Keiko Ozato

Immunodeficiency and Chronic Myelogenous Leukemia-like Syndrome in Mice with a Targeted Mutation of the ICSBP Gene

Interferon consensus sequence binding protein (ICSBP) is a transcription factor of the interferon (IFN) regulatory factor (IRF) family. Mice with a null mutation of ICSBP exhibit two prominent phenotypes related to previously described activities of the IRF family. The first is enhanced susceptibility to virus infections associated with impaired production of IFN(gamma). The second is deregulated hematopoiesis in both ICSBP-/- and ICSBP+/- mice that manifests as a syndrome similar to human chronic myelogenous leukemia. The chronic period of the disease progresses to a fatal blast crisis characterized by a clonal expansion of undifferentiated cells. Normal mice injected with cells from mice in blast crisis developed acute leukemia within 6 weeks of transfer. These results suggest a novel role for ICSBP in regulating the proliferation and differentiation of hematopoietic progenitor cells.

TRIM family proteins and their emerging roles in innate immunity

The superfamily of tripartite motif-containing (TRIM) proteins is conserved throughout the metazoan kingdom and has expanded rapidly during vertebrate evolution; there are now more than 60 TRIM proteins known in humans and mice. Many TRIM proteins are induced by type I and type II interferons, which are crucial for many aspects of resistance to pathogens, and several are known to be required for the restriction of infection by lentiviruses. In this Review, we describe recent data that reveal broader antiviral and antimicrobial activities of TRIM proteins and discuss their involvement in the regulation of pathogen-recognition and transcriptional pathways in host defence.

The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis

Previous in vitro studies showed that the bromodomain binds to acetyllysines on histone tails, leading to the proposal that the domain is involved in deciphering the histone code. However, there is little in vivo evidence supporting 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. Here, 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. This 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 support, salt solubility of Brd4 was markedly reduced upon increased histone acetylation. This change also required both bromodomains. In peptide binding assays, Brd4 avidly bound 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. These observations indicate that Brd4 specifically recognizes acetylated histone codes, and this recognition is passed onto the chromatin of newly divided cells.

Global Nature of Dynamic Protein-Chromatin Interactions In Vivo: Three-Dimensional Genome Scanning and Dynamic Interaction Networks of Chromatin Proteins

ABSTRACT Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic biophysical properties, such as off rate constants, residence time, and bound fraction, of a wide range of chromatin proteins of diverse functions in vivo. We demonstrate that most chromatin proteins have a high turnover on chromatin with a residence time on the order of seconds, that the major fraction of each protein is bound to chromatin at steady state, and that transient binding is a common property of chromatin-associated proteins. Our results indicate that chromatin-binding proteins find their binding sites by three-dimensional scanning of the genome space and our data are consistent with a model in which chromatin-associated proteins form dynamic interaction networks in vivo. We suggest that these properties are crucial for generating high plasticity in genome expression.

H-2RIIBP (RXR beta) heterodimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes.

H‐2RIIBP (RXR beta) is a member of the nuclear hormone receptor superfamily that activates transcription of MHC class I genes in response to retinoic acid (RA). Using chemical cross‐linking, co‐immunoprecipitation, gel mobility shift and streptavidin‐biotin DNA precipitation assays, we show that H‐2RIIBP formed heterodimers with thyroid hormone (T3) and RA receptors (T3R alpha and RAR alpha). H‐2RIIBP heterodimer formation required a conserved sub‐domain of its C‐terminal region, occurred independently of target DNA and was much more efficient than either T3R alpha/RAR alpha heterodimer or H‐2RIIBP homodimer formation. Heterodimers displayed enhanced binding to target DNA elements and contacted DNA in a manner distinct from that of homodimers. A functional role for heterodimers in vivo was demonstrated by synergistic enhancement of MHC class I transcription following co‐transfection of H‐2RIIBP with T3R alpha or RAR alpha. We provide biochemical evidence that H‐2RIIBP formed heterodimers with several naturally occurring nuclear proteins. The results suggest that H‐2RIIBP, by virtue of its ability to heterodimerize, enhances combinatorial diversity and versatility in gene regulation mediated by nuclear hormone receptors.

Monoclonal Antibodies To Mouse Major Histocompatibility Complex Antigens: Iv. A Series Of Hybridoma Clones Producing Anti-h-2d Antibodies And An Examination Of Expression Of H-2d Antigens On The Surface Of These Cells

As part of our continuing effort to produce a library of hybridoma antibodies specific for the products of the mouse major histocompatibility complex (MHC), nine antibodies reacting with antigens of the H-2d haplotype have been produced by cell fusion between immune spleen cells and the SP2/0.Ag.14 cell, of H-2d origin. Serological characterization revealed that seven antibodies reacted with H-2 antigens and two with Ia antigens. Of the anti-H-2 antibodies, four detected private specificities of H-2Kd or H-2Dd antigens and three detected public specificities of H-2d and other haplotypes. One of the anti-la antibodies detected a private Ia specificity corresponding to Ia.23 and the other detected a previously undescribed public specificity. Anti-H-2d hybridoma cells represent a potential “autoreactive” situation in that the antibodies produced by the cells should react with their own H-2 antigens unless expression of the corresponding H-2d antigens in these cells was altered. In order to examine whether H-2d antigens continued to be expressed on these anti-H-2d hybridoma cells, binding of 125I-labeled monoclonal anti-H-2Kd and/or H-2Dd antibodies was studied. Among the four hybridoma clones tested, three bound specifically three independent 125I-labeled anti-H-2d antibodies, including two cases in which binding of autologous antibodies was detected. The last clone did not bind any of the anti-H-2d antibodies, although it bound an anti-H-2k antibody, indicating selective loss of H-2d antigens. These observations demonstrate that neither loss nor retention of H-2d antigen expression on the cell surface is obligatory in hybridoma cells producing anti-H-2d antibodies.

Displacement of sequence-specific transcription factors from mitotic chromatin.

The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBP, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs.

Interaction of the Bovine Papillomavirus E2 Protein with Brd4 Tethers the Viral DNA to Host Mitotic Chromosomes

The papillomavirus E2 protein tethers viral genomes to host mitotic chromosomes to ensure genome maintenance. We have identified the bromodomain protein Brd4 as a major cellular interacting partner of the bovine papillomavirus E2. Brd4 associates with mitotic chromosomes and colocalizes with E2 on mitotic chromosomes. The site of E2 binding maps to the C-terminal domain of Brd4. Expression of this C-terminal Brd4 domain functions in a dominant-negative manner to abrogate the colocalization of E2 with Brd4 on mitotic chromosomes, to block association of the viral episomes with Brd4, and to inhibit BPV-1 DNA-mediated cellular transformation. Brd4 also associates with HPV16 E2, indicating that Brd4 binding may be a shared property of all papillomavirus E2 proteins. The interaction of E2 with Brd4 is required to ensure the tethering of viral genomes to the host mitotic chromosomes for persistence of viral episomes in PV-infected cells.

IFN Regulatory Factor-4 and -8 Govern Dendritic Cell Subset Development and Their Functional Diversity

Dendritic cells (DCs) are bone marrow (BM)-derived APCs central to both innate and adaptive immunity. DCs are a heterogeneous cell population composed of multiple subsets with diverse functions. The mechanism governing the generation of multiple DC subsets is, however, poorly understood. In this study we investigated the roles of closely related transcription factors, IFN regulatory factor (IRF)-4 and IRF-8, in DC development by analyzing IRF-4−/−, IRF-8−/−, and IRF-4−/−IRF-8−/− (double-knockout) mice. We found that IRF-4 is required for the generation of CD4+ DCs, whereas IRF-8 is, as reported previously, essential for CD8α+ DCs. Both IRFs support the development of CD4−CD8α− DCs. IRF-8 and, to a lesser degree, IRF-4 contribute to plasmacytoid DC (PDC) development. Thus, the two IRFs together regulate the development of all conventional DCs as well as PDCs. Consistent with these findings, IRF-4, but not IRF-8, was expressed in CD4+ DCs, whereas only IRF-8 was expressed in CD8α+ DCs. CD4−CD8α− DCs and PDCs expressed both IRFs. We also demonstrate in vitro that GM-CSF-mediated DC differentiation depends on IRF-4, whereas Fms-like tyrosine kinase 3 ligand-mediated differentiation depends mainly on IRF-8. Gene transfer experiments with double-knockout BM cells showed that both IRFs have an overlapping activity and stimulate a common process of DC development. Nonetheless, each IRF also possesses a distinct activity to stimulate subset-specific gene expression, leading to the generation of functionally divergent DCs. Together, IRF-4 and IRF-8 serve as a backbone of the molecular program regulating DC subset development and their functional diversity.

Brd4 coactivates transcriptional activation of NF-κB via specific binding to acetylated RelA

ABSTRACT Acetylation of the RelA subunit of NF-κB, especially at lysine-310, is critical for the transcriptional activation of NF-κB and the expression of inflammatory genes. In this study, we demonstrate that bromodomains of Brd4 bind to acetylated lysine-310. Brd4 enhances transcriptional activation of NF-κB and the expression of a subset of NF-κB-responsive inflammatory genes in an acetylated lysine-310-dependent manner. Bromodomains of Brd4 and acetylated lysine-310 of RelA are both required for the mutual interaction and coactivation function of Brd4. Finally, we demonstrate that Brd4 further recruits CDK9 to phosphorylate C-terminal domain of RNA polymerase II and facilitate the transcription of NF-κB-dependent inflammatory genes. Our results identify Brd4 as a novel coactivator of NF-κB through specifically binding to acetylated lysine-310 of RelA. In addition, these studies reveal a mechanism by which acetylated RelA stimulates the transcriptional activity of NF-κB and the NF-κB-dependent inflammatory response.