Masataka Kasai

Dnmt3a binds deacetylases and is recruited by a sequence‐specific repressor to silence transcription

The Dnmt3a DNA methyltransferase is essential for mammalian development and is responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing. Here, we show that Dnmt3a associates with RP58, a DNA‐binding transcriptional repressor protein found at transcriptionally silent heterochromatin. Dnmt3a acts as a co‐repressor for RP58 in a manner that does not require its de novo methyltransferase activity. Like other characterized co‐repressors, Dnmt3a associates with the histone deacetylase HDAC1 using its ATRX‐homology domain. This domain of Dnmt3a represents an independent transcriptional repressor domain whose silencing functions require HDAC activity. These results identify Dnmt3a as a co‐repressor protein carrying deacetylase activity and show that Dnmt3a can be targeted to specific regulatory foci via its association with DNA‐binding transcription factors.

A Systems Approach Reveals that the Myogenesis Genome Network Is Regulated by the Transcriptional Repressor RP58

We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos--a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoDs ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.

The Translin Ring Specifically Recognizes DNA Ends at Recombination Hot Spots in the Human Genome

We previously showed that consensus sequences exist at the chromosomal breakpoints in lymphoid malignancies and that these sequences are specifically recognized by a novel DNA binding protein, Translin. In the present study, the native form of Translin was established to be a ring-shaped structure by electron microscopy and crystallographic studies. It was also determined that this multimeric Translin formed by the subunits is responsible for its binding to target sequences situated only at single-stranded DNA ends. Furthermore, DNA-damaging reagents were found to initiate a signaling pathway for the active nuclear transport of Translin. The results support the hypothesis that staggered breaks occur at recombination hot spots and Translin has a pivotal function in recognition of the generated single-stranded DNA ends.

Isolation and characterization of a cDNA encoding a Translin-like protein, TRAX

Translin is a DNA binding protein which specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies. To investigate its functional significance at such recombination hotspots, we examined whether Translin interacts with other proteins using a yeast two‐hybrid system and identified an associated 33 kd protein partner, TRAX, with extensive amino acid homology. The TRAX protein was established to contain bipartite nuclear targeting sequences in its N‐terminal region, suggesting a possible role in the selective nuclear transport of Translin protein lacking any nuclear targeting motifs.

RP58 Associates with Condensed Chromatin and Mediates a Sequence-specific Transcriptional Repression

An approximately 120-amino acid domain present generally at the NH2 termini, termed the POZ domain, is highly conserved in various proteins with zinc finger DNA binding motifs. We have isolated a novel protein sharing homology with the POZ domain of a number of zinc finger proteins, including the human BCL-6 protein. By using a binding site selection technique (CAST), a high affinity binding site of the protein was determined to be (A/C)ACATCTG(G/T)(A/C), containing the E box core sequence motif. The protein was shown to repress transcription from a promoter linked to its target sequences and was hence named RP58 (RepressorProtein with a predicted molecular mass of 58kDa). Immunogold electron microscopic study revealed that almost all RP58 is localized in condensed chromatin regions. These observations demonstrate for the first time that a protein mediating a sequence-specific transcriptional repression associates with highly condensed chromatin. We suggest that RP58 may be involved in a molecular link between sequence-specific transcriptional repression and the organization of chromosomes in the nucleus.

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

Abstract The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-γ and TNF-α) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.

The DNA binding activity of Translin is mediated by a basic region in the ring‐shaped structure conserved in evolution

DNA binding proteins, for the most part, function as dimers or tetramers which recognize their target sequences. Here we show that Translin, a novel single‐stranded DNA end binding protein, forms a ring‐shaped structure conserved throughout evolution and that this structure is responsible for its DNA binding activity. Point mutations at Leu184 and Leu191 in the leucine zipper motif of human Translin resulted in loss of the multimeric structure and abrogation of DNA binding. Point mutations at R86, H88, H90 to T86, N88, N90 in one of the basic regions, however, completely inhibited the DNA binding activity without affecting the multimeric structure. These results support the view that the DNA binding domain of Translin is formed in the ring‐shaped structure in combination with its basic region (amino acids 86–97) polypeptides.

Asialo GM1 as a cell-surface marker detected in acute lymphoblastic leukemia.

The heterologous antiserum raised against brain-associated T-cell antigen may react against some immature cells distinct from mature T cells.1 2 3 4 5 6 7 8 Studies in our laboratory have identifie...

RP58 Regulates the Multipolar-Bipolar Transition of Newborn Neurons in the Developing Cerebral Cortex

Accumulating evidence suggests that many brain diseases are associated with defects in neuronal migration, suggesting that this step of neurogenesis is critical for brain organization. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here, we identified the zinc-finger transcriptional repressor RP58 as a key regulator of neuronal migration via multipolar-to-bipolar transition. RP58(-/-) neurons exhibited severe defects in the formation of leading processes and never shifted to the locomotion mode. Cre-mediated deletion of RP58 using in utero electroporation in RP58(flox/flox) mice revealed that RP58 functions in cell-autonomous multipolar-to-bipolar transition, independent of cell-cycle exit. Finally, we found that RP58 represses Ngn2 transcription to regulate the Ngn2-Rnd2 pathway; Ngn2 knockdown rescued migration defects of the RP58(-/-) neurons. Our findings highlight the critical role of RP58 in multipolar-to-bipolar transition via suppression of the Ngn2-Rnd2 pathway in the developing cerebral cortex.

The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex.

The neocortex and the hippocampus comprise several specific layers containing distinct neurons that originate from progenitors at specific development times, under the control of an adequate cell-division patterning mechanism. Although many molecules are known to regulate this cell-division patterning process, its details are not well understood. Here, we show that, in the developing cerebral cortex, the RP58 transcription repressor protein was expressed both in postmitotic glutamatergic projection neurons and in their progenitor cells, but not in GABAergic interneurons. Targeted deletion of the RP58 gene led to dysplasia of the neocortex and of the hippocampus, reduction of the number of mature cortical neurons, and defects of laminar organization, which reflect abnormal neuronal migration within the cortical plate. We demonstrate an impairment of the cell-division patterning during the late embryonic stage and an enhancement of apoptosis of the postmitotic neurons in the RP58-deficient cortex. These results suggest that RP58 controls cell division of progenitor cells and regulates the survival of postmitotic cortical neurons.