The gene for acute myeloid leukemia-1 (AML-1) is one of the most frequently translocated genes in human cancer. It is targeted by t(8;21) and t(3;21) in AML and by t(12;21) in acute lymphocytic leukemia (39). AML-1 is also indirectly targeted by inv(16), which disrupts core binding factor beta, an AML-1-interacting protein. AML-1 binds the enhancer core motif (TGT/cGGT) and regulates a variety of viral and cellular genes in concert with other factors (31). t(8;21) is one of the most frequent translocations found in AML, comprising 10 to 15% of cases with discernible translocations (39). The t(8;21) fusion protein AML-1/ETO acts as a repressor of transcription in transient-transfection assays (10,31,32,43). When expressed during development, the t(8;21) fusion protein yielded the same phenotype as AML-1 deficiency (37, 45).Although eight-twenty-one (ETO; also known as MTG8 [myeloid tumor gene 8] [8,34]) was identified at the breakpoint of t(8;21), little is known about the normal function of the protein. ETO is the human homologue of the Drosophila Nervy protein (9), and it shares four homologous domains with the Nervy protein. These include a region with extensive homology to a Drosophila coactivator, transcription-activating factor 110 (TAF110), a predicted hydrophobic heptad repeat (HHR), a small domain with no other homology, termed the Nervy domain (27), and the MYND (myeloid-Nervy-DEAF-1 [12]) domain. The MYND domain is present in numerous human, murine, Caenorhabditis elegans, and Drosophila proteins and contains two putative zinc finger (ZF) motifs (9,12,31). ETO is expressed in hematopoietic cells and in the brain, but another closely related family member is ubiquitously expressed (19). A third closely related factor, MTG16, is fused to AML-1 by t(16;21) (20).AML-1 is a site-specific DNA binding protein that can both activate and repress transcription (2, 28, 36). The t(8;21) fusion protein AML-1/ETO contains the N-terminal 177 amino acids of AML-1, including the DNA binding domain, fused to nearly all of ETO (7,8,34). The fusion protein inhibits AML-1-dependent transactivation (10, 32). AML-1/ETO also repressed both basal transcription and Ets-1-dependent activation of the multidrug resistance 1 promoter (27). Similarly, AML-1/ETO inhibited both AML-1 and C/EBP␣-dependent transactivation of the neutrophil protein 3 (NP-3) promoter (44). AML-1/ETO-mediated repression is dependent on both the DNA binding domain of AML-1 and ETO sequences (24). AML-1/ETO acts at substoichiometric levels and thus does not compete with AML-1 for DNA binding sites within promoters, nor does it act to "squelch" transcription (24). Thus, we hypothesized that ETO recruits a corepressor or normally functions as a corepressor to inhibit transcription (30,31,33).Several corepressor proteins have been recently described that associate with histone deacetylases (HDACs) to repress transcription (3,5,13,17,38,40,42). The nuclear hormone corepressor N-CoR was identified through interactions with the thyroid hormone receptor and associa...

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Single-nucleotide polymorphisms (SNPs) represent a new form of functional marker, particularly when they are derived from expressed sequence tags (ESTs). A bioinformatics strategy was developed to discover SNPs within a large wheat EST database and to demonstrate the utility of SNPs in genetic mapping and genetic diversity applications. A collection of > 90000 wheat ESTs was assembled into contiguous sequences (contigs), and 45 random contigs were then visually inspected to identify primer pairs capable of amplifying specific alleles. We estimate that homoeologue sequence variants occurred 1 in 24 bp and the frequency of SNPs between wheat genotypes was 1 SNP/540 bp (theta = 0.0069). Furthermore, we estimate that one diagnostic SNP test can be developed from every contig with 10-60 EST members. Thus, EST databases are an abundant source of SNP markers. Polymorphism information content for SNPs ranged from 0.04 to 0.50 and ESTs could be mapped into a framework of microsatellite markers using segregating populations. The results showed that SNPs in wheat can be discovered in ESTs, validated, and be applied to conventional genetic studies.

Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that function as activators or repressors. Although both transcription factors share a common domain involved in forming multimers, we demonstrate that Sp1 and Sp3 form separate complexes in estrogen-dependent human breast cancer cells. Sp1 and Sp3 complexes associate with histone deacetylases (HDACs) 1 and 2. Although most HDAC2 is not phosphorylated in the breast cancer cells, HDAC2 bound to Sp1 and Sp3 and cross-linked to chromatin in situ is highly enriched in a phosphorylated form that has a reduced mobility in SDS-polyacrylamide gels. We show that protein kinase CK2 is associated with and phosphorylates HDAC2. Alkaline phosphatase treatment of HDAC2 and Sp1 and Sp3 complexes reduced the associated HDAC activity. Protein kinase CK2 is up-regulated in several cancers including breast cancer, and Sp1 and Sp3 have key roles in estrogen-induced proliferation and gene expression in estrogen-dependent breast cancer cells. CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities and dynamic chromatin remodeling of estrogenregulated genes.Remodeling of chromatin structure mediated by ATP-driven chromatin-remodeling complexes and histone-modifying enzymes has a crucial role in gene expression. Acetylation of the core histones favors decondensation of the chromatin fiber by preventing interfiber interactions, whereas the unacetylated histone state contributes to chromatin condensation (1, 2). Dynamic histone acetylation catalyzed by histone deacetylases (HDAC) 1 and histone acetyltransferases allows the chromatin fiber to rapidly oscillate from the condensed and decondensed states (3, 4). In mammalian cells three classes of HDACs are identified. Class I HDACs, such as HDAC1 and HDAC2, are homologous to yeast RPD3, whereas class II HDACs are similar to yeast HDA1. Class III HDACs are related to yeast SIR2 (5, 6). HDAC1 and -2 are components of large multisubunit complexes called Sin3 or NuRD, which are recruited by transcriptional factors such as Mad, YY1, and Rb (5, 7-9).Mammalian cells ubiquitously express Sp1 and Sp3. Sp3 has three isoforms, a long (L-Sp3) and two short forms (M1-Sp3, M2-Sp3) that are the products of differential translational initiation (10). Sp3 may act as a repressor or an activator, with the short forms acting only as repressors (10). The protein structure of L-Sp3 is very similar to that of Sp1, except that Sp3 has a repression domain located N-terminal to the zinc finger DNAbinding domain (11). It has been reported that the relative levels of Sp3 forms change with differentiation, with the differentiated Caco-2 cells expressing more long than short forms (12). Further, alterations in the relative levels of Sp1 to Sp3 have been recorded, with Sp3 levels being greater than Sp1 in primary keratinocytes (13).In this study we investigated the association of histone deacetylase with Sp3 in human breast cancer cells. We ...

Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified as Hairy-interacting proteins required for embryonic segmentation and Hairymediated transcriptional repression. While both dCtBP and Groucho are required for proper Hairy function, their properties are very different. As would be expected for a co-repressor, reduced Groucho activity enhances the hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. We show here that dCtBP can function as either a co-activator or co-repressor of transcription in a context-dependent manner. The regions of dCtBP required for activation and repression are separable. We find that mSin3A-histone deacetylase complexes are altered in the presence of dCtBP and that dCtBP interferes with both Groucho and Mad transcriptional repression. Similar to CtBP's role in attenuating E1A's oncogenicity, we propose that dCtBP can interfere with corepressor-histone deacetylase complexes, thereby attenuating transcriptional repression. Hairy defines a new class of proteins that requires both CtBP and Groucho co-factors for proper function.