Katsuhito Ohno

Transcriptional regulation of the Drosophila rfc1 gene by the DRE–DREF pathway

The DNA replication‐related element (DRE) is a common 8‐bp sequence (5′‐TATCGATA) found in the promoters of many DNA replication‐related genes, to which DRE‐binding factor (DREF) specifically binds to activate transcription. Replication factor C (RFC) is an essential five‐subunit complex in DNA replication, the largest subunit being RFC140. We first identified the gene (rfc1) encoding the Drosophila RFC140 (dRFC140) protein and then isolated a mutant. The phenotypes suggested that the gene is essential for cell‐cycle progression, and immunocytochemical studies also indicated a relation between its expression and the cell cycle. The rfc1 gene contains three DRE‐like sequences in its 5′‐flanking region, one of them perfectly matching DRE and the other two demonstrating a match in seven of eight nucleotides. These sequences were named DRE1 (−63 to −69), DRE2 (−378 to −385), and DRE3 (−1127 to −1134), respectively. Immunostaining of polytene chromosomes in third‐instar larvae using anti‐DREF sera detected a specific band in 82E2 of 3R chromosome, containing the rfc1 gene region. Band‐mobility shift assays using Drosophila Kc cell nuclear extracts revealed that DREF binds to DRE1, ‐2, and ‐3 in vitro, and chromatin immunoprecipitation using anti‐DREF IgG confirmed that this occurs in vivo. Luciferase transient expression assays in S2 cells further suggested that DREs in the rfc1 promoter are involved in transcriptional regulation of the gene. Moreover, rfc1 promoter activity was reduced by 38% in DREF double‐stranded RNA‐treated S2 cells. These results indicate that DREF positively regulates the rfc1 promoter.

Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF).

The transcription factor DREF regulates proliferation-related genes in Drosophila. With two-hybrid screening using DREF as a bait, we have obtained a clone encoding a protein homologous to human myelodysplasia/myeloid leukemia factor 1 (hMLF1). We termed the protein Drosophila MLF (dMLF); it consists of a polypeptide of 309 amino acid residues, whose sequence shares 23.1% identity with hMLF1. High conservation of 54.2% identity over 107 amino acids was found in the central region. The dMLF gene was mapped to 52D on the second chromosome by in situ hybridization. Interaction between dMLF and DREF in vitro could be confirmed by glutathione S-transferase pull-down assay, with the conserved central region appearing to play an important role in this. Northern blot hybridization analysis revealed dMLF mRNA levels to be high in unfertilized eggs, early embryos, pupae and adult males, and relatively low in adult females and larvae. This fluctuation of mRNA during Drosophila development is similar to that observed for DREF mRNA, except in the pupa and adult male. Using a specific antibody against the dMLF, we performed immunofluorescent staining of Drosophila Kc cells and showed a primarily cytoplasmic staining, whereas DREF localizes in the nucleus. However, dMLF protein contains a putative 14-3-3 binding motif involved in the subcellular localization of various regulatory molecules, and interaction with DREF could be regulated through this motif. The transgenic fly data suggesting the genetic interaction between DREF and dMLF support this possibility. Characterization of dMLF in the present study provides the molecular basis for analysis of its significance in Drosophila.

CDNA CLONING AND EXPRESSION DURING DEVELOPMENT OF DROSOPHILA MELANOGASTER MCM3, MCM6 AND MCM7

cDNAs encoding three Drosophila melanogaster MCM proteins, DmMCM3, DmMCM6 and DmMCM7, candidates of DNA replication-licensing factors, were cloned and sequenced. The deduced amino-acid sequences displayed 60, 59 and 68% identities with the respective Xenopus laevis homologues, XMCM3, XMCM6 and XMCM7. Six members of the D. melanogaster MCM family were found to share 31-36% identities in their amino-acid sequences, and to possess the five common domains carrying conserved amino-acid sequences as reported with X. laevis MCM proteins. DmMCM3, DmMCM6 and DmMCM7 genes were mapped to the 4F region on the X chromosome, the 6B region on the X chromosome and the 66E region on the third chromosome, respectively, by in situ hybridization. Contents of their mRNAs were proved to be high in unfertilized eggs and early embryos (0-4h after fertilization), then decrease gradually by the 12h time point, with only low levels detected at later stages of development except in adult females. This fluctuation pattern is similar to those of genes for proteins involved in DNA replication, such as DNA polymerase alpha and proliferating cell nuclear antigen, suggesting that expression of DmMCM genes is under the regulatory mechanism which regulates expression of other genes involved in DNA replication.

Successful Transfer of Localized Autoimmunity with Positively Selected CD4+ Cells to scid Mice Lacking Functional B Cells

T-cell function of athymic BALB/c-nu/nu (nude) mice can be corrected by implantation of a embryonic rat thymus graft (TG) under the renal capsule (TG nude mice). However, multiple organ-localized autoimmune diseases, such as oophoritis, Sjögrens syndrome like disease and gastritis, develop spontaneously in TG nude mice. Transfer of spleen cells from TG nude mice with such diseases leads to multiple localized autoimmune lesions with appearance of the corresponding autoantibodies in the recipient C.B-17-scid (SCID) mice. In the present study, removal of CD90+ or CD4+, but not CD8+ cells eliminated the transfer activity. Positively selected CD4+ cells proved capable of inducing lesions without the appearance of organ-specific autoantibodies, although the grade of lesions was lower than that in recipient mice that received untreated or CD8-depleted spleen cells. Target organs demonstrating CD4+ cell infiltration, generally expressed major histocompatibility complex (MHC) class II antigen (Ia) on their parenchymal cells. Injection of sera from TG nude mice with autoantibodies to SCID mice did not induce any pathogenic features in the autoantibody-target organs, although deposition of immunoglobulins in the corresponding target organs was observed. In such cases, no Ia antigens were expressed on the parenchymal cells. The data thus indicate that effector CD4+ cells can induce autoimmunity without B-cell help but that cooperation with functional B cells induces more severe tissue damage.

Genetic screening for modifiers of the DREF pathway in Drosophila melanogaster: identification and characterization of HP6 as a novel target of DREF.

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. By genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express DREF in the eye discs, we identified 24 genes that suppressed and 12 genes that enhanced the rough eye phenotype when heterozygous for mutations. Five genes, HP6, pigeon, lace, X box binding protein 1 and guftagu were found to carry replication-related element (DRE) sequences in their 5′-flanking regions. Of these, the HP6 gene carries two sequences that match seven out of eight nucleotides of DRE and two additional sequences that match six out of eight nucleotides of DRE in the 5′-flanking region. Band mobility shift assays using Drosophila Kc cell nuclear extracts demonstrated DREF binding to two of these sites and chromatin immunoprecipitation using anti-DREF antibodies confirmed that this occurs in vivo. Knockdown of DREF in Drosophila S2 cells decreased the HP6 mRNA level. The results, taken together, indicate that DREF directly regulates expression of the HP6 gene. HP6 mRNA was detected throughout development by RT-PCR with highest levels in adult males. In addition, immunostaining analyses revealed colocalization of HP6 and DREF in nuclei at the apical tips in the testes.

Genetic link between p53 and genes required for formation of the zonula adherens junction

Ectopic expression of human p53 in Drosophila eye imaginal disc cells induces apoptosis and results in a rough eye phenotype in the adult flies. We have screened Drosophila stocks to identify mutations that enhance or suppress the p53‐induced rough eye phenotype. One of the dominant enhancers of the p53‐induced rough eye phenotype corresponds to a loss‐of‐function mutation of the crumbs gene, which is essential for the biogenesis of the zonula adherens junction and the establishment of apical polarity in epithelial cells. Enhancement of p53‐induced apoptosis in the eye imaginal discs by a half‐reduction of the crumbs gene dose was confirmed by a TUNEL method. Furthermore, mutations of genes for Shotgun (Drosophila E‐cadherin) and Armadillo (Drosophilaβ‐catenin), the two main components of the adherens junction, also strongly enhanced the p53‐induced rough eye phenotype. These results suggest that human p53 senses subtle abnormality at the adherens junction or in signals derived from the junction, and consequently induces apoptosis to remove abnormal cells from tissue. Thus p53 likely plays a role as a guardian of the tissue not only by sensing the damaged DNA, but also by sensing signals from the adherens junction.

The myeloid leukemia factor interacts with COP9 signalosome subunit 3 in Drosophila melanogaster

The human myeloid leukemia factor 1 (hMLF1) gene was first identified as an NPM–hMLF1 fusion gene produced by chromosomal translocation. In Drosophila, dMLF has been identified as a protein homologous to hMLF1 and hMLF2, which interacts with various factors involved in transcriptional regulation. However, the precise cellular function of dMLF remains unclear. To generate further insights, we first examined the behavior of dMLF protein using an antibody specific to dMLF. Immunostaining analyses showed that dMLF localizes in the nucleus in early embryos and cultured cells. Ectopic expression of dMLF in the developing eye imaginal disc using eyeless‐GAL4 driver resulted in a small‐eye phenotype and co‐expression of cyclin E rescued the small‐eye phenotype, suggesting the involvement of dMLF in cell‐cycle regulation. We therefore analyzed the molecular mechanism of interactions between dMLF and a dMLF‐interacting protein, dCSN3, a subunit of the COP9 signalosome, which regulates multiple signaling and cell‐cycle pathways. Biochemical and genetic analyses revealed that dMLF interacts with dCSN3 in vivo and glutathione S‐transferase pull‐down assays revealed that the PCI domain of the dCSN3 protein is sufficient for this to occur, possibly functioning as a structural scaffold for assembly of the COP9 signalosome complex. From these data we propose the possibility that dMLF plays a negative role in assembly of the COP9 signalosome complex.