Kyungsoo Park

The SWI/SNF-like BAF Complex Is Essential for Early B Cell Development

During the process of B cell development, transcription factors, such as E2A and Ebf1, have been known to play key roles. Although transcription factors and chromatin regulators work in concert to direct the expression of B lineage-specific genes, little is known about the involvement of regulators for chromatin structure during B lymphopoiesis. In this article, we show that deletion of Srg3/mBaf155, a scaffold subunit of the SWI/SNF-like BAF complex, in the hematopoietic lineage caused defects at both the common lymphoid progenitor stage and the transition from pre–pro-B to early pro-B cells due to failures in the expression of B lineage-specific genes, such as Ebf1 and Il7ra, and their downstream target genes. Moreover, mice that were deficient in the expression of Brg1, a subunit of the complex with ATPase activity, also showed defects in early B cell development. We also found that the expression of Ebf1 and Il7ra is directly regulated by the SWI/SNF-like BAF complex. Thus, our results suggest that the SWI/SNF-like BAF complex facilitates early B cell development by regulating the expression of B lineage-specific genes.

Proteomic analysis of differential protein expression in response to epidermal growth factor in neonatal porcine pancreatic cell monolayers

We have proposed that porcine neonatal pancreatic cell clusters (NPCCs) may be a useful alternative source of cells for islet transplantation, and that monolayer cultures might provide an opportunity to manipulate the cells before transplantation. In addition we previously identified 10 genes up‐regulated by epidermal growth factor (EGF) in cultured porcine NPCC monolayers. We have now analyzed the intracellular signaling pathways activated by EGF and searched for proteins differentially expressed following EGF treatment of the monolayers, using two‐dimensional gel electrophoresis (2‐DE) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). EGF treatment resulted in phosphorylation of both Erk 1/2 and Akt, as well as increased cell proliferation. Five unknown and 13 previously identified proteins were differentially expressed in response to EGF. EGF treatment increased the expression of several structural proteins of epithelial cells, such as cytokeratin 19 and plakoglobin, whereas vimentin, the intermediate filament protein of mesenchymal cells, and non‐muscle myosin alkali chain isoform 1, decreased. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 factor, which promotes epithelial cell proliferation, and hemoglobin alpha I & II also increased, whereas cyclin A1, immunoglobulin heavy chain, apolipoprotein A1, 5,10‐ethylenetetrahydrofolated reductase (5,10‐MTHFR), angiotensin‐converting enzyme 2 (ACE2), co‐lipase II precursor, and NAD+ isocitrate dehydrogenase (NAD+ IDH) alpha chain proteins decreased. Our results show that EGF stimulates proliferation of pancreatic epithelial cells by simultaneously activating the MAPK and PI‐3K pathways. HnRNP A2/B1, hemoglobin, cyclin A1, and ACE2 may play roles in the proliferation of epithelial cells in response to EGF.

RORγt-specific transcriptional interactomic inhibition suppresses autoimmunity associated with TH17 cells

Significance TH17 cells are a subset of CD4+ T helper cells that secrete the cytokine IL-17 and play a role in autoimmunity. RORγt is identified as a key transcription factor driving the TH17 differentiation. Sequence analysis indicated that transcription factor contains several conserved DNA-binding domain and isotype-specific domain that we termed transcription modulation domain (TMD). We designed a novel therapeutics, tRORγt-TMD, to deliver RORγt-TMD efficiently into the nucleus of the cells that regulates TH17 cell functions and TH17-mediated autoimmune diseases. With the same concept, tTbet-TMD also can regulate TH1 functions. In conclusion, tRORγt-TMD/tTbet-TMD can be novel and highly specific therapeutics for the treatment of TH17/TH1-mediated inflammatory disease and further allows us to discover new function of RORγt/Tbet in animals without genetic alteration. The nuclear hormone receptor retinoic acid-related orphan receptor gamma t (RORγt) is a transcription factor (TF) specific to TH17 cells that produce interleukin (IL)-17 and have been implicated in a wide range of autoimmunity. Here, we developed a novel therapeutic strategy to modulate the functions of RORγt using cell-transducible form of transcription modulation domain of RORγt (tRORγt-TMD), which can be delivered effectively into the nucleus of cells and into the central nerve system (CNS). tRORγt-TMD specifically inhibited TH17-related cytokines induced by RORγt, thereby suppressing the differentiation of naïve T cells into TH17, but not into TH1, TH2, or Treg cells. tRORγt-TMD injected into experimental autoimmune encephalomyelitis (EAE) animal model can be delivered effectively in the splenic CD4+ T cells and spinal cord-infiltrating CD4+ T cells, and suppress the functions of TH17 cells. The clinical severity and incidence of EAE were ameliorated by tRORγt-TMD in preventive and therapeutic manner, and significant reduction of both infiltrating CD4+ IL-17+ T cells and inflammatory cells into the CNS was observed. As a result, the number of spinal cord demyelination was also reduced after tRORγt-TMD treatment. With the same proof of concept, tTbet-TMD specifically blocking TH1 differentiation improved the clinical incidence of rheumatoid arthritis (RA). Therefore, tRORγt-TMD and tTbet-TMD can be novel therapeutic reagents with the natural specificity for the treatment of inflammatory diseases associated with TH17 or TH1. This strategy can be applied to treat various diseases where a specific transcription factor has a key role in pathogenesis.

The SWI/SNF chromatin remodeling complex regulates germinal center formation by repressing Blimp-1 expression

Significance Germinal center (GC) response is central for generation of memory B cells and plasma cells that produce high-affinity antibodies, which are crucial for protective immunity against various foreign antigens. Even though key genetic factors for the GC formation are known, it is largely unknown how this process is controlled by epigenetic factors. Here we have demonstrated that the activity of SWI/SNF chromatin remodeling complex is required for the development of both GC B cells and follicular helper T cells. The SWI/SNF complex modulates Bcl-6–mediated Blimp-1 repression, and thus plays as a key mediator in the GC response. Our results provide fundamental insights into the epigenetic regulation in the GC reaction. Germinal center (GC) reaction is crucial in adaptive immune responses. The formation of GC is coordinated by the expression of specific genes including Blimp-1 and Bcl-6. Although gene expression is critically influenced by the status of chromatin structure, little is known about the role of chromatin remodeling factors for regulation of GC formation. Here, we show that the SWI/SNF chromatin remodeling complex is required for GC reactions. Mice lacking Srg3/mBaf155, a core component of the SWI/SNF complex, showed impaired differentiation of GC B and follicular helper T cells in response to T cell-dependent antigen challenge. The SWI/SNF complex regulates chromatin structure at the Blimp-1 locus and represses its expression by interacting cooperatively with Bcl-6 and corepressors. The defect in GC reactions in mice lacking Srg3 was due to the derepression of Blimp-1 as supported by genetic studies with Blimp-1–ablated mice. Hence, our study identifies the SWI/SNF complex as a key mediator in GC reactions by modulating Bcl-6–dependent Blimp-1 repression.

Foxp3+ regulatory T cells ensure B lymphopoiesis by inhibiting the granulopoietic activity of effector T cells in mouse bone marrow

Foxp3+ Treg cells are crucial for maintaining T‐cell homeostasis, but their role in B‐cell homeostasis remains unclear. Here, we found that Foxp3 mutant scurfy mice had fewer B‐lineage cells and progenitors, including common lymphoid progenitors and lymphoid‐primed multipotent progenitors, but higher myeloid‐lineage cell numbers in BM compared with WT littermates. Homeostasis within the HSC compartment was also compromised with apparent expansion of long‐ and short‐term HSCs. This abnormality was due to the lack of Treg cells, but not to the Treg‐cell extrinsic functions of Foxp3 or cell‐autonomous defects. Among cytokines enriched in the BM of scurfy mice, IFN‐γ affected only B lymphopoiesis, but GM‐CSF, TNF, and IL‐6 collectively promoted granulopoiesis at the expense of B lymphopoiesis. Neutralization of these three cytokines reversed the hematopoietic defects on early B‐cell progenitors in scurfy mice. Treg cells ensured B lymphopoiesis by reducing the production of these cytokines by effector T cells, but not by directly affecting B lymphopoiesis. These results suggest that Treg cells occupy an important niche in the BM to protect B‐lineage progenitor cells from excessive exposure to a lymphopoiesis‐regulating milieu.

Enhanced mitochondrial glutamine anaplerosis suppresses pancreatic cancer growth through autophagy inhibition

Cancer cells use precursors derived from tricarboxylic acid (TCA) cycle to support their unlimited growth. However, continuous export of TCA cycle intermediates results in the defect of mitochondrial integrity. Mitochondria glutamine metabolism plays an essential role for the maintenance of mitochondrial functions and its biosynthetic roles by refilling the mitochondrial carbon pool. Here we report that human pancreatic ductal adenocarcinoma (PDAC) cells have a distinct dependence on mitochondrial glutamine metabolism. Whereas glutamine flux into mitochondria contributes to proliferation of most cancer cells, enhanced glutamine anaplerosis results in a pronounced suppression of PDAC growth. A cell membrane permeable α-ketoglutarate analog or overexpression of glutamate dehydrogenase lead to decreased proliferation and increased apoptotic cell death in PDAC cells but not other cancer cells. We found that enhanced glutamine anaplerosis inhibits autophagy, required for tumorigenic growth of PDAC, by activating mammalian TORC1. Together, our results reveal that glutamine anaplerosis is a crucial regulator of growth and survival of PDAC cells, which may provide novel therapeutic approaches to treat these cancers.

Foxp3 expression in induced regulatory T cells is stabilized by C/EBP in inflammatory environments.

Proper control of immune responses by Foxp3+ regulatory T cells at inflamed sites is crucial for the prevention of immunopathology. TGF‐β‐induced Foxp3+ regulatory T (Treg) cells are generated in inflammatory environments as well as in steady‐state conditions. Inflammatory cytokines such as IFN‐γ and IL‐4 have an antagonistic effect on Treg cell conversion. However, it is not known how naive CD4+ T cells overcome the inhibitory environment in inflamed sites to differentiate into Treg cells. Here, we show that CCAAT/enhancer‐binding protein (C/EBP) functions as a safeguard that enhances Treg cell generation by dampening the inhibitory effect of IFN‐γ and IL‐4 on Foxp3 expression. We find that C/EBPβ is induced by retinoic acid and binds to the methyl‐CRE sequence in the Foxp3 TSDR to sustain its expression. C/EBPβ‐transduced iTreg cells show more potent suppressive activity in mouse disease models. We also reveal that C/EBPβ‐transduced human iTreg cells exhibit more enhanced suppressor function. These results establish C/EBP as a new molecular target for enhancing the formation and stability of Treg cells in inflammatory environments.