To elucidate the functional role of Satb1 in T cell development and differentiation

Abstract

As the early thymic precursors migrate from the bone marrow to the thymus, the T lineage commitment is initiated. Determined by the levels of surface receptors CD4 and CD8, the development of T cells is divided into a number of temporally and physiologically different phases(Germain, 2002). The precursors, which do not possess CD4 and CD8, are referred to as double negative (DNs). Upon migration from the cortical segments of the medulla, DN thymocytes evolve to become double positive (DP) cells (Rothenberg et al., 2008). DPs are chosen based on a selection procedure and have a full αβTCR on their cell surface. Thymocytes that have undergone positive selection go on to become CD4 or CD8 single positive thymocytes (Singer and Bosselut, 2004). These thymocytes after maturation go to the secondary organs such as spleen and lymph nodes so they may multiply and react to antigenic cues to build a strong immune response (Lancaster et al., 2018). A fundamental set of factors play vital functions in this sequence of events by effectively triggering or blocking particular genes in conjunction with external stimuli. Though they regulate distinct genes, several of these TFs also play a role in the development during the later T cell stages (Rothenberg, 2014; Yui and Rothenberg, 2014). One such factor is SATB1. Special AT rich binding protein (SATB1) is a transcription factor and chromatin organizer that is enriched in T cells (Galande et al., 2007). Nuclear protein SATB1 expression occurs first in the DN4 stage and then increased in the DP and CD4 phases. It has been previously demonstrated that in naïve CD4 T lymphocytes (Gottimukkala et al., 2016; Stephen et al., 2017), TCR stimulation positively regulates SATB1. Additionally, it was shown that SATB1 in CD4 cells has a "bimodal" distribution (Gottimukkala et al., 2016). The need for several transcriptionally active units is decreased when SATB1 loops the MHC class-I locus into a transcriptionally active area (Kumar et al., 2007). It has also been demonstrated that Th2 specific cytokine genes are regulated transcriptionally and looped (Cai et al., 2006). Recent research using SATB1 null mice (Alvarez et al., 2000) as well as conditional knockout (cKO) has demonstrated that SATB1 deficiency partially affects positive selection (Kondo et al., 2016). Furthermore, it has been demonstrated that SATB1 is necessary for both the derepression of the CD8a gene and ThPOK expression (Kakugawa et al., 2017). Moreover, it has also been shown that SATB1 directly binds to the promoter region of PD1 to control its expression (Stephen et al., 2017). The molecular processes behind SATB1's involvement in thymocyte formation and naïve T cell activation remain unclear despite a plethora of investigations. 17 My doctoral study focuses on the importance of SATB1 in maintaining the gene regulation during T cell development via TCR activation by maintaining appropriate 3D chromatin architecture. The work done during the period is divided into four chapters, summarized below: 1. Satb1 regulates genome conformation and gene expression along with the cohesin complex in T cells In this study, we demonstrate that the T lineage-enriched chromatin organizer Satb1 shares majority of its binding sites across the genome in conjunction with the Cohesin complex as well as Ctcf in DP thymocytes. Genomic analyses revealed that Satb1 and Smc1a share multiple features such as binding motif, genomic locations and TSS binding. We confirm that Satb1 and Smc1a interact in vivo as well as in a purified setup. Satb1 shows strong interaction with the C-terminal regions of Smc1a, while Smc1a shows a preference for the ULD/PDZ domain of Satb1. Moreover, Satb1 and Smc1a synergistically regulate the expression of CD3, a key surface receptor on T cells (Gascoigne et al., 2011), via directly binding its promoter. In the periphery, we observe that CD4+ T cells exhibit dysregulated activation phenotype in Satb1 and Smc1a KO cells, indicating their collaborative effect on TCR signaling. Collectively, we demonstrate a molecular mechanism by which Satb1 mediates chromatin looping via its dynamic DNA binding and recruitment of the Cohesin complex to orchestrate 3D genome organization during T cell differentiation. 2. Identification and characterization of liquid-like condensate formation by Satb1 in thymocytes We describe the ability of Satb1 to undergo condensate formation in this chapter. First, we determine the level of atypical characteristics in Satb1's amino acid composition. Next, we evaluated the biophysical characteristics of phase separation in vitro, including dependency on concentration, nucleic acid binding, and fluidity. Additionally, we investigated the particular domains implicated in Satb1's demixing behavior. We demonstrate that Satb1 produces nuclear foci in several phases of thymocyte development, which is consistent with phase behavior. In DP thymocytes, we also examined the dynamic nature of these foci. We show that nuclear foci of Satb1 co-localize with those of Smc1a, which also exhibit foci-like patterning. In contrast, Ctcf exhibits a more dispersed localization throughout different phases of T cell development. Finally, we demonstrate that the phase shifts of Satb1 in vitro is significantly influenced by certain amino acid residues of Satb1 linked to neurological disorders 18 and cancer. The capacity of Satb1 to undergo condensate formation may collectively increase its role in the control of transcription and genomic organization. 3. Role of Satb1 in regulating TCR-dependent genes in conjunction with NFAT family proteins during T cell development Development of thymocytes is highly dependent on TCR signaling, which receive the signal in a spatiotemporally controlled manner (Love et al., 2000). SATB1, a T-lineage enriched chromatin organizer, controls the expression of various genes important in thymocytes development, and it is under the control of TCR signal (Gottimukkala et al., 2016; Patta et al., 2020). SATB1-deficient thymocytes do not develop beyond double positive (DP) stage and SATB1 knockout (KO) CD4+ T cells undergo apoptosis upon TCR activation (Kondo et al., 2016), underscoring the crucial role of SATB1 in thymic selection. The mechanisms by which SATB1 mediates its regulatory function in developing thymocytes is only partly understood. We observed that in developing as well as naïve T-cells, expression of Satb1 is contingent on the strength of TCR stimuli in a linear manner. Furthermore, upon TCR activation of thymocytes, SATB1 interacts with nuclear factor of activated T-cells (NFAT), which we observe to be important for regulation of the transcriptional activity of numerous T cell specific genes such as Irf4, Dusp22 and IL2. Additionally, SATB1 has reduced occupancy on many of the target genes –which we observe to be enriched upon T cell activation –if NFAT is inhibited. We therefore conclude that these two proteins cooperate to regulate T cell specific key genes upon TCR mediated activation. 4. Dynamic modulation in the transcriptome of γδ T cells upon T cell activation and cross-talks with Notch signaling Gamma delta (γδ) T cells, especially the Vγ9Vδ2 subtype have been implicated in cancer therapy and thus have earned the spotlight in the past decade (Zhao et al., 2018). Although one of the most important properties of γδ T cells is their activation by phosphoantigens which are, intermediates of the Mevalonate and Rohmer pathway of isoprenoid biosynthesis (Correia et al., 2009; Rigau et al., 2020), such as IPP and HDMAPP respectively, the global effects of such treatments on Vγ9Vδ2 T cells remain elusive. Here, we used the high-throughput transcriptomics approach to elucidate the transcriptional changes in human Vγ9Vδ2 T cells upon HDMAPP, IPP and anti-CD3 treatments in combination with IL2 cytokine stimulation. These activation treatments exhibited a dramatic surge in transcription with distinctly enriched pathways. We further assessed the transcriptional dynamics upon inhibition of Notch signaling coupled with activation treatments. We observed that the metabolic processes are most 19 affected upon Notch inhibition via GSI-X. The key effector genes involved in gamma-delta cytotoxic function were downregulated upon Notch blockade even in combination with activation treatment, thus, showing a transcriptional interplay between TCR signaling and Notch signaling in Vγ9Vδ2 T cells. Upon Notch inhibition, genes regulated by SATB1 upon γδ T cell activation via IPP, are dysregulated, suggesting its role at the juncture of both the pathways. Collectively, we show how activation of TCR signaling by phosphoantigens or anti- CD3 changes the transcriptional status of Vγ9Vδ2 T cells along with IL2 stimulation, and how blockade of Notch signaling can affect this activation. References