![]() ![]() Conversely, overexpression of a GFPZFP36L1 transgene reduces cell cycle entry. Furthermore, DCKO thymocytes have elevated expression of positive cell cycle regulators and show increased cycling and DDR pathway activation in vivo. Our results show that DN3 thymocytes lacking Zfp36l1/l2 closely share gene expression profiles with postselection DN3b wild-type thymocytes, despite having reduced VDJ recombination of Trbv gene segments and being icTCRβ negative. We integrate RNA sequencing (RNA-seq) gene expression data with individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) ( 25) to identify RBP binding positions within their mRNA targets. In this report, we combine the detailed phenotypic analyses of early thymocytes from DCKO mice with genome-wide approaches to identify the molecular mechanisms regulated by the RBPs. A better understanding of the spectrum of mRNAs bound by Zfp36l1/l2 in thymocytes is necessary to elucidate the molecular mechanisms through which they regulate the development and proliferative properties of thymocytes. However, the details of how the β-selection checkpoint is circumvented remain unknown. These tumors are dependent on Notch1, for which expression is increased following the release of its mRNA from posttranscriptional repression by Zfp36l1/l2. By contrast, the conditional deletion of both Zfp36l1 and Zfp36l2 (double conditional KO ) in thymocytes results in the bypass of the β-selection checkpoint and development of T cell acute lymphoblastic leukemia (T-ALL) ( 24). ![]() Although the development of B cells lacking both Zfp36l1 and Zfp36l2 is impaired, these mice do not develop B cell malignancy. During early B cell development, Zfp36l1/l2 act redundantly to enforce quiescence and enable recombination of the Ig genes ( 23). Constitutive knockout (KO) of Zfp36 leads to viable animals that develop an autoimmune disease caused by the overexpression of the proinflammatory cytokine TNF ( 17– 19), whereas Zfp36l1- or Zfp36l2-null mice die in utero or shortly after birth due to disorganized vasculature or anemia, respectively ( 20– 22). As such, many mRNAs have been proposed as targets of ZFP36 family proteins, although few have been shown to be physiologically relevant ( 16). These RBPs bind to A/U-rich elements (AREs) in the 3′ untranslated region (UTR) of mRNA and promote RNA decay ( 16). The ZFP36 family of RNA-binding proteins (RBP) comprises three gene family members in humans and four in mice. Remarkably, the activation of these pathways has been linked to the promotion of thymocyte differentiation ( 14, 15) as well as transformation. Atm and DNA-dependent kinase catalytic subunit are also responsible for the activation of the Chk1 and Chk2 protein kinases, which phosphorylate multiple downstream effectors, including Cdc25a and p53, leading to cell cycle arrest and DSB resolution/repair ( 12, 13). p-H2AFX then recruits other DDR factors to the break site and stabilizes cleaved DNA ends prior to joining ( 8– 11). A critical target of these kinases is histone variant H2AFX, which is phosphorylated (p-H2AFX) at the site of DNA damage ( 7). These lead to activation of ataxia-telangiectasia–mutated (Atm), DNA-dependent kinase catalytic subunit, and Atm- and Rad3-related (Atr) ( 5, 6). During VDJ recombination, double-strand DNA breaks (DSBs) are formed by the RAG complex and activate the DNA damage response (DDR) pathway. This dramatically expands the pool of thymocytes with successful Trb rearrangements, which can progress to the double-positive (DP) stage of development ( 2). ![]() They are selected by a process known as the β-selection checkpoint at which icTCRβ positive DN3b cells undergo a proliferative burst and have an increased metabolic state as shown by CD98 expression ( 3, 4). The DN3 stage is further divided into DN3a and DN3b cells, the latter of which have successfully recombined the V, D, and J gene segments of the Trb locus and express intracellular (ic)TCRβ. During the first stages of T cell differentiation in mice, CD4 −CD8 − double-negative (DN) thymocytes can be subdivided into DN1–4 populations based on surface expression of CD44 and CD25 ( 1– 3). These checkpoints ensure TCR reactivity and prevent transformation into highly proliferative states. Early thymocyte development occurs in multiple quasi-discrete stages during which cells are required to pass through stringent developmental checkpoints. ![]()
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