Hideto Hoshino

Hemoprotein Bach1 regulates enhancer availability of heme oxygenase‐1 gene

Heme oxygenase‐1 (HO‐1) protects cells from various insults including oxidative stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have been the focus of studies on the inducible expression of ho‐1. Here we show that a heme‐binding factor, Bach1, is a critical physiological repressor of ho‐1. Bach1 bound to the multiple Maf recognition elements (MAREs) of ho‐1 enhancers with MafK in vitro and repressed their activity in vivo, while heme abrogated this repressor function of Bach1 by inhibiting its binding to the ho‐1 enhancers. Gene targeting experiments in mice revealed that, in the absence of Bach1, ho‐1 became expressed constitutively at high levels in various tissues under normal physiological conditions. By analyzing bach1/nrf2 compound‐deficient mice, we documented antagonistic activities of Bach1 and Nrf2 in several tissues. Chromatin immunoprecipitation revealed that small Maf proteins participate in both repression and activation of ho‐1. Thus, regulation of ho‐1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor–activator antagonism.

The transcriptional programme of antibody class switching involves the repressor Bach2

Activated B cells differentiate to plasma cells to secrete IgM or, after undergoing class switch recombination (CSR), to secrete other classes of immunoglobulins. Diversification of antibody function by CSR is important for humoral immunity. However, it remains unclear how the decision for the bifurcation is made. Bach2 is a B-cell-specific transcription repressor interacting with the small Maf proteins whose expression is high only before the plasma cell stage. Here we show that Bach2 is critical for CSR and somatic hypermutation (SHM) of immunoglobulin genes. Genetic ablation of Bach2 in mice revealed that Bach2 was required for both T-cell-independent and T-cell-dependent IgG responses and SHM. When stimulated in vitro, Bach2-deficient B cells produced IgM, as did wild-type cells, and abundantly expressed Blimp-1 (refs 9, 10) and XBP-1 (ref. 11), critical regulators of the plasmacytic differentiation, indicating that Bach2 was not required for the plasmacytic differentiation itself. However, they failed to undergo efficient CSR. These findings define Bach2 as a key regulator of antibody response and provide an insight into the orchestration of CSR and SHM during plasma cell differentiation.

Identification of Bach2 as a B-cell-specific partner for small maf proteins that negatively regulate the immunoglobulin heavy chain gene 3' enhancer.

Maf family transcription factors are important regulators in various differentiation systems. Putative Maf recognition elements (MAREs) are found in the 3′ enhancer region of the immunoglobulin heavy chain (IgH) gene. These elements are bound in B‐cell extracts by a heterodimeric protein complex containing both Bach2 and a small Maf protein. Analysis of normal hematopoietic cells revealed that Bach2 is specifically expressed in B cells. Bach2 is abundantly expressed in the early stages of B‐cell differentiation and turned off in terminally differentiated cells. Bach2 acts together with MafK as a negative effector of the IgH 3′ enhancer and binds to the co‐repressor SMRT (silencing mediator of retinoid and thyroid receptor). Hence the Bach2–small‐Maf heterodimer may represent the first example of a B‐cell lineage, and of a developmental stage‐restricted negative effector of the MARE in the IgH 3′ enhancer region.

A combinatorial code for gene expression generated by transcription factor Bach2 and MAZR (MAZ-related factor) through the BTB/POZ domain.

ABSTRACT Bach2 is a B-cell- and neuron-specific transcription repressor that forms heterodimers with the Maf-related oncoproteins. We show here that Bach2 activates transcription by interacting with its novel partner MAZR. MAZR was isolated by the yeast two-hybrid screen using the BTB/POZ domain of Bach2 as bait. Besides the BTB/POZ domain, MAZR possesses Zn finger motifs that are closely related to those of the Myc-associated Zn finger (MAZ) protein. MAZR mRNA was coexpressed with Bach2 in B cells among hematopoietic cells and in developing mouse limb buds, suggesting a cooperative role for MAZR and Bach2 in these cells. MAZR forms homo- and hetero-oligomers with Bach2 through the BTB domain, which oligomers bind to guanine-rich sequences. Unlike MAZ, MAZR functioned as a strong activator of the c-myc promoter in transfection assays with B cells. However, it does not possess a typical activation domain, suggesting a role for it as an unusual type of transactivator. The fgf4 gene, which regulates morphogenesis of limb buds, contains both guanine-rich sequences and a Bach2 binding site in its regulatory region. In transfection assays using fibroblast cells, the fgf4 gene was upregulated in the presence of both MAZR and Bach2 in a BTB/POZ domain-dependent manner. The results provide a new perspective on the function of BTB/POZ domain factors and indicate that BTB/POZ domain-mediated oligomers of transcription factors may serve as combinatorial codes for gene expression.

Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging

Taking advantage of the phenomenon of bioluminescence resonance energy transfer (BRET), we developed a bioluminescent probe composed of EYFP and Renilla reniformis luciferase (RLuc)—BRET-based autoilluminated fluorescent protein on EYFP (BAF-Y)—for near-real-time single-cell imaging. We show that BAF-Y exhibits enhanced RLuc luminescence intensity and appropriate subcellular distribution when it was fused to targeting-signal peptides or histone H2AX, thus allowing high spatial and temporal resolution microscopy of living cells.

Enhanced Beetle Luciferase for High-Resolution Bioluminescence Imaging

We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin α by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc.

Repression of PML Nuclear Body-Associated Transcription by Oxidative Stress-Activated Bach2

ABSTRACT Several lines of evidence suggest that gene expression is regulated not only by the interaction between transcription factors and DNA but also by the higher-order architecture of the cell nucleus. PML bodies are one of the most prominent nuclear substructures which have been implicated in transcription regulation during apoptosis and stress responses. Bach2 is a member of the BTB-basic region leucine zipper factor family and represses transcription activity directed by the 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element. Bach2 forms nuclear foci associated with PML bodies upon oxidative stress. Here, we demonstrate that transcription activity associated with PML bodies is selectively repressed by the recruitment of Bach2 around PML bodies. Fluorescence recovery after photobleaching experiments revealed that Bach2 showed rapid turnover in the nuclear foci. The Bach2 N-terminal region including the BTB domain is essential for the focus formation. Sumoylation of Bach2 is required for the recruitment of the protein around PML bodies. These observations represent the first example of modulation of transcription activity associated with PML bodies by a sequence-specific transcription factor upon oxidative stress.

14-3-3 regulates the nuclear import of class IIa histone deacetylases.

Class IIa histone deacetylases (HDACs) form complexes with a class of transcriptional repressors in the nucleus. While screening for compounds that could block the association of HDAC4 with the BTB domain-containing transcriptional repressor Bach2, we discovered that phorbol 12-myristate 13-acetate (PMA) induced the cytoplasmic retention of HDAC4 mutants lacking a nuclear export signal (NES). Although PMA treatment and PKD overexpression has been proposed to facilitate the nuclear export of class IIa HDACs by creating 14-3-3 binding sites containing phosphoserines, our experiments using HDAC mutants demonstrated that PMA greatly reduces nuclear import. PMA treatment repressed the NLS activity in a manner dependent on 14-3-3 binding. These results suggest that nuclear HDAC4 is not tethered in the nucleus, but instead shuttles between the nucleus and the cytoplasm. Phosphorylation-induced 14-3-3 binding biases the balance of nucleo-cytoplasmic shuttling toward the cytoplasm by inhibiting nuclear import.

From Metal–Organic Framework to Intrinsically Fluorescent Carbon Nanodots

Highly photoluminescent carbon nanodots (CNDs) were synthesized for the first time from metal-organic framework (MOF, ZIF-8) nanoparticles. Coupled with fluorescence and non-toxic characteristics, these carbon nanodots could potentially be used in biosafe color patterning.

Current advanced bioluminescence technology in drug discovery

Background:Bioluminescence technology is based on the luciferin–luciferase reaction and is generally well known as a reporter gene assay system that uses firefly luciferase. It has revolutionized the field of transcriptional analysis owing to its usability and quantitative capability. Several methods for transcription analysis have emerged in the past two decades. Recently, novel bioluminescence techniques that differ from typical approaches were developed for the detection of transcriptional regulation or direct protein–protein interactions. Objective: As each method has its own characteristics, this review summarizes the latest bioluminescence methods that are applicable to the field of drug discovery research. Methods: Considering the diversity of related techniques, this review covers several aspects that have been divided into the following classes: variation of reporter gene assays, secretion properties, protein–protein interaction assays in living cells and bioluminescence imaging of living cells. Results/conclusions: The practical application of several luciferins and/or luciferases and the generation of novel applications by incorporating fluorescent molecules into bioluminescence techniques will become increasingly important because bioluminescence technology has a significant potential depending on how we use it.