Sung H. Jeon

SRG3 Interacts Directly with the Major Components of the SWI/SNF Chromatin Remodeling Complex and Protects Them from Proteasomal Degradation

The mammalian SWI/SNF complex is an evolutionarily conserved ATP-dependent chromatin remodeling complex that consists of nine or more components. SRG3, a murine homologue of yeast SWI3, Drosophila MOIRA, and human BAF155, is a core component of the murine SWI/SNF complex required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. Here we report that SRG3 interacts directly with other components of the mammalian SWI/SNF complex such as SNF5, BRG1, and BAF60a. The SWIRM domain and the SANT domain were required for SRG3-SNF5 and SRG3-BRG1 interactions, respectively. In addition, SRG3 stabilized SNF5, BRG1, and BAF60a by attenuating their proteasomal degradation, suggesting its general role in the stabilization of the SWI/SNF complex. Such a stabilization effect of SRG3 was not only observed in the in vitro cell system, but also in cells isolated from SRG3 transgenic mice or knock-out mice haploinsufficient for the Srg3 gene. Taken together, these results suggest the critical role of SRG3 in the post-transcriptional stabilization of the major components of the SWI/SNF complex.

BAF60a Interacts with p53 to Recruit the SWI/SNF Complex

To understand the tumor-suppressing mechanism of the SWI/SNF chromatin remodeling complex, we investigated its molecular relationship with p53. Using the pREP4-luc episomal reporter, we first demonstrated that p53 utilizes the chromatin remodeling activity of the SWI/SNF complex to initiate transcription from the chromatin-structured promoter. Among the components of the SWI/SNF complex, we identified BAF60a as a mediator of the interaction with p53 by the yeast two-hybrid assay. p53 directly interacted only with BAF60a, but not with other components of the SWI/SNF complex, such as BRG1, SRG3, SNF5, or BAF57. We found out that multiple residues at the amino acid 108–150 region of BAF60a were involved in the interaction with the tetramerization domain of p53. The N-terminal fragment of BAF60a containing the p53-interacting region as well as small interfering RNA for baf60a inhibited the SWI/SNF complex-mediated transcriptional activity of p53. The uncoupling of p53 with the SWI/SNF complex resulted in the repression of both p53-dependent apoptosis and cell cycle arrest by the regulation of target genes. These results suggest that the SWI/SNF chromatin remodeling complex is involved in the suppression of tumors by the interaction with p53.

Notch1 confers a resistance to glucocorticoid-induced apoptosis on developing thymocytes by down-regulating SRG3 expression

We previously have reported that SRG3 is required for glucocorticoid (GC)-induced apoptosis in the S49.1 thymoma cell line. Activation of Notch1 was shown to induce GC resistance in thymocytes. However, the specific downstream target of Notch1 that confers GC resistance on thymocytes is currently unknown. We found that the expression level of SRG3 was critical in determining GC sensitivity in developing thymocytes. The expression of SRG3 also was down-regulated by the activated form of Notch1 (NotchIC). The promoter activity of the SRG3 gene also was down-regulated by NotchIC. Expression of transgenic SRG3 resulted in the restoration of GC sensitivity in thymocytes expressing transgenic Notch1. These results suggest that SRG3 is the downstream target of Notch1 in regulating GC sensitivity of thymocytes.

CD7 expression and galectin-1-induced apoptosis of immature thymocytes are directly regulated by NF-κB upon T-cell activation

CD7, one of the galectin-1 receptors, has crucial roles in galectin-1-mediated apoptosis of activated T-cells and T-lymphoma progression in peripheral tissues. In this study, we showed that CD7 promoter activity was increased by NF-kappaB and that this activity was synergistic when Sp1 was co-expressed in the immature T-cell line L7. Site-directed mutagenesis analysis of the CD7 promoter indicated that NF-kappaB specifically bound to the NF-kappaE2 site in cooperation with Sp1. Overexpression of E12 or Twist2 proteins negatively regulated NF-kappaB-mediated activity of the CD7 proximal promoter. In addition, CD7 expression was down-regulated by treatment with the p38 MAPK inhibitor SB20358, or the MSK1 inhibitor H-89. These signaling pathway inhibitors prevented galectin-1-mediated apoptosis of immature T-cells. From these results, we concluded that the regulation of CD7 gene expression through NF-kappaB activation induced by TCR/CD28 might have significant implications for T-cell homeostasis.

T Cell Receptor Signaling Inhibits Glucocorticoid-induced Apoptosis by Repressing the SRG3 Expression via Ras Activation

Activation of T cell antigen receptor (TCR) signaling inhibits glucocorticoid (GC)-induced apoptosis of T cells. However, the detailed mechanism regarding how activated T cells are protected from GC-induced apoptosis is unclear. Previously, we have shown that the expression level of SRG3, a murine homolog of BAF155 in humans, correlated well with the GC sensitivity of T cells either in vitro or in vivo. Intriguingly, the expression of SRG3 decreased upon positive selection in the thymus. Here we have shown that TCR signaling inhibits the SRG3 expression via Ras activation and thereby renders primary thymocytes and some thymoma cells resistant to GC-mediated apoptosis. By using pharmacological inhibitors, we have shown that Ras-mediated down-regulation of the SRG3 gene expression is mediated by MEK/ERK and phosphatidylinositol 3-kinase pathways. Moreover, TCR signals repressed the SRG3 transcription through the putative binding sites for E proteins and Ets family transcription factors in the proximal region of the SRG3 promoter. Introduction of mutations in these elements rendered the SRG3 promoter immune to the Ras or TCR signals. Taken together, these observations suggest that TCR signals result in GC desensitization in immature T cells by repressing SRG3 gene expression via Ras activation.

E2A/HEB and Id3 proteins control the sensitivity to glucocorticoid-induced apoptosis in thymocytes by regulating the SRG3 expression.

The E protein family transcription factors encoded by the E2A and HEB genes are known to play critical roles in the coordinate regulation of lymphocyte development. Previous studies have shown that T cell receptor (TCR) signals rapidly induce Id3, a dominant negative antagonist of E2A activity and allow thymocytes to survive selection events in the thymus. Here we show that SRG3 acts as a novel downstream target of E2A/HeLa E box-binding (HEB) complex and modulates glucocorticoid (GC) susceptibility in thymocytes in response to TCR signals. We have identified a putative E box element in the SRG3 promoter that is required for optimal promoter activity. The transcription factors E2A and HEB specifically associate with the E box element. Moreover, E2A-HEB heterodimers cooperated to activate SRG3 transcription, which was inhibited by the expression of Id proteins. TCR-mediated signals rapidly induced Id3 via MEK/ERK activation and thereby kept the E2A/HEB complex from binding to the E box element in the SRG3 promoter. Retroviral transduction of Id3 also repressed the SRG3 expression by inhibiting the E box binding activity of the E2A/HEB complex. Intriguingly, enforced Id3 expression conferred thymocyte resistance to GCs, which could be overcome by the overexpression of SRG3. Taken together, these results suggest that Id3 may enhance the viability of immature thymocytes by at least rendering them resistant to GCs through SRG3 down-regulation.

Activation of natural killer T cells inhibits the development of induced regulatory T cells via IFNγ.

Recent reports have provided evidence for cross-talk between regulatory T (Treg) cells and natural killer T (NKT) cells. However, it is unclear whether NKT cells play a role in the differentiation of Treg cells. By employing NKT cell-abundant Vα14 TCR transgenic (Tg) and NKT cell-deficient CD1d knock-out (KO) mice, we examined the effects of NKT cells on the in vitro differentiation of induced Treg (iTreg) cells with IL2 and TGFβ. We found that iTreg induction from CD1d KO mice was significantly increased compared to the control. Also, the addition of isolated NKT cells from Vα14 TCR Tg mice to naïve CD4(+) T cells from CD1d KO mice during iTreg differentiation caused a remarkable reduction of iTreg cells. Through IFNγ neutralization, we showed that this reduction was mediated by IFNγ. Furthermore, the main source of IFNγ during iTreg differentiation was NK1.1(-)CD4(+)Foxp3(-) T cells. This finding implied that early-activated NKT cells induced Th1-type cells and subsequently underwent apoptosis. Taken together, our results suggest that NKT cells inhibit the in vitro development of iTreg cells by increasing IFNγ.

Down-Regulation of the SWI/SNF Chromatin Remodeling Activity by TCR Signaling Is Required for Proper Thymocyte Maturation

The process of thymocyte development requires an exquisite regulation of many genes via transcription factors and chromatin remodeling activities. Even though the SWI/SNF chromatin remodeling complex has been thought to play important roles during thymocyte development, its known function is very limited. In this study, we show that the SWI/SNF chromatin remodeling activity is finely regulated during thymocyte maturation process, especially during thymocyte selections. We found that TCR signaling directly down-regulates mBRG1 and SWI3-related gene, the core components of murine SWI/SNF complex, during thymocyte maturation. Constitutive expression of SWI3-related gene in developing thymocytes attenuated the down-regulation of the SWI/SNF complex and resulted in a change in the expression of genes such as linker for activation of T cells and casitas B lineage lymphoma, which affected the TCR-mediated intracellular signaling pathway. The defects in TCR signaling resulted in the disruption of both positive and negative selections in specific TCR transgenic mice systems. Our results state, for the first time, that the chromatin remodeling activity needs to be finely controlled for proper thymocyte selection and maturation processes.

Intrinsic dorsoventral patterning and extrinsic EGFR signaling genes control glial cell development in the Drosophila nervous system

Dorsoventral patterning and epidermal growth factor receptor (EGFR) signaling genes are essential for determining neural identity and differentiation of the Drosophila nervous system. Their role in glial cell development in the Drosophila nervous system is not clearly established. Our study demonstrated that the dorsoventral patterning genes, vnd, ind, and msh, are intrinsically essential for the proper expression of a master glial cell regulator, gcm, and a differentiation gene, repo, in the lateral glia. In addition, we showed that esg is particularly required for their expression in the peripheral glia. These results indicate that the dorsoventral patterning and EGFR signaling genes are essential for identity determination and differentiation of the lateral glia by regulating proper expression of gcm and repo in the lateral glia from the early glial development. In contrast, overexpression of vnd, msh, spi, and Egfr genes repressed the expression of Repo in the ventral neuroectoderm, indicating that maintenance of correct columnar identity along the dorsoventral axis by proper expression of these genes is essential for restrictive formation of glial precursor cells in the lateral neuroectoderm. Therefore, the dorsoventral patterning and EGFR signaling genes play essential roles in correct identity determination and differentiation of lateral glia in the Drosophila nervous system.