Tomohiko Kanno

The oncoprotein Bcl-3 directly transactivates through κB motifs via association with DNA-binding p50B homodimers

Bcl-3 is an I kappa B-related protein with ankyrin repeat motifs. Its gene is located at a site of recurrent translocations in a subset of B cell chronic lymphocytic leukemias. Bcl-3 associates tightly with p50B (NFKB2, p52) homodimers in cells, and together these proteins form a ternary complex with DNA at kappa B sites. Such an association functionally leads to a novel and potent form of transactivation through the kappa B motif: the tethering of Bcl-3 to DNA via the p50B homodimers allows Bcl-3 to transactivate directly, while p50B homodimers alone cannot. Transactivation mediated by Bcl-3 requires two cooperating domains located amino- and carboxy-terminal to the ankyrin domain. Bcl-3 is localized to the nucleus, and a Bcl-3-p50B complex is detected in certain lymphoid cells. Our data reveal a novel role for Bcl-3, distinct from that of the inhibitor I kappa B. The results have implications for tumorigenesis.

Interaction and Functional Cooperation of PEBP2/CBF with Smads SYNERGISTIC INDUCTION OF THE IMMUNOGLOBULIN GERMLINE Cα PROMOTER

Smads are signal transducers for members of the transforming growth factor-β (TGF-β) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of α and β subunits. The α subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian α subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-β/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2αC/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Cα promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-β/BMP signaling.

Selective Recognition of Acetylated Histones by Bromodomain Proteins Visualized in Living Cells

Acetylation and other modifications on histones comprise histone codes that govern transcriptional regulatory processes in chromatin. Yet little is known how different histone codes are translated and put into action. Using fluorescence resonance energy transfer, we show that bromodomain-containing proteins recognize different patterns of acetylated histones in intact nuclei of living cells. The bromodomain protein Brd2 selectively interacted with acetylated lysine 12 on histone H4, whereas TAF(II)250 and PCAF recognized H3 and other acetylated histones, indicating fine specificity of histone recognition by different bromodomains. This hierarchy of interactions was also seen in direct peptide binding assays. Interaction with acetylated histone was essential for Brd2 to amplify transcription. Moreover association of Brd2, but not other bromodomain proteins, with acetylated chromatin persisted on chromosomes during mitosis. Thus the recognition of histone acetylation code by bromodomains is selective, is involved in transcription, and potentially conveys transcriptional memory across cell divisions.

TGF-β and retinoic acid induce the microRNA miR-10a, which targets Bcl-6 and constrains the plasticity of helper T cells

Distinct CD4+ T cell subsets are critical for host defense and immunoregulation. Although these subsets can act as terminally differentiated lineages, they have been increasingly noted to demonstrated plasticity. MicroRNAs are factors that control T cell stability and plasticity. Here we report that naturally occurring regulatory T cells (Treg cells) had high expression of the microRNA miR-10a and that miR-10a was induced by retinoic acid and transforming growth factor-β (TGF-β) in inducible Treg cells. By simultaneously targeting the transcriptional repressor Bcl-6 and the corepressor Ncor2, miR-10a attenuated the phenotypic conversion of inducible Treg cells into follicular helper T cells. We also found that miR-10a limited differentiation into the TH17 subset of helper T cells and therefore represents a factor that can fine-tune the plasticity and fate of helper T cells.

Intrinsic transcriptional activation-inhibition domains of the polyomavirus enhancer binding protein 2/core binding factor alpha subunit revealed in the presence of the beta subunit.

ABSTRACT A member of the polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is composed of PEBP2αB1/AML1 (as the α subunit) and a β subunit. It plays an essential role in definitive hematopoiesis and is frequently involved in the chromosomal abnormalities associated with leukemia. In the present study, we report functionally separable modular structures in PEBP2αB1 for DNA binding and for transcriptional activation. DNA binding through the Runt domain of PEBP2αB1 was hindered by the adjacent carboxy-terminal region, and this inhibition was relieved by interaction with the β subunit. Utilizing a reporter assay system in which both the α and β subunits are required to achieve strong transactivation, we uncovered the presence of transcriptional activation and inhibitory domains in PEBP2αB1 that were only apparent in the presence of the β subunit. The inhibitory domain keeps the full transactivation potential of full-length PEBP2αB1 below its maximum potential. Fusion of the transactivation domain of PEBP2αB1 to the yeast GAL4 DNA-binding domain conferred transactivation potential, but further addition of the inhibitory domain diminished the activity. These results suggest that the activity of the α subunit as a transcriptional activator is regulated intramolecularly as well as by the β subunit. PEBP2αB1 and the β subunit were targeted to the nuclear matrix via signals distinct from the nuclear localization signal. Moreover, the transactivation domain by itself was capable of associating with the nuclear matrix, which implies the existence of a relationship between transactivation and nuclear matrix attachment.

Requirement of Runx1/AML1/PEBP2alphaB for the generation of haematopoietic cells from endothelial cells.

Recent studies revealing that endothelial cells derived from E8.5‐E10.5 mouse embryos give rise to haematopoietic cells appear to correspond to previous histological observations that haematopoietic cell clusters are attached to the ventral aspect of dorsal aorta in such a way as if they were budding from the endothelial cell layer. Gene disruption studies have revealed that Runx1/AML1 is required for definitive haematopoiesis but not for primitive haematopoiesis, but the precise stage of gene function is not yet known.

The oncoprotein Bcl-3 can facilitate NF-kappa B-mediated transactivation by removing inhibiting p50 homodimers from select kappa B sites.

Previously we have proposed a role for Bcl‐3 in facilitating transactivation through kappa B sites by counteracting the inhibitory effects of bound, non‐transactivating homodimers of the p50 subunit of NF‐kappa B. Such homodimers are abundant for example in nuclei of unstimulated primary T cells. Here we extend the model and provide new evidence which fulfills a number of predictions. (i) Bcl‐3 preferentially targets p50 homodimers over NF‐kappa B heterodimers since the homodimers are completely dissociated from kappa B sites at concentrations of Bcl‐3 which do not affect NF‐kappa B. (ii) Select kappa B sites associate very strongly and stably with p50 homodimers, completely preventing binding by NF‐kappa B. Such kappa B sites are likely candidates for regulation by p50 homodimers and Bcl‐3. (iii) Bcl‐3 and p50 can be co‐localized in the nucleus, a requirement for active removal of homodimers from their binding sites in vivo. (iv) The ankyrin repeat domain of Bcl‐3 is sufficient for the reversal of p50 homodimer‐mediated inhibition, correlating with the ability of this domain alone to inhibit p50 binding to kappa B sites in vitro. Our data support the model that induction of nuclear Bcl‐3 may be required during cellular stimulation to actively remove stably bound p50 homodimers from certain kappa B sites in order to allow transactivating NF‐kappa B complexes to engage. This exact mechanism is demonstrated with in vitro experiments.

BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones

Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb and has been proposed to release RNA polymerase II (Pol II) from promoter-proximal pausing. We show that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function. BRD4 interacts with elongating Pol II complexes and assists Pol II in progression through hyperacetylated nucleosomes by interacting with acetylated histones via bromodomains. On active enhancers, the BET inhibitor JQ1 antagonizes BRD4-associated eRNA synthesis. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by facilitating transcript elongation both at enhancers and on gene bodies independently of P-TEFb.

Kinetic analysis of human T-cell leukemia virus type I Tax-mediated activation of NF-kappa B.

The human T-cell leukemia virus type I (HTLV-I) Tax protein induces the expression of cellular genes, at least in part, by activating the endogenous NF-kappa B transcription factors. Induced expression of cellular genes is thought to be important for transformation of T cells to continued growth, a prelude to the establishment of adult T-cell leukemia. However, neither underlying mechanisms nor kinetics of the Tax-mediated activation of NF-kappa B are understood. We have analyzed a permanently transfected Jurkat T-cell line in which the expression of Tax is entirely dependent on addition of heavy metals. The initial NF-kappa B binding activity seen after induction of Tax is due almost exclusively to p50/p65 heterodimers. At later times, NF-kappa B complexes containing c-Rel and/or p52 accumulate. The early activation of p50/p65 complexes is a posttranslational event, since neither mRNA nor protein levels of NF-kappa B subunits had increased at that time. We demonstrate for the first time a Tax-induced proteolytic degradation of the NF-kappa B inhibitor, I kappa B-alpha, which may trigger the initial nuclear translocation of NF-kappa B. As nuclear NF-kappa B rapidly and potently stimulates resynthesis of I kappa B-alpha, the steady-state level of I kappa B-alpha does not significantly change. Thus, the dramatic Tax-induced increase in the I kappa B-alpha turnover may continually weaken inhibition and activate NF-kappa B. Additional, distinct actions of Tax may contribute further to the high levels of NF-kappa B activity seen.

A novel transcript encoding an N-terminally truncated AML1/PEBP2 alphaB protein interferes with transactivation and blocks granulocytic differentiation of 32Dcl3 myeloid cells.

The gene AML1/PEBP2 alphaB encodes the alpha subunit of transcription factor PEBP2/CBF and is essential for the establishment of fetal liver hematopoiesis. Rearrangements of AML1 are frequently associated with several types of human leukemia. Three types of AML1 cDNA isoforms have been described to date; they have been designated AML1a, AML1b, and AML1c. All of these isoforms encode the conserved-Runt domain, which harbors the DNA binding and heterodimerization activities. We have identified a new isoform of the AML1 transcript, termed AML1 deltaN, in which exon 1 is directly connected to exon 4 by alternative splicing. The AML1 deltaN transcript was detected in various hematopoietic cell lines of lymphoid to myeloid cell origin, as revealed by RNase protection and reverse transcriptase PCR analyses. The protein product of AML1 deltaN lacks the N-terminal region of AML1, including half of the Runt domain, and neither binds to DNA nor heterodimerizes with the beta subunit. However, AML1 deltaN was found to interfere with the transactivation activity of PEBP2, and the molecular region responsible for this activity was identified. Stable expression of AML1 deltaN in 32Dcl3 myeloid cells blocked granulocytic differentiation in response to granulocyte colony-stimulating factor. These results suggest that AML1 deltaN acts as a modulator of AML1 function and serves as a useful tool to dissect the functional domains in the C-terminal region of AML1.