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Developmental Gene Regulation of the Immune System
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Keiko
Ozato, PhD, Head, Section on
Molecular Genetics of Immunity Anup
Dey, PhD, Research Fellow Andrea
Farina, PhD, Visiting Fellow Moon
Kyoo Jang, PhD, Visiting Fellow Ji-Young
Kim, PhD, Visiting Fellow Hee
Jeong Kong, PhD, Visiting Fellow Kazuyuki
Mochizuki, PhD, Visiting Fellow Hiroko
Natsume, MD, PhD, Visiting Fellow Anna
Lisa Remoli, PhD, Visiting Fellow Prafullerkmur
Tailor, PhD, Visiting Fellow Hideki
Tsujimura, MD, PhD, Visiting Fellow Akira
Nishiyama, PhD, Guest Researcher Ranmal
Samarasinghe, BS, Postbaccalaureate
Fellow |
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We
work on 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
growth and differentiation of myeloid cells. IRF-8 plays a critical role in
the development of macrophages and dendritic cells, which are responsible for
eliciting innate immunity. In addition, 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 investigate the bromodomain protein
Brd4, a transcription factor that binds to chromatin and associates with
chromosomes during mitosis. We found that papillomaviruses take advantage of
Brd4’s property to associate with mitotic chromosomes in that viral
genomes are tethered to cellular chromosomes through Brd4 to partition their
genomes into the newly divided cells. We also visualized real-time behaviors
of Brd4 and the closely related Brd2. We showed that the Brd proteins
interact with the core histones H3 and H4 by binding to acetylated lysine
residues of the histone’s tail peptides and to acetyl chromatin through
the bromodomains in a highly transient manner. The high mobility of Brd
proteins appears to be an integral aspect of their function. Role
of IRF-8 in cell growth and innate immunity Kim, Kong,
Laricchia-Robbio,a Samarasinghe, Tailor, Tamura, Tsujimura,
Remoli; in collaboration with Calame, Levi, Sher, Wolfe, Xiong Previously,
we found that IRF-8 knockout mice develop a leukemia-like disease that
resembles chronic myelogenous leukemia (CML). We established bone marrow
progenitor cells from IRF-8−/− mice that grow continuously
in culture, similar to leukemia. IRF-8 gene transfer into these cells
altered the cells’ growth properties: they ceased to grow but
differentiated into functional macrophages. Thus, IRF-8 has a role in growth
inhibition in these cells. The growth inhibition coincided with repression of
the c-myc protooncogene, a gene linked to growth stimulation. We found
that IRF-8 represses c-myc expression through induction of BRIMP1, a
transcription repressor that associates with histone deacetylases. Similarly,
IRF-8 was found to induce the cdk inhibitor P15ink4b, revealing
another mechanism by which IRF-8 inhibits hematopoietic cell growth. IRF-8 is
required for the expression of genes important for innate immunity. These
genes are expressed in macrophages and dendritic cells (DC) and include
cytokines that confer broad antimicrobial activities. They are induced in
response to signaling by toll-like receptors (TLR) activated by various
pathogens. In particular, induction of type I interferons (IFNs alpha/beta)
as well as of the recently found type III IFN (gamma) in DC depends on IRF-8.
IRF-8 is also required for the induction of IL-12, another cytokine required
for type II IFN (gamma) expression. Thus, IRF-8 regulates induction of all
three types of IFN gene. Other genes involved in establishing innate immunity
such as those encoding nitric oxide synthase and NRAMP1 are also found to be
regulated by IRF-8. IRF-8 controls innate immunity by directly acting on the
transcription of target genes as well as by controlling TLR signaling
pathways. We showed that IRF-8−/− DCs are
defective in TLR9 signaling mediated by unmethylated CpG DNA. Our results
illustrate multiple roles of IRF-8 in eliciting innate immunity. Schmidt M, Bies J, Tamura T, Ozato K, Wolff L. IRF-8 in
combination with PU.1 upregulates p15IHK4b tumor suppressor expression in
murine myeloid cells. Blood 2004;103:4142-4149. Tamura T, Kong H, Tunyaplin C, Calame K, Ozato K. ICSBP/IRF-8
inhibits mitogenic activity of p210 Bcr/Abl in differentiating myeloid
progenitor cells. Blood 2003;102:4547-4554. Tsujimura H, Tamura T, Gongora C, Aliberti
J, Reis e Sousa C, Sher A, Ozato K. ICSBP/IRF-8 retrovirus transduction
rescues dendritic cell development. Blood 2003;101:961-969. 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. Tsujimura H, Tamura T, Ozato K. ICSBP/IRF-8 drives the
development of type I interferon producing plasmacytoid dendritic cells. J
Immunol Cutting Edge 2003;170:1131-1135. Real-time
interactions of bromodomain proteins with chromatin in living cells Dey, Farina, Jang,
Kanno T,b Kanno Y,c Nishiyama, Mochizuki; in
collaboration with Howley, Lenardo, Siegel Both
Brd2 and Brd4 carry bromodomains and the ET domain, another conserved motif.
A notable feature of Brd2 and Brd4 is that they associate with chromosomes
during mitosis. Although most other nuclear regulatory proteins interact with
chromatin during interphase, Brd2 and Brd4 are dissociated from chromatin
during mitosis, coinciding with the general transcriptional shut-down during
mitosis. Given the chromosomal retention of Brd2/Brd4, these proteins may
play a role in the inheritance of epigenetic memory. The property of Brd4,
i.e., that it associates with mitotic chromosomes, is used by the bovine
papillomavirus (BPV). In collaboration with the Howley laboratory, we showed
that the BPV transactivator protein E2 binds to Brd4 and loads the viral
genome onto cellular chromosomes, thereby allowing equal partitioning of the
viral genome into two daughter cells. By
using several live-cell technologies, we visualized the real-time interaction
of Brd2 and Brd4 with chromatin in the nucleus. Fluorescence resonance energy
transfer (FRET) is based on the principle that the CFP fluorophore, when
excited, can transfer energy to a nearby YFP fluorophore, prompting the
emission of YFP signals. FRET therefore permits the detection of
protein-protein interactions in live cells. CFP-labeled Brd2 and YFP-labeled
histones (all the core histones H2A, H2B, H3, H4, and H1) were co-expressed
in HeLa cells, and FRET signals were visualized on a single-cell basis by
flow cytometry. We detected FRET signals only with H4, not with other
histones, indicating that Brd2 specifically interacts with H4. The finding
that no adventitious FRET signals were observed with other core histones in
the nucleosome illustrates the high specificity of protein-histone
recognition detected by FRET. Deletion of the 28 amino acid H4 tail and
mutation of acetylatable lysine residues within the H4 tails abolished FRET
signals. Furthermore, mutation of the K12 residue, but not of other lysine
residues, eliminated FRET signals. These experiments and in vitro
peptide binding assays showed that Brd2 binds to chromatin through its
interaction with acetylated K12 of histone H4. FRET analysis performed with
TAFII250 and chimeric proteins in which Brd2 bromodomains were exchanged with
those of TAFII250 showed that it is the bromodomain that determines the
specificity of acetyl histone recognition. We
employed the bifluorescence complementation (BiFC) assay, another live-cell
technology, in order to visualize still further the Brd2-H4 interaction. In this
method, YFP is split and linked to N- and C-terminal halves, which are fused
to partner proteins. Interaction of the partners complements YFP, producing
fluorescent signals. The advantage of BiFC is that it allows detection of the
intracellular site of protein-protein interaction. We observed BiFC signals
on mitotic chromosomes showing that the Brd2-H4 interaction persists during
mitosis. Fluorescent
recovery after photobleaching was a third method with which we visualized
real-time interaction of Brd4 with chromatin. This method can detect the
mobility of a nuclear protein that is regulated by its interaction with
chromatin. Brd4 was highly mobile in the nucleus and transiently bound to
chromatin. The mobility of Brd4 was significantly delayed when chromatin was
hyperacetylated following treatment with histone deacetylase inhibitors.
Mathematical analysis of fluorescence recovery data showed that
Brd4-chromatin interactions follow two-phase kinetics with the average
residence time of 8 seconds for the faster-moving component and 39 seconds
for the slow-moving component. The overall mobility pattern was similar to
that of BRG1 and other chromatin-binding proteins. Our studies highlight the
dynamic interactions of Brd4 with acetyl chromatin, which are likely to be a
basis for the role of Brd4 in epigenetic memory and cell growth regulation. Dey A, Chitsaz F, Abbasi A, Misteli T,
Ozato K. The double bromodomain protein Brd4 binds to acetylated chromatin
during interphase and mitosis. Proc Nat Acad Sci USA 2003;100:8758-8763. 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. 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. 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. 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;117:349-360. aLeopddo Laricchia-Robio, PhD,
former Courtesy Contractor bTomohiko Kanno, MD, PhD, former
Guest Researcher cYuka
Kanno, MD, PhD, former Guest Researcher Collaborators Kathryn Calame, PhD, Howard Hughes Medical
Institute, Peter M. Howley, MD, Michael Lenardo, PhD, Laboratory of
Immunology, NIAID, Ben-Zion Levi, PhD, Technion-Israel
Institute of Technology, Tom Misteli, PhD, Laboratory of Receptor
Biology and Gene Expression, NCI, Alan Sher, PhD, Laboratory of Parasitic Diseases, NIAID, Richard Siegel, PhD, Laboratory of
Immunology, NIAID, Linda Wolfe, PhD, Laboratory of Cell
Biology, NCI, Huabao Xiong, MD, PhD, City University of
New York, Mount Sinai School of Medicine, New York, NY
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