DEVELOPMENTAL GENE REGULATION OF THE IMMUNE SYSTEM
, Head, Section on Molecular Genetics of Immunity
Tomohiko Kanno, MD, PhD, Staff Scientist
Anup Dey, PhD, Research Fellow
Akira Nishiyama, PhD, Research Fellow
Toru Atsumi, PhD, Visiting Fellow
Tsung Hsien Chang, PhD, Visiting Fellow
Anu Ghosh, PhD, Visiting Fellow
Sukhendu Gosh, PhD, Visiting Fellow
JiYoung Kim, PhD, Visiting Fellow
Prafullerkumur Tailor, PhD, Visiting Fellow
Matthew Smith, BS, Postbaccalaureate Fellow
We study transcription factors and chromatin-binding proteins that control the development of innate immunity. For example, we work on IRF8, a DNA-binding protein that we previously cloned. IRF8 is essential for the development of macrophages and dendritic cells (DCs). DCs, which provide immediate resistance against infection by producing large amounts of type I interferons (IFNs), are critical for the establishment of innate immunity and ensuing adaptive immunity. Extending our earlier observations that IRF8 is essential for DC development, we found that IRF8 activates a diverse set of genes involved in host defense, including PML (promyelocytic leukemia) and type I IFN genes in differentiated DCs. We also work on a bromodomain protein called Brd4. Brd4 binds to acetylated chromatin during interphase and mitosis and is implicated in the maintenance of transcriptional memory across cell division. We found that treatment of cells with antimitotic drugs temporarily abrogates the interaction of Brd4 with chromatin, despite the fact that such drugs have no effect on histone acetylation patterns. Our results indicate that mitotic stress signals control Brd4-chroamatin interactions. Analysis of cells with low Brd4 expression showed that Brd4 plays an important role in protecting cells against mitotic stress and is required for accurate cell division.
IRF-8 is ubiquitinated by a novel IFN-inducible E3 ligase and stimulates cytokine gene transcription in macrophages
IRF8 is a member of the IFN regulatory factor (IRF) family that is expressed only in the immune system, including in macrophages and DCs. Macrophages and DCs produce various pro-inflammatory cytokines and confer antiviral and antimicrobial activities on the host, thereby playing an essential role in innate immunity. We have found that IRF-8 is subject to post-translational modification during interferon and viral stimulation. In particular, IRF8, among other nuclear proteins, was ubiquitinated in macrophages after IFNg treatment. Interestingly, some IRF8 was sumoylated before IFNg treatment, but sumoylation diminished after IFNg treatment. We have identified a novel E3 ligase called Ro52 that ubiquitinates IRF8 in activated macrophages. Ro52 is a protein of about 50 kDa present in many cells, mostly localized to the cytoplasm. Both IFNa/b and IFNg induce Ro52 expression. Although its function has not been fully understood, Ro52 has long been known as an auto-antigen characteristic of patients with autoimmune diseases such as systemic lupus erythematosus and Sjögren’s disease. Ro52 is a member of the large tripartite motif (TRIM) protein family and is composed of distinct domain structures, including the N-terminal RING domain, which is a signature motif for ubiquitin E3 ligase. Ro52 is also contains zinc-finger, coiled-coil, and C-terminal SPRY domains. The SPRY domain represents a motif involved in restricting viral replication, including that of HIV. We found that, through the SPRY domain, Ro52 interacts with IRF8 in macrophages after IFNg treatment and that such interaction leads to ubiquitination of IRF8. Thus, our work identifies Ro52 as an IFNg-inducible E3 ligase.
In agreement with studies of other E3 ligases, we discovered that Ro52 itself was ubiquitinated when incubated with E1 and E2 enzymes in vitro. The classic paradigm links ubiquitination to proteasome-mediated protein degradation. However, recent studies provide ample evidence that ubiquitination plays additional, diverse roles, including signaling and transcriptional activation. To assess the biological role of Ro52-mediated IRF8 ubiquitination, we exogenously expressed Ro52 and IRF8 in macrophage cell lines and tested for induction of the cytokine IL12p40. By promoting IFNg production, this cytokine is essential for host resistance to certain pathogens. Expression of IL12p40 has previously been shown to require IRF8. We found that full-length Ro52, but not truncated Ro52 lacking either the RING or SPRY domain, enhanced IL12p40 transcript expression, but we did not observe such enhancement in the absence of IRF8. The results indicate that ubiquitin modification enhances transcriptional activity of IRF8, although several critical issues regarding Ro52 remain unresolved, such as (1) what other functions, if any, Ro52 has; (2) what other substrates Ro52 ubiquitinates; and (3) identification of the molecular mechanism of Ro52-mediated IRF8 ubiquitination. We will work to resolve these issues in the coming year by using Ro52 knockout mice recently constructed in collaboration with Herbert Morse.
Kong HJ, Anderson DE, Lee CH, Jang MK, Tamura T, Tailor P, Cho HK, Cheong J, Xiong H, Morse HC, Ozato K. Cutting edge: autoantigen Ro52 is an interferon inducible E3 ligase that ubiquitinates IRF-8 and enhances cytokine expression in macrophages. J Immunol 2007;179:26-30.
Ozato K, Uno K, Iwakura Y. Another road to interferon: Yasuichi Nagano’s journey. J Interferon Cytokine Res 2007;27:349-52.
Tamura T, Tailor P, Yamaoka K, Kong HJ, Tsujimura H, O’Shea JJ, Singh H, Ozato K. IFN regulatory factor-4 and -8 govern dendritic cell subset development and their functional diversity. J Immunol 2005;174:2573-81.
Tamura T, Thotakura P, Tanaka TS, Ko MSH, Ozato K. Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages. Blood 2005;106:1938-47.
Zhao J, Kong HJ, Li H, Huang B, Yang M, Zhu C, Bogunovic M, Zheng F, Mayer L, Ozato K, Unkeless J, Xiong H. IRF-8/interferon (IFN) consensus sequence-binding protein is involved in Toll-like receptor (TLR) signaling and contributes to the cross-talk between TLR and IFN-gamma signaling pathways. J Biol Chem 2007;281:10073-80.
IRF8 is required for expression of PML and stimulates interferon gene transcription in dendritic cells
By using microarray analysis, we compared gene expression patterns in macrophages derived from wild-type or Irf8 knockout (KO) mice. In some cases, we extended our analysis to DCs by inducing several genes after stimulation by IFN and toll-like receptor (TLR) ligands in cells from wild-type mice. However, a significant fraction of the genes was not induced in cells from Irf8 KO mice. Among them were PML protein and type I IFNs. Both genes were strongly induced in wild-type DCs after TLR stimulation, but the expression was absent in Irf8 KO DCs. PML is a member of the TRIM family that forms a distinct nuclear structure called the nuclear body. In some types of leukemia, the PML gene is fused to other genes and acts as an oncogene. On the other hand, the unfused PML is thought to counteract leukemogenesis. Although PML has the RING domain, its E3 ligase activity has not been found. However, the PML RING domain is reported to be involved in PML sumoylation and nuclear body formation. Recent studies indicate that PML plays a role in restricting replication of herpes virus and cytomegalovirus and thus contributes to innate immunity. We found that PML expression is high in wild-type DCs, where we detected intense nuclear body formation. In contrast, Irf8KO DCs showed no nuclear bodies, suggesting that PML plays a role in DC function. We also found that re-introduction of the Irf8 gene rescues the expression of PML as well as the formation of nuclear bodies in DCs. For two reasons, the IRF8-PML link found in our study may open a new horizon in our understanding of myelogenous leukemia. First, several laboratories have shown that IRF8 expression is reduced in the peripheral blood of leukemia patients and might be a contributing factor to leukemogenesis. Second, a collaborative study re-investigated IRF8 expression in leukemia patients and extended the analysis to PML expression. Results indicated that, in leukemia patients, expression of PML was significantly lower than that in healthy donors and that such reduced PML expression precisely correlated with that of IRF expression, suggesting a role for IRF8 in mediating a tumor suppressor role of PML.
Chen L, Calomeni E, Wen J, Ozato K, Shen R, Gao JX. Natural killer dendritic cells are an intermediate of developing dendritic cells. J Leukoc Biol 2007;81:1422-33.
Dror N, Alter-Koltunoff M, Azriel A, Amariglio N, Jacob-Hirsch J, Zeligson S, Morgenstern A, Tamura T, Hauser H, Rechavi G, Ozato K, Levi BZ. Identification of IRF-8 and IRF-1 target genes in activated macrophages. Mol Immunol 2007;44:338-46.
Dror N, Rave-Harel N, Burchert A, Azriel A, Tamura T, Tailor P, Neubauer A, Ozato K, Levi BZ. Interferon regulatory factor-8 is indispensable for the expression of promyelocytic leukemia and the formation of nuclear bodies in myeloid cells. J Biol Chem 2007;282:5633-40.
Samarasinghe R, Tailor P, Tamura T, Kaisho T, Akira S, Ozato K. Induction of an anti-inflammatory cytokine, IL-10, in dendritic cells after toll-like receptor signaling. J Interferon Cytokine Res 2006;26:893-900.
Tailor P, Tamura T, Ozato K. IRF family proteins and type I interferon induction in dendritic cells. Cell Res 2006;16:134-40.
The role of Brd4 in mitotic stress response
Brd4 is a double bromodomain protein of about 200 kDa and is expressed in the nucleus of most cell types. It belongs to the BET family of bromodomain proteins conserved throughout eukaryotes, which includes yeast bdf1 and bdf2 as well as Drosophila Fsh. Mammalian species express not only Brd4 but also Brd2, Brd3, and Brdt1. A notable feature of the BET family proteins is their association with mitotic chromosomes. During mitosis, chromatin is condensed and hypoacetylated. In addition, transcription factors dissociate from chromatin and are released, resulting in the general cessation of transcription involving RNA polymerases I, II, and III. Although many bromodomain proteins are released from chromatin during mitosis, Brd4 and other BET family proteins remain associated with condensed chromosomes. We have observed Brd4-chromosome interactions from embryonic stem cells to activated lymphocytes. In a recent collaboration, we examined Brd4 in mammalian oocytes and found that it is associated with meiotic chromosomes, although we do not fully understand the mechanism by which Brd4 persists on mitotic chromosomes. Furthermore, while the functional significance of Brd4’s chromosomal retention remains elusive, some evidence suggests that Brd4 and other BET family proteins are involved in holding transcriptional memory during mitosis for inheritance by cells of the next generation.
With the aim of gaining insight into the biology of Brd4 during mitosis, we studied the behavior of Brd4 in abnormal mitosis. Our efforts were prompted by several lines of evidence indicating that Brd4 plays a role in cell growth affecting G1/S transition as well as G2/M stage. We began our work with a number of antitubulin drugs that arrest cells at mitosis. The drugs interfere with dynamic tubulin assembly/disassembly, resulting in the disruption of mitotic spindle formation and thus cause mitotic arrest. To our surprise, treatment with antitubulin drugs completely abrogated the Brd4-chromosome interaction. In the presence of the drugs, most of Brd4 was localized outside condensed chromosomes in live cells with exogenous Brd4 fused to GFP as well as in fixed cells with endogenous Brd4. All tested antitubulin agents yielded essentially the same results, including derivatives of colchicine, the oldest drug in the group. More recently developed drugs of the colchicine group include taxol and nocodazole, which also inhibited Brd4 binding to mitotic chromosomes. The former is used for treatment of various solid tumors. Laboratories make extensive use of the latter drug to study cell cycle and division because its reversible antimitotic effect is a convenient feature for laboratory experiments.
We observed the effect of antitubulin drugs in all cultured cells subjected to testing, although the effect was reversed when nocodazole was removed from the culture by simple washing. While Brd4 was displaced from chromosomes as long as nocodazole was present in the media, Brd4 was reloaded onto chromosomes within 30 minutes of the removal of the drug. Furthermore, within 60 to 90 minutes of nocodazole wash-out, wild-type cells expressing normal levels of Brd4 resumed mitosis and produced two new daughter cells. However, cells expressing less Brd4 (owing to disruption of one Brd4 allele [Brd4+/−]) were not able to reload Brd4 onto chromosomes after nocodazole removal and were defective in the ensuing cell division. That is, many of the Brd4+/− cells did not resume mitosis, resulting in a significantly fewer number of daughter cells. The data indicate that the Brd4-chromosome interaction plays an important role in normal progression of mitosis and protects cells from adverse effects of antitubulin drugs. Our data further indicate that Brd4 is recruited to the promoters of genes that regulate cell cycle progression, presumably by binding to chromatin. Some agents, including antitubulin drugs, appear to affect recruitment patterns. Clearly, study of genome-wide Brd4 recruitment is warranted.
Chen L, Shen R, Ye Y, Pu XA, Liu X, Duan W, Wen J, Zimmerer J, Wang Y, Liu Y, Lasky LC, Heerema NA, Perrotti D, Ozato K, Kuramochi-Miyagawa S, Nakano T, Yates AJ, Carson WE, Lin H, Barsky SH, Gao JX. Precancerous stem cells have the potential for both benign and malignant differentiation. PLoS ONE 2007;2:293.
Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K. The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell 2005;19:523-34.
Nagashima T, Maruyama T, Furuya M, Kajitani T, Uchida H, Masuda H, Ono M, Arase T, Ozato K, Yoshimura Y. Histone acetylation and subcellular localization of chromosomal protein BRD4 during mouse oocyte meiosis and mitosis. Mol Hum Reprod 2007;13:141-8.
Nishiyama A, Dey A, Miyazaki J, Ozato K. Brd4 is required for recovery from antimicrotubule drug-induced mitotic arrest: preservation of acetylated chromatin. Mol Biol Cell 2006;17:814-23.
Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, Zhou Q. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol Cell 2005;19:535-45.
COLLABORATORS
Jian-Xin Gao, MD, PhD, Ohio State University Medical Center, Columbus, OH
Bruce Howard, MD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Tatiana Karpova, PhD, Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD
Toru Kubota, PhD, Japan National Institute of Infectious Diseases, Tokyo, Japan
Ben-Zion Levi, PhD, Technion-Israel Institute of Technology, Haifa, Israel
James McNally, PhD, Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD
Herbert Morse, MD, Laboratory of Immunopathology, NIAID, Rockville, MD
Tomohiko Tamura, MD, PhD, Tokyo University, Tokyo, Japan
Huabao Xiong, MD, PhD, City University of New York, Mount Sinai School of Medicine, New York, NY
For further information, contact ozatok@mail.nih.gov.
