We work on nuclear factors that regulate gene expression in the immune system. We previously isolated IRF-8/ICSBP, a DNA-specific transcription factor expressed in hematopoietic cells, and showed that it plays a critical role in the development of macrophages and dendritic cells, the cells important for innate immunity. IRF-8 is required for the expression of cytokines such as type I interferons and IL-12, which confer host resistance against pathogens. We also study the bromodomain protein Brd4, which binds to chromatin and associates with chromosomes during mitosis and is thought to play a role in the inheritance of epigenetic memory. Analysis with live cell technologies showed that Brd4 and the related Brd2 bind preferentially to acetylated lysine residues of histones H3 and H4 in vivo. Our search for proteins that interact with Brd4 identified P-TEFb, the cyclinT/Cdk9 heterodimer, as a stable partner of Brd4. We found that an inactive P-TEFb is converted to a functionally active form upon interaction with Brd4. Using chromatin immunoprecipitation and reporter assays, we have shown that Brd4 recruits P-TEFb to the HIV LTR promoter, and further that P-TEFb phosphorylates the C-terminal domain of RNA polymerase II, thereby stimulating transcription in vivo.
The IRF-4/IRF-8 paradigm in dendritic cell development
Tamura, Kong, Tailor, Kim, Remoli, Kubota, Samarasinghe; in collaboration with Levi, McNally, Morse, Xiong
Dendritic cells (DCs), the cells that spot pathogen infection and confer innate immunity in the body, are a heterogeneous cell population that can be classified into four subsets with different surface marker expression and function: plasmacytoid DCs (pDCs), CD8 alpha+ DCs, CD4+ DCs, and double negative DCs. Given that they produce high levels of type I interferons (IFNs), which are important for host defense, pDCs have attracted intense attention. The other three DC subtypes present antigens to T cells and produce various cytokines. We found that the two transcription factors IRF-4 and IRF-8, which are selectively expressed in immune cells, are differentially expressed in DCs; IRF-8 was expressed at high levels in pDC and CD8alpha+ cells while IRF-4 was expressed at relatively low levels in these cells. Conversely, IRF-4 expression was high in CD4+ and double negative cells, with IRF-8 expression low in these cells. By studying IRF-4–/–, IRF-8–/–, and double knockout (IRF-4–/–IRF-8–/–) mice, we found that IRF-4 and IRF-8 are differentially required for the generation of DC subsets, thus correlating with the factors’ expression patterns; namely, IRF-8 is required for pDC and CD8alpha+ cells while IRF-4 is important for the other DC subsets. In line with the defects in single IRF knockout mice, double knockout mice have few DCs. We found that IRF-4 and IRF-8 regulate expression of a subset of specific genes that dictate the function of each subset. For example, IRF-8–/– DCs were defective in the expression of type I IFN, TLR3, and IL-12p40, whereas these genes are expressed in IRF-4–/– DCs. On the other hand, IRF-4–/– DCs were defective in integrin alpha expression. Retroviral-based rescue experiments supported the idea that IRF-4 and IRF-8 regulate subset-specific genes. Transfer of IRF-4 or IRF-8 into DCs derived from double knockout mice restored expression of corresponding target genes. Our results establish that IRF-4 and IRF-8 are a backbone of DC subset development and that they control the expression of subset-specific DC functions by regulating critical target genes.
To delineate further the range of target genes controlled by IRF-8, we carried out microarray analysis by using our macrophage differentiation model of Tot2 cells. In this model, IRF-8–/– Tot2 progenitor cells develop into macrophages upon IRF-8 gene transfer. We found that IRF-8 directly activates several lysosomal enzyme genes (cystatin C, cathepsin C, lysozyme M, and prosaposin). To study the mechanism by which IRF-8 activates promoter activity of these genes, we constructed reporters based on a self-inactivating retroviral system that contains promoters of the above genes. The activity of these reporters lent itself to assessment in live cells by flow cytometry. We found that IRF-8 stimulates transcription from these promoters by binding to novel cis-regulatory elements together with the transcription factor PU.1. The elements with the consensus of GAAANN(N)GGAA are viewed as a composite element accommodating cooperative binding of two proteins belonging to the IRF and Ets families.
Ishii KJ, Ito S, Tamura T, Hemmin H, Conover J, Ozato K, Akira S, Klinman D. CpG-activated Thy1.2+ dendritic cells protect against lethal Listeria monocytogenesis infection. Eur J Immunol 2005;35:1-9.
Tamura T, Tailor K, Kong HJ, Tsujimura H, Singh H, Ozato K. IRF-4 and IRF-8 govern dendritic cell development and their functional diversity. J Immunol 2005;174:2573-2581.
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;106:1938-1947.
Tsujimura H, Tamura T, Kong H, Nishiyama A, Ishii KT, Klinman D, Ozato K. Toll like receptor 9 signaling activates NFkB through IRF-8 in dendritic cells. J Immunol 2004;172:6820-6827.
Xiong H, Li H, Kong H, Chen Y, Zhao J, Huang B, Gu H, Mayer L, Ozato K, Unkeless JC. Ubiquitin-dependent degradation of IRF-8 mediated by cbl down-regulates IL-12 expression. J Biol Chem 2005;280:23531-23538.
Interaction of the bromodomain protein Brd4 with P-TEFb and regulation of RNA polymerase II–dependent transcription
Jang, Dey, Nishiyama, Natsume, Mochizuki; in collaboration with McBride, Zhou
Brd4 is a mammalian member of the BET family that is conserved from yeast to humans. It is a ubiquitous protein that localizes to the nucleus in interphase. Like other BET proteins, Brd4 carries two bromodomains through which it interacts with acetylated chromatin in vivo. Interaction of Brd4 with chromatin persists during mitosis, a feature that distinguishes Brd4 from other bromodomain proteins and transcription factors. Given that most transcription factors dissociate from chromosomes during mitosis and that transcription stops during mitosis, it has been suggested that Brd4 is involved in epigenetic memory across cell division. Papillomaviruses take advantage of the Brd4’s ability to associate with mitotic chromosomes in that the viral transactivator E2 binds to the C-terminal region of Brd4, enabling the viral genomes to segregate equally between the newly divided daughter cells. Detailed mutation analysis led to the finding that a specific domain of E2 unrelated to the trans-activation activity is important for association with Brd4 and mitotic chromosomes; accordingly, the ability to associate with Brd4 correlated with viral virulence.
To gain insight into the function of Brd4, we searched for proteins that interact with Brd4. Using tandem immunopurification and mass spectrometry, we identified the cyclinT1/Cdk9 complex as a factor that stably associates with Brd4. CyclinT and Cdk9 form a heterodimer called P-TEFb, which phosphorylates the C-terminal domain of RNA polymerase II, critical for elongation of nascent mRNAs. The interaction of Brd4 with P-TEFb was mediated by a small internal region of cyclin T and the bromodomains of Brd4. Using bifluorescence complementation analysis, we were able to demonstrate the Brd4-P-TEFb interaction in living cells. P-TEFb is known to associate with the inhibitory HEXIM1- and 7SKRNA-containing subunit, which inactivates Cdk9 kinase activity. We found that Brd4 binds to P-TEFb, which lacks the inhibitory subunit, and liberates its kinase activity in vivo and in vitro. We examined the role of Brd4 in the expression of the human immunodeficiency virus (HIV) by testing HIV-LTR promoter activity in HeLa cells. We found that Brd4 overexpression increases promoter activity while Brd4 underexpression decreases activity in the absence of Tat, the HIV viral trans-activator. Using chromatin immunoprecipitation analysis, we found that Brd4 binds to the HIV promoter and recruits P-TEFb in vivo; binding of Cdk9 to the promoter was low in Brd4-underexpressed cells while it increased when Brd4 was overexpressed. When histone deacetylase inhibitor increased chromatin acetylation, the recruitment of Brd4 and of P-TEFb to the promoter significantly increased, concomitant with an increase in HIV promoter activity. These results demonstrate that Brd4 recruits P-TEFb to acetylated and transcriptionally competent promoters.
Farina A, Hattori M, Qin J, Nakatani Y, Monato N, Ozato K. Bromodomain protein Brd4 binds to the GTPase activating SPA-1, modulating its activity and subcellular localization. Mol Cell Biol 2004;24:9059-9069.
Jang MK, Mochizuk K, Zhou M, Jeon HS, Brady JN, Ozato K. Bromodomain protein Brd4 is a positive regulatory component of P-TEFb and regulates transcription by RNA polymerase II. Mol Cell 2005;19:523-534.
Kanno T, Kanno Y, Siegel R, Jang M-K, Lenardo M, Ozato K. Selective recognition of acetylated histones by bromodomain proteins visualized in living cells. Mol Cell 2004;13:33-43.
Yang Z, Yik JHN, Chen R, Jang M, Ozato K, Zhou Q. Recruitment of P-TEFb for stimulation of transcriptional elongation by bromodomain protein Brd4. Mol Cell 2005;19:535-545.
You J, Croyle JL, Nishimura A, Ozato K, Howley PM. Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral DNA to host cell mitotic chromosomes. Cell 2004;30:117:349-360.
Publication Related to Other Work
Karginov VA, Nestorovich EM, Moayeri M, Leppla SH, Bezrukov SM. Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design. Proc Natl Acad Sci USA 2005:102:15075-15080.
Collaborators
Ben-Zion Levi, PhD, Technion-Israel Institute of Technology, Haifa, Israel
Alison McBride, PhD, Laboratory of Viral Diseases, NIAID, Bethesda, MD
James McNally, PhD, Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD
Herbert Morse, MD, Laboratory of Immunopathology, NIAID, Rockville, MD
Huabao Xiong, MD, PhD, City University of New York, Mount Sinai School of Medicine, New York, NY
Qiang Zhou, PhD, University of California Berkeley, Berkeley, CA
For further information, contact ozatok@mail.nih.gov.