Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.

Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Krüppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene.

Mammalian orthologues of a yeast regulator of nonsense transcript stability.

All eukaryotes that have been studied to date possess the ability to detect and degrade transcripts that contain a premature signal for the termination of translation. This process of nonsense-mediated RNA decay has been most comprehensively studied in the yeast Saccharomyces cerevisiae where at least three trans-acting factors (Upf1p through Upf3P) are required. We have cloned cDNAs encoding human and murine homologues of Upf1p, termed rent1 (regulator of nonsense transcripts). Rent1 is the first identified mammalian protein that contains all of the putative functional elements in Upf1p including zinc finger-like and NTPase domains, as well as all motifs common to members of helicase superfamily I. Moreover, expression of a chimeric protein, N and C termini of Upf1p, complements the Upf1p-deficient phenotype in yeast. Thus, despite apparent differences between yeast and mammalian nonsense-mediated RNA decay, these data suggest that the two pathways use functionally related machinery.

The conserved role of Krox-20 in directing Hox gene expression during vertebrate hindbrain segmentation.

Transient segmentation in the hindbrain is a fundamental morphogenetic phenomenon in the vertebrate embryo, and the restricted expression of subsets of Hox genes in the developing rhombomeric units and their derivatives is linked with regional specification. Here we show that patterning of the vertebrate hindbrain involves the direct upregulation of the chicken and pufferfish group 2 paralogous genes, Hoxb-2 and Hoxa-2, in rhombomeres 3 and 5 (r3 and r5) by the zinc finger gene Krox-20. We identified evolutionarily conserved r3/r5 enhancers that contain high affinity Krox-20. binding sites capable of mediating transactivation by Krox-20. In addition to conservation of binding sites critical for Krox-20 activity in the chicken Hoxa-2 and pufferfish Hoxb-2 genes, the r3/r5 enhancers are also characterized by the presence of a number of identical motifs likely to be involved in cooperative interactions with Krox-20 during the process of hindbrain patterning in vertebrates.

Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression.

Developmentally regulated genes in Drosophila, which are conserved through evolution, are potential candidates for key functions in biological processes such as cell cycle, programmed cell death, and cancer. We report cloning and characterization of the human homologue of the Drosophila seven in absentia gene (HUMSIAH), which codes for a 282 amino acids putative zinc finger protein. HUMSIAH is localized on human chromosome 16q12-q13. This gene is activated during the physiological program of cell death in the intestinal epithelium. Moreover, human cancer-derived cells selected for suppression of their tumorigenic phenotype exhibit constitutively elevated levels of HUMSIAH mRNA. A similar pattern of expression is also displayed by the p21waf1. These results suggest that mammalian seven in absentia gene, which is a target for activation by p53, may play a role in apoptosis and tumor suppression.

HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro.

After a retrovirus particle is released from the cell, the dimeric genomic RNA undergoes a change in conformation. We have previously proposed that this change, termed maturation of the dimer, is due to the action of nucleocapsid (NC) protein on the RNA within the virus particle. We now report that treatment of a 345-base synthetic fragment of Harvey sarcoma virus RNA with recombinant or synthetic HIV-1 NC protein converts a less stable form of dimeric RNA to a more stable form. This phenomenon thus appears to reproduce the maturation of dimeric retroviral RNA in a completely defined system in vitro. To our knowledge, maturation of dimeric RNA within a retrovirus particle is the first example of action of an "RNA chaperone" protein in vivo. Studies with mutant NC proteins suggest that the activity depends upon basic amino acid residues flanking the N-terminal zinc finger and upon residues within the N-terminal finger, including an aromatic amino acid, but do not require the zinc finger structures themselves.

Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain.

A novel human cDNA encoding a cytosolic 62-kDa protein (p62) that binds to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner has been cloned. The cDNA is composed of 2074 nucleotides with an open reading frame encoding 440 amino acids. Northern analysis suggests that p62 is expressed ubiquitously in all tissues examined. p62 is not homologous to any known protein in the data base. However, it contains a cysteine-rich region resembling a zinc finger motif, a potential G-protein-binding region, a PEST motif, and several potential phosphorylation sites. Using T7-epitope tagged p62 expression in HeLa cells, the expressed protein was shown to bind to the lck SH2 domain. Deletion of the N-terminal 50 amino acids abolished binding, but mutagenesis of the single tyrosine residue in this region had no effect on binding. Thus, the cloned cDNA indeed encodes the p62 protein, which is a phosphotyrosine-independent ligand for the lck SH2 domain. Its binding mechanism is unique with respect to binding modes of other known ligands for SH2 domains.

Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family.

The EVI1 gene, located at chromosome band 3q26, is overexpressed in some myeloid leukemia patients with breakpoints either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26). EVI1 is also expressed as part of a fusion transcript with the transcription factor AML1 in the t(3;21)(q26;q22), associated with myeloid leukemia. In cells with t(3;21), additional fusion transcripts are AML1-MDS1 and AML1-MDS1-EVI1. MDS1 is located at 3q26 170-400 kb upstream (telomeric) of EVI1 in the chromosomal region in which some of the breakpoints 5' of EVI1 have been mapped. MDS1 has been identified as a single gene as well as a previously unreported exon(s) of EVI1 We have analyzed the relationship between MDS1 and EVI1 to determine whether they are two separate genes. In this report, we present evidence indicating that MDS1 exists in normal tissues both as a unique transcript and as a normal fusion transcript with EVI1, with an additional 188 codons at the 5' end of the previously reported EVI1 open reading frame. This additional region has about 40% homology at the amino acid level with the PR domain of the retinoblastoma-interacting zinc-finger protein RIZ. These results are important in view of the fact that EVI1 and MDS1 are involved in leukemia associated with chromosomal translocation breakpoints in the region between these genes.

Krüppel-associated box-mediated repression of RNA polymerase II promoters is influenced by the arrangement of basal promoter elements.

The evolutionarily conserved Krüppel-associated box (KRAB) is present in the N-terminal regions of more than one-third of all Krüppel-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 of a 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.

Truncated WT1 mutants alter the subnuclear localization of the wild-type protein.

WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors. Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins. The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus. In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1 (+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing. Localization to subnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments. Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures.

Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases.

We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1. Here we describe a second methionine aminopeptidase (Met-AP2) in S. cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain. The DNA sequence of MAP2 encodes a protein of 421 amino acids that shows 22% identity with the sequence of yeast Met-AP1. Surprisingly, comparison with sequences in the GenBank data base showed that the product of MAP2 has even greater homology (55% identity) with rat p67, which was characterized as an initiation factor 2-associated protein but not yet shown to have Met-AP activity. Transformants of map1 null cells expressing MAP2 in a high-copy-number plasmid contained 3- to 12-fold increases in Met-AP activity on different peptide substrates. The epitope-tagged suppressor gene product was purified by immunoaffinity chromatography and shown to contain Met-AP activity. To evaluate the physiological significance of Met-AP2, the MAP2 gene was deleted from wild-type and map1 null yeast strains. The map2 null strain, like the map1 null strain, is viable but with a slower growth rate. The map1, map2 double-null strains are nonviable. Thus, removal of N-terminal methionine is an essential function in yeast, as in prokaryotes, but yeast require two methionine aminopeptidases to provide the essential function which can only be partially provided by Met-AP1 or Met-AP2 alone.

Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B-cell lymphoma.

The BCL6 gene encodes a zinc-finger transcription factor and is altered by chromosomal arrangements in its 5' noncoding region in approximately 30% of diffuse large-cell lymphoma (DLCL). We report here that, in 22/30 (73%) DLCL and 7/15 (47%) follicular lymphoma (FL), but not in other tumor types, the BCL6 gene is also altered by multiple (1.4 x 10(-3) -1.6 x 10(-2) per bp), often biallelic, mutations clustering in its 5' noncoding region. These mutations are of somatic origin and are found in cases displaying either normal or rearranged BLC6 alleles indicating their independence from chromosomal rearrangements and linkage to immunoglobulin genes. These alterations identify a mechanism of genetic instability in malignant B cells and may have been selected during lymphomagenesis for their role in altering BCL6 expression.

Alternative translation initiation site usage results in two functionally distinct forms of the GATA-1 transcription factor.

GATA-1 is a zinc-finger transcription factor that plays a critical role in the normal development of hematopoietic cell lineages. In human and murine erythroid cells a previously undescribed 40-kDa protein is detected with GATA-1-specific antibodies. We show that the 40-kDa GATA-1 (GATA-1s) is produced by the use of an internal AUG initiation codon in the GATA-1 transcript. The GATA-1 proteins share identical binding activity and form heterodimers in erythroleukemic cells but differ in their transactivation potential and in their expression in developing mouse embryos.

Identification of a Drosophila muscle development gene with structural homology to mammalian early growth response transcription factors.

In Drosophila, stripe (sr) gene function is required for normal muscle development. Some mutations disrupt embryonic muscle development and are lethal. Other mutations cause total loss of only a single muscle in the adult. Molecular analysis shows that sr encodes a predicted protein containing a zinc finger motif. This motif is homologous to the DNA binding domains encoded by members of the early growth response (egr) gene family. In mammals, expression of egr genes is induced by intercellular signals, and there is evidence for their role in many developmental events. The identification of sr as an egr gene and its pattern of expression suggest that it functions in muscle development via intercellular communication.

Role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes.

We have isolated the NIL1 gene, whose product is an activator of the transcription of nitrogen-regulated genes, by virtue of the homology of its zinc-finger domain to that of the previously identified activator, the product of GLN3. Disruption of the chromosomal NIL1 gene enabled us to compare the effects of Gln3p and of Nil1p on the expression of the nitrogen-regulated genes GLN1, GDH2, and GAP1, coding respectively for glutamine synthetase, NAD-linked glutamate dehydrogenase, and general amino acid permease. Our results show that the nature of GATAAG sequence that serve as the upstream activation sequence elements for these genes determines their abilities to respond to Gln3p and Nil1p. The results further indicate that Gln3p is inactivated by an increase in the intracellular concentration of glutamine and that Nil1p is inactivated by an increase in intracellular glutamate.

The leukemia-associated-protein (LAP) domain, a cysteine-rich motif, is present in a wide range of proteins, including MLL, AF10, and MLLT6 proteins.

We have identified and further characterized a Caenorhabditis elegans gene, CEZF, that encodes a protein with substantial homology to the zinc finger and leucine zipper motifs of the human gene products AF10, MLLT6, and BR140. The first part of the zinc finger region of CEZF has strong similarity to the corresponding regions of AF10 (66%) and MLLT6 (64%) at the cDNA level. As this region is structurally different from previously described zinc finger motifs, sequence homology searches were done. Twenty-five other proteins with a similar motif were identified. Because the functional domain of this motif is potentially disrupted in leukemia-associated chromosomal translocations, we propose the name of leukemia-associated protein (LAP) finger. On the basis of these comparisons, the LAP domain consensus sequence is Cys1-Xaa1-2-Cys2-Xaa9-21-Cys3-Xaa2-4 -Cys4-Xaa4-5-His5-Xaa2-Cys6-Xaa12-46 - Cys7-Xaa2-Cys8, where subscripted numbers represent the number of amino acid residues. We review the evidence that this motif binds zinc, is the important DNA-binding domain in this group of regulatory proteins, and may be involved in leukemogenesis.