We are interested in transcription factors that regulate the development of the immune system. We previously isolated ICSBP/IRF-8, 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. We also showed that the introduction of ICSBP rescues normal growth and differentiation of these cells in ICSBP-null progenitors. Our studies have investigated the effect of ICSBP on the development and maturation of dendritic cells (DCs). We have also identified target genes regulated by ICSBP and characterized ICSBP’s role in controlling target promoters and regulating the activity of chromatin.

We have studied the role of chromatin from another perspective and characterized a bromodomain protein Brd4. Brd4 is a novel member of the conserved BET family and contains a motif indicative of interaction with chromatin. We showed that, consistent with a strong affinity for chromatin, it associates with mitotic chromosomes. Recent FRET and FLIP analyses suggest that Brd4 and a related protein interact with acetylated histones in living cells. Conditional knock-out and antisense/RNAi approaches further elucidate the role of Brd4, particularly in cell growth.

Microarray Identification of Genes Controlled by ICSBP during Macrophage Differentiation and Role of Chromatin
Tamura, Laricchia, Thotokura; in collaboration with Ko
ICSBP-/- mice develop a syndrome similar to human chronic myelogenous leukemia (CML). In addition, ICSBP-/- macrophages are functionally defective, causing the mice to be immunodeficient. By studying bipotential ICSBP-/- myeloid progenitor cell lines, we found that ICSBP drives their differentiation toward mature functional macrophages while causing a complete growth arrest. On the other hand, it inhibits granulocytic differentiation, explaining the CML-like syndrome in ICSBP-/- mice.

To study the molecular mechanism by which ICSBP controls myeloid cell differentiation, we performed a genome-wide gene expression analysis by using the mouse 15K cDNA microarray. To analyze a change in gene expression patterns at an early stage, we used an inducible ICSBP/estrogen receptor (ICSBP/ER) chimera. We identified 27 known genes and 28 unknown genes that displayed a greater than two-fold increase or decrease within four hours of ICSBP expression. Among them, three genes including cystatin C were induced early, even in the presence of cycloheximide, indicating that they are direct targets of ICSBP. By using a newly developed self-inactivating retrovirus-based reporter assay, we observed that ICSBP/ER induces promoter activation. ICSBP/ER down-regulated the expression of the c-myc gene, but cycloheximide inhibited the repression, indicating that ICSBP regulates c-myc gene expression through an ICSBP-inducible molecule or molecules. We found that the transcription factor Blimp-1, which is implicated in macrophage differentiation, growth inhibition, and c-myc repression, was directly induced by ICSBP, suggesting that ICSBP down-regulates c-myc through Blimp-1. The results indicate that ICSBP controls the expression of regulatory genes as well as those involved in macrophage function. Microarray analysis is thus a useful first step toward understanding global changes in gene expression patterns during differentiation of immune cells.

Introduction of ICSBP into progenitor cells causes dramatic alterations in the nuclear architecture, suggesting that ICSBP interacts with chromatin to alter target gene expression. Interaction of ICSBP with chromatin during macrophage differentiation is under study with fluorescent recovery after photobleaching.

Role of ICSBP in the Development of Dendritic Cells
Tsujimura, Tamura, Kong
Dendritic cells (DCs) 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 lack subsets of DCs (pDCs and CD8a+ Dcs). To investigate the role of ICSBP in DC development, we employed a Flt3 ligand-based in vitro culture system. DCs that developed in vitro from ICSBP-/- BM cells were defective in the expression of MHC class II and co-stimulatory molecules, indicating that differentiation of ICSBP-/- progenitors toward immature DCs is impaired. Moreover, -/- DCs displayed a generalized maturation failure and did not express IL-12 p40 mRNA and proteins in response to various maturation signals, consistent with the feature observed with ICSBP-/-DCs in vivo. In addition, -/- DCs failed to increase expression of MHC class II and co-stimulatory molecules upon maturation signals. We show that retroviral introduction of ICSBP restores the ability of -/- progenitors to develop immature DCs that can undergo full maturation upon activation signals. Transduction of the wild-type ICSBP, but not transcriptionally inactive mutants, increased expression of MHC class II and co-stimulatory molecules on immature cells. More important, ICSBP-transduced cells produced IL-12 proteins upon activation, along with increased expression of co-stimulatory molecules. The study identifies ICSBP as a factor critical for both early differentiation and final maturation of DCs.

Interaction of Bromodomain Protein Brd4 with the DNA Replication Machinery and the Role in the G1- S Cell Cycle Progression
Farina, Dey, Jang; in collaboration with Hurwitz
Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains thought to be implicated in interactions with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G1 phase upon mitogenic stimuli. To study the role of Brd4 in cell growth control, we examined the effects of over-expression of Brd4 in NIH-3T3 and HeLa cells. We found that ectopic Brd4 expression inhibits cell cycle progression from G1 to S. Co-immunoprecipitation experiments showed that endogenous and transfected Brd4 interact with the replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis demonstrated that Brd4 directly binds to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletions indicated that both the interaction with RFC-140 and the inhibition of entry into S phase depend on the second bromodomain of Brd4.

FIGRURE 28

Model of Brd4 action

Finally, supporting the functional importance of this interaction, co-transfection of RFC-140 reduced the growth-inhibitory effect of Brd4. Taken together, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC (Fig. 28). A systematic biochemical analysis is under way to identify proteins with which Brd4 forms a complex.

Interaction of Brd4 with Acetylated Chromatin
Kanno T, Kanno Y, Dey; in collaboration with Lenardo, Siegel
To assess the interaction of Brd4 with chromatin, we have carried out analysis of fluorescent loss in photobleaching (FLIP) of transiently transfected GFP-Brd4 in the presence or absence of TSA, a histone deacetylase inhibitor. We found that TSA treatment inhibits loss of fluorescence, indicating that Brd4 preferentially interacts with chromatin. Biochemical analysis with Brd4-nucleosome interactions also supports strong affinity of Brd4 for acetylated histones. Interaction of the BET family proteins with chromatin is also under investigation with the fluorescence resonance energy transfer technique (FRET).

Dynamic Relationship between Histone Acetylases and Deacetylases
Kanno T, Kanno Y; in collaboration with Howard, Lenardo, Siegel
Histone acetylases (HAT) and deacetylases have opposing enzymatic activities. Both enzymes affect chromatin structures and regulate transcription. It is assumed that a mechanism balances the activities of the two enzymes in the cell. We hypothesized that one way to achieve balance is for the two enzymes to interact with each other and function as a single entity. By FRET analysis, we showed that the acetylase PCAF and deacetylase HDAC1 are spatially proximate in living cells compatible with their physical interaction. In agreement, co-immunoprecipitation assays demonstrated that endogenous HDACs are associated with PCAF and another acetylase GCN5 in HeLa cells. We found by glycerol gradient sedimentation analysis that HATs are integrated into a large multi-protein HDAC complex that is distinct from the previously described HDAC complexes containing mSin3A, Mi-2/NRD, or CoREST. The HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed in vitro. The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes in vivo.