Keiko Ozato, PhD, Head, Section on Molecular Genetics of Immunity

Tomohiko Tamura, MD, PhD, Staff Scientist
Anup Dey, PhD, Research Fellow
Andrea Farina, PhD, Visiting Fellow
Moon Kyoo Jang, PhD, Visiting Fellow
Hee Jeong Kong, PhD, Visiting Fellow
Kazuyuki Mochizuki, PhD, Visiting Fellow
Prafullerkmur Tailor, PhD, Visiting Fellow
Leopoldo Laricchia-Robio, PhD, Courtesy Contract
Pratima Thotakura, BS, Postbaccalaureate Fellow
Tomohiko Kanno, MD, PhD, Guest Researcher
Akira Nishiyama, PhD, Guest Researcher
Hideki Tsujimura, MD, PhD, Guest Researcher

We are interested in transcription factors that regulate the development of the immune system. We previously isolated IRF-8/ICSBP, a DNA-specific transcription factor expressed in hematopoietic cells, and showed that it regulates the development of bone marrow progenitor cells to generate macrophages and granulocytes and that it is critical for the development of dendritic cells (DCs) and the production of the type I interferons and IL-12, cytokines that are required for host defense. Using DNA microarray analysis, we have so far identified about 50 genes that are found either to increase or decrease following IRF-8 expression, some of which are important for macrophage/DC function. Studies of target promoters and the chromatin status of ICSBP-null cells suggest that IRF-8 regulates gene expression not only by controlling target promoters but also by regulating the activity of chromatin. We also study the role of chromatin from another perspective: the bromodomain protein Brd4. We showed that it associates with mitotic chromosomes, consistent with a strong affinity for chromatin. Using a proteomic approach, we have isolated a number of proteins that interact with Brd4, such as SPA-1, an activator of the small GTPase Rap, whose function is linked to Ras, a major regulator of mitogenic signaling pathways.

Interferon gamma triggers the interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase HDAC3 to the interferon-responsive element

Tamura, Kanno; in collaboration with Kuwata

ICSBP (IRF-8) is a transcription factor of the IRF family expressed only in the immune system. It is induced in macrophages by interferon gamma (IFNgamma) and critically contributes to macrophage functions. By interacting with an Ets family protein PU.1, ICSBP binds to the IRF/Ets composite element and stimulates transcription. ICSBP binds to another DNA element, the IFN-stimulated response element (ISRE), a common target of the IRF family. Despite much progress in the field, limited knowledge is available as to how ICSBP and other IRF proteins regulate ISRE-dependent transcription in IFNgamma-activated macrophages. Using mass-spectrometry analysis of ISRE-bound proteins in macrophages, we identified TEL, another Ets member, as a factor recruited to the element in an IFNgamma-dependent manner. In vitro analysis with recombinant proteins indicated that the recruitment is attributable to a direct interaction between ICSBP and TEL, which is enhanced by the presence of ISRE. significantly, the interaction with TEL in turn resulted in the recruitment of the histone deacetylase HDAC3 to the ISRE, causing increased repression in IFNgamma-mediated reporter activity through the ISRE. Together, by associating with two different Ets family proteins, ICSBP exerts a dual function in IFNgamma-dependent gene regulation in an immune system-specific manner. 

Kuwata T, Gongora C, Kanno Y, Sakagichi K, Tamura T, Basrur V, Martinez, Appella E, Golub T, Ozato K.

Interferon gamma triggers interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase

HDAC3 to the interferon responsive element. Mol Cell Biol 2002;22:7439-7448.

Masusumi A, Yamakawa Y, Fukazawa H, Ozato K, Komuro K. Interferon regulatory factor-2 regulates

cell growth through its acetylation. J Biol Chem 2003;11:25401-25407.

Tamura T, Ozato K. ICSBP (IRF-8): its regulatory roles in the development of myeloid cells. J Inter-

feron Cytokine Res 2002;22:145-152.

Tsujimura H, Nagamura-Inoue T, Tamura T, Ozato K. ICSBP/IRF-8 guides bone marrow progenitor

cells towards the macrophage lineage. J Immunol 2002;169:1261-1269.

Xiong H, Zhu V, Li H, Chen F, Mayer L, Ozato K, Unkeless JC, Plevy SE. Complex formation of the

IFN consensus binding protein (ICSBP) with IR-1 is essential for murine macrophage

IFNgamma-induced iNOS gene expression. J Biol Chem 2003;278:2271-2277.

Role of ICSBP in the development of interferon-producing plasmacytoid dendritic cells

Tsujimura, Tamura, Kong, Tailor

DCs, a newly identified cell type important for establishing innate and adaptive immunity, develop from bone marrow (BM) progenitor cells and mature in response to external signals to elicit functions important for innate and adaptive immunity. We found that ICSBP-/- mice, in addition to their inability to produce type I interferons and IL-12, lack subsets of DCs (pDCs and CD8alpha+ DCs). To investigate the role of ICSBP in DC development, we employed an Flt3 ligand-based in vitro culture system. DCs developed in vitro from ICSBP-/- BM cells were defective in the expression of MHC class II and costimulatory molecules, indicating that differentiation of ICSBP-/- progenitors toward immature DCs is impaired. Moreover, -/- DCs displayed a generalized maturation failure and did not express type I interferon and IL-12 p40 mRNA in response to various maturation signals, consistent with the feature observed with ICSBP-/- DCs in vivo. Further, -/- DCs failed to increase expression of MHC class II and costimulatory molecules upon maturation signals. We showed that retroviral introduction of ICSBP restores the ability of -/- progenitors to develop plasmacytoid DCs that undergo full maturation upon activation signals. Transduction of the wild-type ICSBP, but not transcriptionally inactive mutants, increased expression of MHC class II and costimulatory molecules on immature cells. More important, ICSBP-transduced cells produced interferons and IL-12 proteins upon activation, along with increased expression of costimulatory molecules. Thus, introduction of ICSBP rescued not only the development of macrophages and DCs but also the expression of both cytokine genes. Our study identifies ICSBP as a factor critical for both early differentiation and final maturation of DCs.

Aliberti J, Schulz O, Pennington DJ, Tsujimura H, Reis e Sousa C, Ozato K, Sher A. Essential role

for ICSBP in the in vivo development of murin CD8alpha+ dendritic cells. Blood 2003;101:305-310.

Masumi A, Tamaoki S, Wang I-M, Ozato, K, Komuro K. IRF-8/ICSBP and IRF cooperatively stimulate

mouse IL-12 promoter activity in macrophages. FEBS Lett 2002;531:348-353.

Ozato K, Tsujimura H, Tamura T. Toll-like receptor signaling and regulation of cytokine gene expression

in the immune system. BioTechniques 2002;October suppl:66-73.

Tsujimura H, Tamura T, Gongora C, Aliberti J, Reis e Sousa C, Sher A, Ozato K. ICSBP/IRF-8 retro-

virus transduction rescues dendritic cell development. Blood 2003;101:961-969.

Tsujimura H, Tamura T, Ozato K. Cutting edge: ICSBP/IRF-8 drives the development of type I interferon
producing plasmacytoid dendritic cells. J Immunol 2003;170:1131-1135.

Bromodomain protein Brd4 binding to and modulation of the activity and subcellular localization of the GTPase-activating SPA-1

Farina, Dey

Brd4, a novel bromodomain-containing protein that binds to chromatin, regulates cell growth and affects cell cycle progression at multiple stages. By mass-spectrometry analysis, we identified the GTPase-activating protein (GAP) SPA-1 as a factor that interacts with Brd4 in human cells. Brd4 and SPA-1 bound to each other in vitro through specific domains. SPA-1 regulates mitogenic signaling pathways by activating Rap1 and Rap2. Consistent with the functional importance of the interaction, Brd4 enhanced Rap GAP activity of SPA-1 in vivo and in vitro. SPA-1 localizes to the cytoplasm and, to a lesser degree, in the nucleus while Brd4 resides in the nucleus. We show that ectopic expression of Brd4 redirects subcellular localization of SPA-1 and vice versa, indicating that Brd4 and SPA-1 mutually influence their localization in the cell. Our work reveals a novel link between Brd4 and GTPase-dependent mitogenic signaling pathways. Functional analysis of Brd4-SPA-1 interaction indicates that it is part of growth-regulatory activity of Brd4 in G1/S transition. We are pursuing further elucidation of the role of this factor by conditional knock-out and antisense/RNAi approaches.

Yamagoe S, Kanno T, Kanno Y, Sasaki S, Siegel RM, Lenardo M, Humphrey G, Wang Y, Nakatani

Y, Howard BH, Ozato K. Interaction of histone acetylases and deacetylases in vivo. Mol Cell Biol 2003;23:1025-1033.

Double bromodomain protein Brd4 binding to acetylated chromatin during interphase and mitosis

Dey, Ozato; in collaboration with Misteli

Previous studies on RXR led to the identification and characterization of Brd4. Recent FRAP and FLIP analyses combined with biochemical studies showed that Brd4 interacts with acetylated chromatin through the bromodomains in living cells as a consequence of its high affinity for acetylated core histones H4 and H3, leading to the proposal that the domain is involved in deciphering the histone code. However, little in vivo evidence supports the binding of bromodomains to acetylated chromatin in the native environment. Brd4 is a member of the BET family that carries two bromodomains. It associates with mitotic chromosomes, a feature characteristic of the family (Figure 18.1). We studied the interaction of Brd4 with chromatin in living cells by photobleaching. Brd4 was mobile and interacted with chromatin with a rapid "on and off" mode of binding. The interaction required both bromodomains. Indicating a preferential interaction with acetylated chromatin, Brd4 became less mobile upon increased chromatin acetylation caused by a histone deacetylase inhibitor.

Providing biochemical supporting evidence, the salt solubility of Brd4 was markedly reduced upon increased histone acetylation. This change also required both bromodomains. In peptide binding assays, Brd4 bound avidly to di- and tetraacetylated histone H4 and diacetylated H3 but weakly or not at all to mono- and unacetylated H3 and H4. By contrast, it did not bind to unacetylated H4 or H3. Further, Brd4 colocalized with acetylated H4 and H3 in noncentromeric regions of mitotic chromosomes. This colocalization also required both bromodomains. Our observations indicate that Brd4 specifically recognizes acetylated histone codes, with such recognition passed on to the chromatin of newly divided cells.

Dey A, Chitsaz F, Abbasi A, Misteli T, Ozato K. The double bromodomain protein Brd4 binds to

acetylated chromatin during interphase and mitosis. Proc Natl Acad Sci USA 2003:100 8758-8763.

Lefevbre B, Ozato K. Retinoic acid receptors. Wiley Encyclopedia of Molecular Medicine. John Wiley

& Sons. 2002;2775-2779.

Lefevbre B, Brand C, Lefevbre P, Ozato K. Chromosomal integration of retinoic acid responsive

elements prevent cooperative activation of RAR and RXR. Mol Cell Biol 2002;22:1446-1459.

Lefevbre B, Ozato K, Lefevbre P. Phosphorylation of histone H3 is functionally linked to retinoic acid

receptor beta promoter activation. EMBO Rep 2002;3:335-340.

Maruyama T, Dey A, Farina A, Cheong J, Bermudez VP, Tamura T, Sciortino S, Shuman J, Hurwitz

J, Ozato K. A mammalian bromodomain protein Brd4 interacts with the replication factor C and inhibits

progression to S phase. Mol Cell Biol 2002;22:6509-6520.

COLLABORATORS

Kathryn Calame, PhD, Howard Hughes Medical Institute, Columbia University, New York NY 

Todd Golub, MD, PhD, Harvard University, Cambridge MA 

Takeshi Kuwata, MD, PhD, Kitasato University, Tokyo, Japan

Tom Misteli, PhD, Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda MD 

Yoshihiro Nakatani, PhD, Harvard University, Cambridge MA 

Huabao Xiong, MD, PhD, New York University, New York NY 

For further information, contact ozatok@mail.nih.gov