 in adult hematopoiesis. We discovered that megakaryocyte (MK) progenitors and MK-biased HSC were decreased in bone marrow from L2KO/TTP∆ARE mice and exhibited enriched erythroid and decreased MK gene signatures. In addition, L2KO/TTP∆ARE HSC failed to reconstitute hematopoiesis upon non-competitive transplantation, and showed molecular features of stress and reduced cycling. Thus, TTP can assume some functions of ZFP36L2 in a genetic dose-dependent manner, but ZFP36L2 may be specifically required for the maintenance of megakaryopoiesis and HSC function.In summary, our studies provide novel insights into the essential role of ZFP36L2 in the maintenance of HSC throughout both developmental stages and adulthood. We demonstrate that ZFP36L2 is essential for restraining abnormal cell cycling and lymphoid commitment during HSC development, thereby ensuring proper HSC maintenance. Interestingly, our results also suggest that TTP may partially compensate for ZFP36L2 deficiency during the development of the hematopoietic system, but that ZFP36L2 may have specific functions in maintaining megakaryopoiesis and HSC function in adulthood. Overall, our research provides a strong foundation for future studies aimed at elucidating the underlying mechanisms that govern ZFP36L2's function in hematopoiesis, further advancing our understanding of the intricate regulatory paradigm of HSC biology.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich elements in the 3' untranslated regions of certain cellular mRNAs, leading to their deadenylation and destabilization. Studies in knockout mice have demonstrated roles for three of the family members, TTP, ZFP36L1 (L1), and ZFP36L2 (L2), in inflammation, chorioallantoic fusion, and hematopoiesis, respectively. However, little is known about a recently-discovered TTP family member, ZFP36L3 (L3). Although L3 exhibits similar general biochemical functions to other members of the TTP family, initial studies of this family member revealed a number of unique characteristics.

First, L3 does not shuttle between the nucleus and cytoplasm like TTP, L1, and L2. Through studies of L3 deletion mutants, we determined that a nuclear localization signal that resides within the conserved tandem zinc finger domain was functional, although the C-terminal nuclear export sequence was non-functional. We then demonstrated that the unique repeat domain of L3 was responsible for the "full-time" cytoplasmic localization of the protein and was able to override the ability of the nuclear localization signal to direct transport into the nucleus.

In addition, L3 is specifically expressed in rodent yolk sac and placenta, while the other members of the TTP family exhibit relatively ubiquitous expression. We further examined the expression of L3 at both the RNA and protein level. Through northern and western blotting, we demonstrated the expression of L3 during mid-to-late gestation in mouse placenta. We also performed immunostaining of placental sections to demonstrate that this protein is exclusively expressed in the cytoplasm of the labyrinthine trophoblast cells and trophoblast giant cells of the placenta.

L3 most likely binds to and promotes the decay of a certain set of mRNA transcripts. Because of its specific sites of expression, we hypothesized that L3 may regulate the decay of a set of mRNAs that are important for the development or physiology of the placenta. We employed the ribonucleoprotein immunoprecipitation-microarray analysis of mouse placenta lysates to identify possible mRNA targets of L3. Our study identified approximately 400 transcripts that were enriched in immunoprecipitates using a highly specific L3 antibody. Some of these transcripts could be bound and downregulated by L3 in a physiological setting. Our top candidate transcript, based on relative enrichment and sequence analysis, was B-type natriuretic peptide, a hormone well-known for its role in cardiac physiology. We confirmed the expression of B-type natriuretic peptide in mouse placenta through northern blotting and in situ hybridization histochemistry. We also verified the ability of L3 to directly bind to and promote the degradation of this transcript in electrophoretic mobility shift assays and co-transfection assays, respectively.

Lastly, L3 demonstrates a unique migration characteristic in denaturing polyacrylamide gel electrophoresis as compared to TTP, L1, and L2. It migrates as two distinct species of Mr ~90,000 and ~100,000. We investigated the basis for this unusual migration in studies of deletion mutants and serine mutants. We found that both phosphorylation and the presence of the conserved C-terminus are required for the existence of the slower-migrating species. We then focused our study on phosphorylation of the C-terminus and discovered that the phosphorylation of Ser721 may play a role in creating the slower-migrating species. We also identified four other phosphorylated residues with mass spectrometry. Finally, we examined the effect of the C-terminus on the function of L3 and determined that this conserved region is not required for mRNA binding or to promote mRNA deadenylation or degradation in our assays.

The work described in this dissertation increases our understanding of this unique tristetraprolin family member, L3. Additional study of this protein is required to further elucidate its role in the physiology of rodent placenta, and to determine whether this role is subsumed by one of the other TTP family members in the placentas of other mammals.