F.Fuks and W.A.Burgers contributed equally to this workThe 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 corepressor 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 ATRXhomology 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 speci®c regulatory foci via its association with DNA-binding transcription factors.

We have identified a novel gene, Translin, encoding a protein which specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies. The encoded protein, Translin, is a previously undescribed type with no significant similarity to known proteins. In the native form, Translin polypeptides form a multimeric structure which is responsible for its DNA binding activity. Nuclear localization of Translin is limited to lymphoid cell lines, raising the intriguing possibility that nuclear transport of Translin is regulated in a physiologically significant way such that active nuclear transport is associated with the lymphoid specific process known as Ig/TCR gene rearrangement.

Cytotoxic treatment with rabbit antiserum raised against purified glycosphingolipid "asialo GM1" was capable of eliminating natural killer (NK) activity of spleen cells from different inbred mouse strains including CBA/J, C57BL/6, BALB/c, AKR, and athymic nude mice. The anti-asialo GM1 antiserum showed little cross-reactivity with structurally related glycolipids, e.g. GM), GD 1 b and asialo GM2 in the microflocculation test. The specific reactivity of this antiserum with NK cells was confirmed by the quantitative absorption of anti-NK activity with graded amounts of asialo GM1 but not with other glycosphingolipids. The absorption of anti-brain-associated T cell antigen (anti-BAT) with asialo GM1 also effectively diminished its anti-NK activity, leaving the ability to kill T cells intact. This suggests that the antibody to asialo GM1 is responsible for the anti-NK activity contained in the anti-BAT antiserum. In contrast to the extreme sensitivity of NK cells to anti-asialo GM1, alloreactive cytotoxic T killer cells generated in the mixed lymphocyte culture were not killed by anti-asialo GM1 and complement. These results indicate that asialo GM1 is expressed on mouse NK cells in a high concentration.

SUMMARY 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 MyoD’s 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.

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