Developmental Gene Regulation of the Immune System

Keiko Ozato, PhD, Head, Section on Molecular Genetics of Immunity
Tomohiko Kanno, MD, PhD, Staff Scientist
Anup Dey, PhD, Biologist
Prafullakumar Tailor, PhD, Research Fellow
Toru Atsumi, PhD, Visiting Fellow
Tsung Hsien Chang, PhD, Visiting Fellow
Anu Ghosh, PhD, Visiting Fellow
Sukhendu Gosh, PhD, Visiting Fellow
Lakshmi Ramakrishna, PhD, Visiting Fellow
Matthew Smith, BS, Postbaccalaureate Fellow

We study transcription factors and chromatin-binding proteins that control the development of innate immunity. Our aim is to study the activity of IRF8 in the development of dendritic cells (DC)—cells that play essential roles in innate immunity. We showed that IRF8 drives, among other DC subsets, the development of plasmacytoid DCs (pDC) and CD8α⁺ DCs, which produce type I interferons (IFNs) and IL-12, respectively. These cytokines are important for the establishment of an anti-microbial state. Our studies yielded clues to the mechanism of IRF8 action, namely, that (1) IRF8 contributes to high IFN production in DCs by amplifying the feedback phase of transcription and that (2) the naturally occurring IRF8 point mutation alters IRF8’s interaction with partner proteins, thereby altering DC developmental pathways for a specific DC subset. We also work on the bromodomain protein Brd4. Brd4 binds to acetylated chromatin, which is implicated in the maintenance of transcriptional memory. By extending our earlier study showing that Brd4 regulates cell-cycle progression, we found that Brd4 binds to the promoters of many G1 genes and recruits P-TEFb, a kinase that triggers transcriptional elongation. These studies have provided a mechanistic basis for Brd4 action.

Role of IRF8 in type I IFN transcription in DCs

Tailor, Ramakrishna, Atsumi, Chang

The observation that DCs from IRF8 knockout mice produce little type I IFNs led us to study the molecular basis of IFN transcription in DCs. We found that type I IFN mRNA induction follows two-phase kinetics. Induction of the early transcripts, seen within one hour of Newcastle disease virus (NDV) stimulation, was intact in IRF8 knockout DCs. Transcription in the second phase, which peaked at seven hours, produced much higher levels of IFN mRNA. The second transcript induction was completely missing in IRF8 knockout DCs. Both pDCs and conventional DCs (cDCs) induced IFN transcripts in response to NDV infection. The second-phase induction represented an IFN feedback response in that it was also missing in DCs from Ifnar1 knockout mice. IRF3 and IRF7 were normally expressed and translocated into the nucleus upon NDV stimulation in IRF8 knockout DCs. Supporting the view that IRF8 plays a role in transcription, introduction of IRF8 into NIH3T3 fibroblasts led to a large amplification of the otherwise weak second phase of IFN induction. Using chromatin immunoprecipitation (ChIP) analysis, we showed that IRF8 is recruited to the promoters of the Ifnb, Ifna4, and Ifna5 genes (which encode IFNβ, IFNα4, and IFNα5). These promoters have several virus response elements similar to IFN-stimulated response elements (ISREs). Consistent with its role in the second phase, we observed recruitment of IRF8 following that of IRF7 and pol II—IRF7 and pol II were both bound to the promoters at an earlier time. Similarly, IRF8 bound to the IFN promoter after stimulation by TLR (Toll-like receptor) ligands such as CpG DNA and poly IC. We have thus provided a mechanistic basis of IRF8 action by showing that binding of pol II to the IFN promoters declined rapidly after initial recruitment in IRF8 knockout DCs, while pol II binding continued for an extended period in Irf8^wt DCs. We expect that IRF8 supports re-recruitment of pol II, thereby boosting second-phase transcription.

Gabriele L, Ozato K. The role of the interferon regulatory factor (IRF) family in dendritic cell development and function. Cytokine Growth Factor Rev 2007;18:503-510.
Ozato K, Tailor P, Kubota T. The interferon regulatory factor family in host defense: mechanism of action. J Biol Chem 2007;282:20065-20069.
Tailor P, Tamura T, Kong H, Kubota T, Kubota M, Borghi P, Gabriele L, Ozato K. The feedback phase of type I interferon induction in dendritic cells requires interferon regulatory factor 8. Immunity 2007;27:228-239.

Mechanism of IRF8 action as revealed by the BXH2 mutation

Tailor, Ramakrishna, Atsumi, Chang, Smith

The BXH2 recombinant inbred mouse strain displays a phenotype similar to IRF8 knockout mice. BXH2 mice are defective in IL12p40 production and hence highly susceptible to Mycobacterium bovis (BCG). They carry a point mutation in the Irf8/Icsbp gene that changes arginine (R) to cysteine (C) in position 294. The mutation, designated Irf8^R294C, is within the IAD (IRF association domain), which is important for the interaction of IRF8 with partner proteins. We found that CD8α⁺ DCs are selectively missing in these mice, although the mice have normal numbers of pDC s. Consistent with the selective inability to generate CD8α⁺ DCs, FMS-like tyrosine kinase 3 ligand (Flt3L)–based bone marrow (BM) cultures from Irf8^R294C mutants failed to produce this DC subset but generated abundant pDCs. Accordingly, DCs generated from the Irf8^R294C mutants were defective in IL-20p40 production while intact in type I IFN production. This defect could be rescued when the IRF8^wt gene was transferred into the Irf8^R294C BM cells. Electrophoretic mobility shift assays and ChIP experiments showed that IRF8^R294C did not interact with its partners PU.1, SpiB, and IRF1,2 and was not recruited to the promoter of the IL-12p40 and Csc3 (cystatin C) genes, which are expressed mostly in CD8α⁺ DCs. The results demonstrated that IRF8 drives the development of the two DC subsets by two distinct mechanisms and that the one requiring interactions with these partners is essential for CD8α⁺DCs. Our study demonstrates the molecular mechanisms of the developmental pathways that demarcate pDCs and CD8α⁺DCs.

Alter-Koltunoff M, Goren S, Nousbeck J, Feng CG, Sher A, Ozato K, Azriel A, Levi BZ. Innate immunity to intraphagosomal pathogens is mediated by interferon regulatory factor 8 (IRF-8) that stimulates the expression of macrophage-specific nramp1 through antagonizing repression by c-Myc. J Biol Chem 2008;283:2724-2733.
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;282:5633-5640.
Kong HJ, Anderson DE, Lee CH, Jang MK, Tamura T, Tailor P, Cho HK, Cheong J, Xiong H, Morse HC 3rd, 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.
Tailor P, Tamura T, Morse HC 3rd, Ozato K. The BXH2 mutation in IRF8 differentially impairs dendritic cell subset development in the mouse. Blood 2008;111:1942-1948.
Watanabe T, Asano N, Murray PJ, Ozato K, Tailor P, Fuss IJ, Kitani A, Strober W. Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis. J Clin Inv 2008;118:545-559.

Co-recruitment of Brd4, P-TEFb, and pol II drives G1 gene transcription and cell-cycle progression

Dey, Kanno, Ghosh, Smith

Brd4 interacts with P-TEFb and the RNA polymerase II mediator complex. P-TEFb is a kinase composed of Cdk9 and cyclinT and is important for transcriptional elongation of RNA polymerase II. The multiprotein complex mediator associates with polymerase II and contributes to transcription. This interaction pattern suggests that Brd4 is involved in transcriptional regulation.

On the other hand, mounting evidence points to a role for Brd4 in cell growth, including that of cancer cells. Thus, we investigated cell growth by using NIH3T3 cells in which Brd4 was knocked down by short hairpin RNA (shRNA). The use of shRNA was a practical alternative to studying Brd4 knockout mice because the mice die early without allowing for cell culture. Brd4 knockdown cells, when synchronized to G0 by serum starvation, failed to progress through G1 after release and did not enter S-phase. Microarray analysis of synchronized Brd4 knockdown cells showed that, while numerous genes were induced in control shRNA cells during G1 progression, the majority of these genes remained uninduced in Brd4 knockdown cells, indicating that Brd4 regulates cell growth partly through the regulation of G1 genes. We confirmed the role of Brd4 in G1 progression by the rescue of G1 gene expression and restoration of S-phase entry following reintroduction of Brd4. We performed ChIP analysis for G1 genes downregulated in Brd4 knockdown cells, including those encoding cyclin D1, cyclin D2, Orc2, and Mcm2. We found that Brd4 was bound to these and other G1 genes at the transcription start sites when cells progressed from G0 to G1. However, Brd4 did not bind to Tlr9, a gene not expressed in NIH3T3 cells, validating the specificity of our ChIP data. By extending our ChIP experiments, we showed that P-TEFb and pol II were co-recruited along with Brd4 to these genes during G1. The results lend credence to the role of Brd4 in G1 gene regulation.

Crawford NP, Alsarraj J, Lukes L, Walker RC, Officewala JS, Yang HH, Lee MP, Ozato K, Hunter KW. Bromodomain 4 activation predicts breast cancer survival. Proc Natl Acad Sci USA 2008;105:6380-6385.
Mochizuki K, Nishiyama A, Jang MK, Dey A, Ghosh A, Tamura T, Natsume H, Yao H, Ozato K. The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase. J Biol Chem 2008;283:90440-90448.

Intracellular delivery of acetyl histone peptides inhibits native bromodomain-chromatin interactions and impairs mitotic progression

Dey, Ghosh, Smith

Post-translational modification of histone tails regulates the chromatin environment and influences gene expression and cell growth. The bromodomains in Brd4 and other chromatin-binding proteins bind to acetylated histone tails and modulate transcription. Changes in bromodomain-histone interactions are thought to affect epigenetic memory and cellular functions. With the aim of altering Brd4-chromatin interactions in vivo, we took a protein transduction approach to deliver acetylated histone tail peptides into cultured cells. NIH 3T3 and P19 cells efficiently incorporated unacetylated and acetylated histone H3 and H4 peptides when fused to the protein transduction domain (PTD) derived from the Tat protein of the human immunodeficiency virus (HIV). We found that acetyl H4 peptides, but not their unacetylated counterparts, increased the real-time mobility of Brd4, as assessed by fluorescence recovery after photobleaching. Increased Brd4 mobility indicates that the acetylated H4 peptides specifically inhibited Brd4-chromatin interactions in living cells, consistent with previous in vitro observations that the same peptides bind to Brd4. The acetyl-H4 peptides also inhibited the interaction of Brd4 with mitotic chromosomes and compromised subsequent cell growth. In summary, delivery of PTD-mediated histone tail peptides offers a novel means both to manipulate bromodomain-chromatin interactions in vivo and to study their biological significance.

Nishiyama A, Mochizuki K, Mueller F, Karpova T, McNally JG, Ozato K. Intracellular delivery of acetylhistone peptides inhibits native bromodomain-chromatin interactions and impairs mitotic progression. FEBS Lett 2008;582:1501-1507.
Toyama R, Rebbert ML, Dey A, Ozato K, Dawid IB. Brd4 associates with mitotic chromosomes throughout early zebrafish embryogenesis. Dev Dyn 2008;237:1636-1644.

Collaborators

Bruce Howard, MD, Program in Genomics of Differentiation, NICHD, 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, II, MD, Laboratory of Immunopathology, NIAID, Rockville, MD
Tomohiko Tamura, MD, PhD, Tokyo University, Tokyo, Japan

For further information, contact ozatok@mail.nih.gov or visit http://ozatolab.nichd.nih.gov.