Sub-families of related zinc finger protein genes have been defined on the basis of evolutionarily conserved structural features found outside the C2-H2 finger repeats. Such elements include the FAX domain found in a large number of Xenopus ZFPs, the evolutionarily conserved KRAB (Krüppel-associated box) and the ZiN (zinc finger N-terminal) domains. Here we describe a new evolutionarily conserved motif within zinc finger proteins which we have named the leucine rich region (LeR). Since conserved modules in regulatory proteins may specify properties relevant to their action we have determined the functional capabilities of LeR and the KRAB domains in the regulation of gene transcription by fusing relevant regions to a heterologous DNA-binding domain (GAL4 DNA-binding domain). We found that the KRAB-A domain tethered to RNA polymerase II promoters by a GAL4 DNA-binding domain actively represses transcription in a distance-independent manner. KRAB-mediated repression is dependent on the dose of the GAL4-KRAB-A fusion protein and on the presence of GAL4 binding sites on the DNA. Conversely, the LeR domain did not modulate significantly the transcription. Our results indicate that the KRAB domain present in the non-finger region of many ZFP genes quenches transcription possibly due to specific protein-protein interactions between the KRAB-A domain and components of the proximal transcriptional apparatus.

A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous lo the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pot specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.

The t(8;21)(q22;q22) translocation, present in 10 -15% of acute myeloid leukemia (AML) cases, generates the AML1͞ETO fusion protein.To study the role of AML1͞ETO in the pathogenesis of AML, we used the Ly6A locus that encodes the well characterized hematopoietic stem cell marker, Sca1, to target expression of AML1͞ETO to the hematopoietic stem cell compartment in mice. Whereas germ-line expression of AML1͞ETO from the AML1 promoter results in embryonic lethality, heterozygous Sca1 ؉/AML1-ETO ires EGFP (abbreviated Sca ؉/AE ) mutant mice are born in Mendelian ratios with no apparent abnormalities in growth or fertility. Hematopoietic cells from Sca ؉/AE mice have markedly extended survival in vitro and increasing myeloid clonogenic progenitor output over time. Sca ؉/AE mice develop a spontaneous myeloproliferative disorder with a latency of 6 months and a penetrance of 82% at 14 months. These results reinforce the notion that the phenotype of murine transgenic models of human leukemia is critically dependent on the cellular compartment targeted by the transgene. This model should provide a useful platform to analyze the effect of AML1͞ETO on hematopoiesis and its potential cooperation with other mutations in the pathogenesis of leukemia.T he t(8;21)(q22;q22) translocation is one of the most commonly detected karyotypic abnormalities in acute myeloid leukemia (AML). This genetic alteration is found in up to 40% of de novo AML cases of the French-American-British M2 subtype, and in 12-15% of AML cases overall (1, 2). In many instances of AML, t(8;21) is the sole cytogenetic abnormality, suggesting that the translocation plays a key role in transformation. The t(8;21) translocation fuses sequences from the AML1 (RUNX1, CBFA2, and PEBP␣) gene on chromosome 21 to the ETO (MTG8) gene on chromosome 8. This translocation results in production of the AML1͞ETO protein, an in-frame fusion of the N terminus of AML1 and virtually the entire ETO protein. AML1 is the DNAbinding subunit of core-binding factor (CBF), a multimeric transcription factor complex that includes CBF␤ and additional transcriptional coactivators. Mice lacking either Aml1 or Cbf␤ fail to develop definitive intraembryonic hematopoiesis and die midgestation (3-6). Mice heterozygous for an AML1͞ETO allele knocked into the Aml1 locus have an identical phenotype, providing genetic evidence that the AML1͞ETO fusion protein acts as a dominant inhibitor of CBF activity (7,8).Although it is clear that CBF plays a critical role in hematopoietic development, several lines of evidence suggest that expression of its dominant inhibitor AML1͞ETO is not sufficient to cause AML. AML1͞ETO expression is frequently detectable in peripheral blood cells from t(8;21) AML patients for years into durable remission (9, 10). Conversely, clonotypic AML1͞ETO sequences were identified in DNA retrospectively extracted from neonatal blood samples from 5 of 10 children with t(8;21) AML, preceding the development of AML by 5-10 years in these cases (11). Several strains of AML1͞ETO-express...

The evolutionarily conserved Kruppelassociated box (KRAB) is present in the N-terminal regions of more than one-third of all Kruppel-class zinc finger proteins. Recent experiments have demonstrated that the KRAB-A domain tethered to a promoter DNA by connecting to heterologous DNA-binding protein domain or targeted to a promoter-proximal RNA sequence acts as a transcriptional silencing of RNA polymerase II promoters. Here we show that expression of KRAB domain suppresses in vivo the activating function of various defined activating transcription factors, and we demonstrate that the KRAB domain specifically silences the activity of promoters whose initiation is dependent on the presence ofa TATA box. Promoters whose accurate transcription initiation is directed by a pyrimidine-rich initiator element, however, are relatively unaffected. We also report in vitro transcription experiments indicating that the KRAB domain is able to repress both activated and basal promoter activity. Thus, the KRAB domain appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.While much attention has been focused on understanding how cellular transcription factors activate gene transcription, increasing evidence suggests that regulation of many genes is the result of a balance between positive and negative regulatory proteins. However, compared to activators, the number of transcriptional repressors that have been characterized is small. Analyses of the mechanisms of transcriptional repression by sequence-specific DNA-binding negative regulatory Proteins that do not bind DNA directly but instead recognize their appropriate DNA-bound proteins (6, 7)-for example, the adenovirus E1B 55K protein represses p53-mediated activation upon binding to p53 without displacing it from its DNA-binding site (7). In yeast, a2 and MCM1 recruit the SSN6/TUP1 repressor (8, 9). (iv) Proteins that recognize a DNA element that can function in an orientation and distanceindependent manner to block (silence) the formation of an active transcription complex. In this case, the repressor (silencer) could function in a promoter and distance-independent manner (10-12). (v) Transcription factors that activate in one

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Methylation of CpG islands is an established transcriptional repressive mechanism and is a feature of silencing in X chromosome inactivation. Housekeeping genes that are subject to X inactivation exhibit differential methylation of their CpG islands such that the inactive alleles are hypermethylated. In this report, we examine two contrasting X-linked genes with CpG islands for regulation by DNA methylation: SYBL1, a housekeeping gene in the Xq pseudoautosomal region, and GPC3, a tissue-specific gene in Xq26 that is implicated in the etiology of the Simpson-GolabiBehmel overgrowth syndrome. We observed that in vitro methylation of either the SYBL1 or the GPC3 promoter resulted in repression of reporter constructs. In normal contexts, we found that both the Y and inactive X alleles of SYBL1 are repressed and hypermethylated, whereas the active X allele is expressed and unmethylated. Furthermore, the Y and inactive X alleles of SYBL1 were derepressed by treatment with the demethylating agent azadeoxycytidine. GPC3 is also subject to X inactivation, and the active X allele is unmethylated in nonexpressing leukocytes as well as in an expressing cell line, suggesting that methylation is not involved in the tissue-specific repression of this allele. The inactive X allele, however, is hypermethylated in leukocytes, presumably ref lecting early X inactivation events that become important for gene dosage in expressing lineages. These and other data suggest that all CpG islands on Xq, including the pseudoautosomal region, are subject to X inactivation-induced methylation. Additionally, methylation of SYBL1 on Yq may derive from a process related to X inactivation that targets large chromatin domains for transcriptional repression.

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