Hidetaka Seo

A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs.

BACKGROUND Alteration of chromatin structure is a key step in various aspects of DNA metabolism. DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication. For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure. At present, however, only limited data suggest an involvement of DNA unwinding factors in DNA replication. RESULTS We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptides. DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce negative supercoils into DNA at the expense of ATP hydrolysis. Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein. The 140 kDa polypeptide is homologous to yeast Cdc68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin or plasmid DNA. CONCLUSIONS We propose that DUF plays a role in DNA replication in Xenopus egg extracts.

Rapid generation of specific antibodies by enhanced homologous recombination

In the chicken immune system, gene conversion, a type of homologous recombination, primarily contributes to diversification of the immunoglobulin gene. Here, we report on the rapid generation of specific monoclonal antibodies using the chicken DT40 B-cell line undergoing gene conversion. We discovered that the gene conversion frequency at the immunoglobulin locus is increased by treating DT40 cells with a histone deacetylase inhibitor, trichostatin A (TSA), thereby generating diversity at the immunoglobulin locus in the majority of treated cells. This indicates that TSA treatment accelerates the autonomous diversification of surface IgMs on DT40 cells. We took advantage of this effect to select DT40 cells producing specific antibodies with antigen-conjugated magnetic beads. This autonomously diversifying library (ADLib) selection system enables the quick establishment (∼1 week from a diversifying library) of various clones producing monoclonal IgMs with enough specificity and affinity for immunological assays, and is applicable to various biotechnologies including rational protein design.

p97 ATPase, an ATPase involved in membrane fusion, interacts with DNA unwinding factor (DUF) that functions in DNA replication

DNA unwinding factor (DUF) unwinds duplex DNA and is supposed to function in DNA replication in Xenopus egg extracts. Here we report the isolation and analysis of a DUF‐interacting factor. By immunoprecipitation, we found that p97 ATPase (p97) interacts with DUF in Xenopus egg extracts. This interaction was confirmed by the in vitro binding of purified p97 with DUF. When sperm chromatin was added to Xenopus egg extracts to construct nuclei active in DNA replication, p97 was incorporated into the nuclei. These data suggest that the complex of DUF and p97 may function in DNA replication.

Regulation of histone H4 acetylation by transcription factor E2A in Ig gene conversion

Recent studies implicate the transcription factor E2A in Ig diversification such as somatic hypermutation or gene conversion (GCV). GCV also requires active Ig transcription, expression of the activation-induced deaminase (AID) and a set of homologous recombination factors. We have disrupted the E2A gene in the chicken B-cell line DT40 and found greatly diminished rate of GCV without changes in the levels of transcripts from AID and Ig heavy chain or Ig light chain (IgL) genes. However, chromatin immunoprecipitation analysis revealed that the loss of E2A accompanies drastically reduced acetylation levels of the histone H4 in rearranged IgL locus. Furthermore, the defects in GCV were restored by trichostatin A treatment, which raised H4 acetylation to the normal levels. Thus, E2A may contribute to GCV by maintaining histone acetylation, which could be a prerequisite for targeting or full deaminase function of AID.

An ex vivo method for rapid generation of monoclonal antibodies (ADLib system)

Here, we describe a protocol for using the ADLib (Autonomously Diversifying Library) system to rapidly generate specific monoclonal antibodies using DT40, a chicken B-cell line that undergoes constitutive gene conversion at both light- and heavy-chain immunoglobulin loci. We previously developed the ADLib system on the basis of our finding that gene conversion in DT40 cells was enhanced by treatment of the cells with a histone deacetylase inhibitor, trichostatin A (TSA). TSA treatment evolves a diversified library of DT40 cells (ADLib), in which each cell has different surface IgM specificity. Antigen-specific DT40 cells are selected from ADLib using antigen-conjugated magnetic beads, and their specificity can be examined by various immunological assays, using culture supernatant containing secreted IgM. The whole process from selection to screening can be completed in about 1 week. Thus, the ADLib system will accelerate biological studies, including drug discovery and design.

Modulation of immunoglobulin gene conversion frequency and distribution by the histone deacetylase HDAC2 in chicken DT40.

Modifications of histones are reportedly associated with the regulation of immunoglobulin (Ig) gene diversification mechanisms, but the extent of their involvement in promoting sequence alterations at the Ig variable (V) regions still remains to be elucidated. We have previously demonstrated that Ig gene conversion in the B cell line DT40 is accompanied by the local hyperacetylation of histones, and that its frequency is highly increased in cells treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). In this report, we describe the enhancing effects of the homozygous deletion of HDAC2 (HDAC2–/–) on Ig gene conversion. Remarkably, sequence analysis revealed that the distribution of the gene conversion tracts induced throughout the Ig V regions in HDAC2–/– was significantly different from the diversification patterns in TSA‐treated wild‐type cultures. Furthermore, we found that the effects of HDAC2–/– and of the treatment with TSA were additive as regards histone acetylation, Ig gene transcription, gene conversion frequency and distribution of gene conversion tracts. These results underscore the potential participation of HDAC‐mediated histone acetylation in Ig diversification, but also suggest a specific role of HDAC2 to control the spatial targeting of Ig gene conversion.

Incorporation of DUF/FACT into chromatin enhances the accessibility of nucleosomal DNA.

DNA unwinding factor (DUF) was discovered as an essential DNA replication factor in Xenopus egg extracts. DUF consists of an HMG protein and a homolog of Cdc68p/Spt16p, and has the capability of unwinding dsDNA. Here we have examined the interaction of DUF with chromatin. DUF was incorporated into chromatin assembled from sperm heads and from plasmid DNA in egg extracts. It was revealed that the chromatin assembled in egg extracts immunodepleted of DUF is less sensitive to micrococcal nuclease (NNase) digestion than that assembled in control extracts, indicating that chromatin containing DUF has more decompact structure than that without DUF. Also we found that DUF has a high affinity for core histones in vitro. We suggest that the function of DUF may be to make the chromatin structure accessible to replication factors.

Modulation of Immunoglobulin Gene Conversion in Chicken DT40 by Enhancing Histone Acetylation, and its Application to Antibody Engineering

Abbreviations: MAb: monoclonal antibody; Ig: immunoglobulin; HAT: histone acetyltransferase; IgH: Ig heavy; IgL: Ig light; TSA: Trichostatin A; HDAC: histone deacetylase; ADLib: Autonomously Diversifying Library; ELISA: enzyme linked immunosolvent assay; FACS; fluorescence activated cell sorting; GPCR: GTP-binding protein coupled receptors; FITC: Fluorescein isothiocyanate; DNP; dinitrophenol. Biotechnology and Genetic Engineering Reviews Vol. 24, 179-194 (2007)

B Cell-Based Seamless Engineering of Antibody Fc Domains.

Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody’s Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the ‘ADLib system’). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs’ Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors.