Su Hwa Jang

Supplementary Figures S1-S7 from p31comet Induces Cellular Senescence through p21 Accumulation and Mad2 Disruption

Supplementary Figures S1-S7 from p31comet Induces Cellular Senescence through p21 Accumulation and Mad2 Disruption

Follicular Th (Tfh) cells are key CD4 ⁺ Th cells involved in regulating B cell differentiation in the germinal center. Mice with conditional KO (CKO) of B lymphocyte-induced maturation protein-1 (BLIMP-1) expression in CD11c ^hi DCs ( Prdm1 CKO) spontaneously develop a lupus-like disease. We found an increase in the number of Tfh cells in secondary lymphoid organs in lupus-prone Prdm1 -CKO mice. B cells stimulated with Tfh cells become Ab-secreting cells (ASCs); there was an increase in ASC differentiation mediated by Tfh cells from Prdm1 -CKO mice compared with Tfh cells from control mice. This was mainly due to the increased expression of molecules within the IL-23/IL-17 pathway in Tfh cells from Prdm1 -CKO mice. There was an increased frequency of IL-17A ⁺ Tfh cells in Prdm1 -CKO mice. These Tfh cells secrete IL-17A and an increased amount of IL-21. Furthermore, neutralizing IL-17A and IL-21 reduced ASC differentiation induced by Tfh cells. Lastly, we found the expression of IL-1β and IL-6 was increased in BLIMP-1–deficient DCs, which are key for Th17 induction. Altogether, the lack of BLIMP-1 expression in DCs induces not only an increased number of Tfh cells, but also functionally distinct Tfh cells that lead to increased ASC differentiation.

Proper expression of the transcription factor, Positive regulatory domain 1 (PRDM1), is required for maintaining homeostasis of human monocyte derived-dendritic cells (MO-DCs). The molecular mechanisms and gene targets of PRDM1 in B and T lymphocytes have been identified. However, the function of PRDM1 in dendritic cells (DCs) remains unclear. We investigate co-regulators of PRDM1 in MO-DCs identified by mass spectrometry (MS) and co-immunoprecipitation (Co-IP). Notably, non-POU domain-containing octamer-binding protein (NonO) was found to be a PRDM1 binding protein in the nucleus of MO-DCs. NonO is recruited to the PRDM1 binding site in the promoter region of IL-6. Knockdown of NonO expression by siRNA lessened suppression of IL-6 promoter activity by PRMD1 following LPS stimulation. While NonO binding to PRDM1 was observed in human myeloma cell lines, an effect of NonO on IL-6 expression was not observed. Thus, loss of NonO interrupted the inhibitory effect of PRDM1 on IL-6 expression in MO-DCs, but not plasma cells. Moreover, MO-DCs with low expression of PRDM1 or NonO induce an increased number of IL-21-producing T_FH-like cells in vitro. These data suggest that low level of PRDM1 and NonO lead to enhanced activation of MO-DCs and the regulation of MO-DC function by PRDM1 is mediated through cell lineage-specific mechanisms.

Cathepsin S is responsible for cleaving the invariant chain Ii for the presentation of peptides by major histocompatibility complex class II on DCs. Diamond and colleagues show that the transcription factor Blimp-1 negatively regulates expression of cathepsin S and influences selection of the TFH cell repertoire and autoantibody-producing B cells. Aberrant population expansion of follicular helper T cells (TFH cells) occurs in patients with lupus. An unanswered question is whether an altered repertoire of T cell antigen receptors (TCRs) is associated with such expansion. Here we found that the transcription factor Blimp-1 (encoded by Prdm1) repressed expression of the gene encoding cathepsin S (Ctss), a cysteine protease that cleaves invariant chains and produces antigenic peptides for loading onto major histocompatibility complex (MHC) class II molecules. The increased CTSS expression in dendritic cells (DCs) from female mice with dendritic cell–specific conditional knockout of Prdm1 (CKO mice) altered the presentation of antigen to CD4+ T cells. Analysis of complementarity-determining region 3 (CDR3) regions containing the β-chain variable region (Vβ) demonstrated a more diverse repertoire of TFH cells from female CKO mice than of those from wild-type mice. In vivo treatment of CKO mice with a CTSS inhibitor abolished the lupus-related phenotype and reduced the diversity of the TFH cell TCR repertoire. Thus, Blimp-1 deficiency in DCs led to loss of appropriate regulation of Ctss expression in female mice and thereby modulated antigen presentation and the TFH cell repertoire to contribute to autoimmunity.

A SNP identified as rs548234, which is found in PRDM1 , the gene that encodes BLIMP1, is a risk allele associated with systemic lupus erythematosus (SLE). BLIMP1 expression was reported to be decreased in women with the PRDM1 rs548234 risk allele compared with women with the nonrisk allele in monocyte-derived DCs (MO-DCs). In this study, we demonstrate that BLIMP1 expression is regulated by the binding of Kruppel-like factor 4 (KLF4) to the risk SNP. KLF4 is highly expressed in MO-DCs but undetectable in B cells, consistent with the lack of altered expression of BLIMP1 in B cells from risk SNP carriers. Female rs548234 risk allele carriers, but not nonrisk allele carriers, exhibited decreased levels of BLIMP1 in MO-DCs, showing that the regulatory function of KLF4 is influenced by the risk allele. In addition, KLF4 directly recruits histone deacetylases (HDAC4, HDAC6, and HDAC7), established negative regulators of gene expression. Finally, the knock down of KLF4 expression reversed the inhibitory effects of the risk SNP on promoter activity and BLIMP1 expression. Therefore, the binding of KLF4 and the subsequent recruitment of HDACs represent a mechanism for reduced BLIMP1 expression in MO-DCs bearing the SLE risk allele rs548234.

Telomere uncapping is thought to be the fundamental cause of replicative cellular senescence, but the cellular machineries mediating this process have not been fully understood. In the present study, we present the role of Sp1 transcription factor in the state of telomere uncapping using the TRF2^ΔBΔM-induced senescence model in human diploid fibroblasts. We observed that the expression of Sp1 is down-regulated in the TRF2^ΔBΔM-induced senescence, which was mediated by ATM and p38 MAPK. In addition, overexpression of Sp1 prevented the TRF2^ΔBΔM-induced senescence. Among transcriptional targets of Sp1, expression levels of nuclear transport genes such as karyopherin α, Nup107, and Nup50 were down-regulated in the TRF2^ΔBΔM-induced senescence, which was prevented by Sp1 overexpression. Moreover, inhibition of the nuclear transport by wheat germ agglutinin (an import inhibitor) and leptomycin B (an export inhibitor) induced premature senescence. These results suggest that Sp1 is an anti-senescence transcription factor in the telomere uncapping-induced senescence and that down-regulation of Sp1 leads to the senescence via down-regulation of the nuclear transport.

The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31^comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31^comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31^comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31^comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31^comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31^comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31^comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31^comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31^comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31^comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.