Four of the seven zinc fingers of the Evi-1 myeloid-transforming gene are required for sequence-specific binding to GA(C/T)AAGA(T/C)AAGATAA.

Expression of the Evi-1 gene is activated in murine myeloid leukemias by retroviral insertions and in human acute myelogenous leukemia by translocations and inversions involving chromosome band 3q26 where the gene resides. Aberrant expression of the Evi-1 gene has been shown to interfere with myeloid differentiation, which is proposed to be the basis for its role in leukemias. The Evi-1 gene encodes a 145-kDa DNA-binding protein containing two domains of seven and three Cys2-His2 zinc fingers. Previous studies identified a portion of the consensus DNA-binding sequence for the first domain of zinc fingers. The experiments presented here extend these studies and demonstrate that the first domain recognizes a consensus of 15 nucleotides consisting of GA(C/T)AAGA(T/C)AAGATAA. The first three fingers of the first domain do not detectably bind DNA but contribute to the binding by conferring a relative specificity for GACAA verses GATAA in the first position. The first three fingers also contribute to optimal binding of the 15-nucleotide consensus sequence.

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Four of the seven zinc fingers of the Evi-1 myeloid-transforming gene are required for sequence-specific binding to GA(C/T)AAGA(T/C)AAGATAA

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4291-4300.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4291-4300

Author(s):

R Delwel ◽

T Funabiki ◽

B L Kreider ◽

K Morishita ◽

J N Ihle

Keyword(s):

Dna Binding ◽

Specific Binding ◽

Zinc Fingers ◽

Consensus Sequence ◽

Myelogenous Leukemia ◽

Chromosome Band ◽

Aberrant Expression ◽

Relative Specificity ◽

Acute Myelogenous ◽

Binding Sequence

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Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.1940-1949.1992 ◽

1992 ◽

Vol 12 (5) ◽

pp. 1940-1949

Author(s):

A D Keller ◽

T Maniatis

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Specific Binding ◽

Regulatory Element ◽

Zinc Fingers ◽

Transcriptional Repressor ◽

Gene Promoter ◽

Binding Activity ◽

Necessary And Sufficient ◽

Sequence Requirements

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.

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The t(8;21) fusion protein interferes with AML-1B-dependent transcriptional activation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.1974 ◽

1995 ◽

Vol 15 (4) ◽

pp. 1974-1982 ◽

Cited By ~ 283

Author(s):

S Meyers ◽

N Lenny ◽

S W Hiebert

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Chimeric Protein ◽

Cell Receptor ◽

Subcellular Fractionation ◽

Myelogenous Leukemia ◽

Core Motif ◽

Acute Myelogenous ◽

Beta Enhancer ◽

A Site

The AML-1/CBF beta transcription factor complex is targeted by both the t(8;21) and the inv(16) chromosomal alterations, which are frequently observed in acute myelogenous leukemia. AML-1 is a site-specific DNA-binding protein that recognizes the enhancer core motif TGTGGT. The t(8;21) translocation fuses the first 177 amino acids of AML-1 to MTG8 (also known as ETO), generating a chimeric protein that retains the DNA-binding domain of AML-1. Analysis of endogenous AML-1 DNA-binding complexes suggested the presence of at least two AML-1 isoforms. Accordingly, we screened a human B-cell cDNA library and isolated a larger, potentially alternatively spliced, form of AML1, termed AML1B. AML-1B is a protein of 53 kDa that binds to a consensus AML-1-binding site and complexes with CBF beta. Subcellular fractionation experiments demonstrated that both AML-1 and AML-1/ETO are efficiently extracted from the nucleus under ionic conditions but that AML-1B is localized to a salt-resistant nuclear compartment. Analysis of the transcriptional activities of AML-1, AML-1B, and AML-1/ETO demonstrated that only AML-1B activates transcription from the T-cell receptor beta enhancer. Mixing experiments indicated that AML-1/ETO can efficiently block AML-1B-dependent transcriptional activation, suggesting that the t(8;21) translocation creates a dominant interfering protein.

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Heterogeneity in CBF beta/MYH11 fusion messages encoded by the inv(16)(p13q22) and the t(16;16)(p13;q22) in acute myelogenous leukemia

Blood ◽

10.1182/blood.v85.12.3695.bloodjournal85123695 ◽

1995 ◽

Vol 85 (12) ◽

pp. 3695-3703 ◽

Cited By ~ 75

Author(s):

SA Shurtleff ◽

S Meyers ◽

SW Hiebert ◽

SC Raimondi ◽

DR Head ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Pcr Analysis ◽

Chain Gene ◽

Genomic Locus ◽

Acute Myelogenous

Inv(16)(p13q22) is one of the most frequent chromosomal rearrangements found in acute myelogenous leukemia (AML), representing approximately 16% of documented karyotypic abnormalities. The inv(16) breakpoints have been cloned and shown to involve the non-DNA binding component of the AML-1 transcription factor complex termed core binding factor beta gene (CBF beta) on 16q and the smooth muscle myosin heavy chain gene (MYH11) on 16p. In this study, we analyzed 37 cases of inv(16)-containing AML and 4 cases with t(16;16)(p13;q22) for expression of the CBF beta/MYH11 chimeric message by reverse transcription-polymerase chain reaction (PCR) analysis. CBF beta/MYH11 chimeric messages were detected in 33 of 37 cases with the inv(16) and in the 4 t(16;16)-containing cases. Sequence analysis of PCR products showed extensive breakpoint heterogeneity in both CBF beta and MYH11. In addition to the previously described breakpoint in CBF beta at nucleotide (nt) 495 (amino acid 165), we have identified a second novel fusion point at nt 399 (amino acid 133) in 7% of the cases. Similarly, a unique breakpoint site was identified in MYH11 at nt 1098, as well as at three previously characterized sites at nts 994, 1201, and 1921. Of the 4 PCR-negative cases, 2 of 3 tested lacked CBF beta rearrangements by Southern blot analysis, suggesting the possible involvement of a different genomic locus in some cases with cytogenetic evidence of inv(16). To assess whether the portions of CBF-beta contained within the CBF beta/MYH11 chimeric products retain the ability to interact with their heterodimeric DNA-binding partner AML-1, we performed in vitro DNA- binding analysis. Recombinant CBF-beta polypeptides consisting of the N-terminal 165 amino acids retained their ability to interact with AML-1, whereas mutants containing only the N-terminal 133 amino acids interacted with AML-1 less efficiently. These data suggest that different CBF beta/MYH11 products may vary subtly in their biologic activities.

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Oligomerization of ETO Is Obligatory for Corepressor Interaction

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.156-163.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 156-163 ◽

Cited By ~ 73

Author(s):

Jinsong Zhang ◽

Bruce A. Hug ◽

Eric Y. Huang ◽

Clarice W. Chen ◽

Vania Gelmetti ◽

...

Keyword(s):

Zinc Fingers ◽

Chromosomal Translocation ◽

Dna Binding Protein ◽

Myelogenous Leukemia ◽

Common Mechanism ◽

Dimerization Domain ◽

Acute Myelogenous ◽

Affinity Interaction ◽

Oligomerization Domain

ABSTRACT Nearly 40% of cases of acute myelogenous leukemia (AML) of the M2 subtype are due to a chromosomal translocation that combines a sequence-specific DNA binding protein, AML1, with a potent transcriptional repressor, ETO. ETO interacts with nuclear receptor corepressors SMRT and N-CoR, which recruit histone deacetylase to the AML1-ETO oncoprotein. SMRT–N-CoR interaction requires each of two zinc fingers contained in C-terminal Nervy homology region 4 (NHR4) of ETO. However, here we show that polypeptides containing NHR4 are insufficient for interaction with SMRT. NHR2 is also required for SMRT interaction and repression by ETO, as well as for inhibition of hematopoietic differentiation by AML1-ETO. NHR2 mediates oligomerization of ETO as well as AML1-ETO. Fusion of NHR4 polypeptide to a heterologous dimerization domain allows strong interaction with SMRT in vitro. These data support a model in which NHR2 and NHR4 have complementary functions in repression by ETO. NHR2 functions as an oligomerization domain bringing together NHR4 polypeptides that together form the surface required for high-affinity interaction with corepressors. As nuclear receptors also interact with corepressors as dimers, oligomerization may be a common mechanism regulating corepressor interactions.

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DNA-binding specificity of NGFI-A and related zinc finger transcription factors.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2275 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2275-2287 ◽

Cited By ~ 225

Author(s):

A H Swirnoff ◽

J Milbrandt

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Target Genes ◽

Zinc Fingers ◽

Consensus Sequence ◽

Binding Specificity ◽

Early Gene ◽

Dna Binding Domains ◽

Binding Domains ◽

Random Site

NGFI-A is the prototypic member of a family of immediate-early gene-encoded transcription factors which includes NGFI-C, Egr3, and Krox20. These proteins possess highly homologous DNA-binding domains, composed of three Cys2-His2 zinc fingers, and all bind to and activate transcription from the sequence GCGGGGGCG. We used a PCR-mediated random site selection protocol to determine whether other sites could be bound by these proteins and the extent to which their binding site preferences are similar or different. The high-affinity consensus sites generated from the selection data are similar, and the combined consensus sequence is T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase letters indicate bases selected less frequently). Using gel shift assays, we found that sequences that diverge from the consensus were bound by NGFI-A, confirming that there is greater variability in binding sites than has generally been acknowledged. We also provide evidence that protein-DNA interactions not noted, or whose importance was not apparent from the X-ray cocrystal structure of the NGFI-A zinc fingers complexed with DNA, contribute significantly to the binding energy of these proteins and confirm that an optimal site is at least 10 instead of 9 nucleotides in length. In contrast to the similarities in binding specificity among these proteins we found that while NGFI-A, Egr3, and Krox20 have comparable DNA binding affinities and kinetics of dissociation, the affinity of NGFI-C is more than threefold lower. This could result in differential regulation of target genes in cells where NGFI-C and the other proteins are coexpressed. Furthermore, we show that this affinity difference is a property not of the zinc fingers themselves but rather of the protein context of the DNA-binding domain.

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Critical DNA Binding Interactions of the Insulator Protein CTCF

Journal of Biological Chemistry ◽

10.1074/jbc.m706213200 ◽

2007 ◽

Vol 282 (46) ◽

pp. 33336-33345 ◽

Cited By ~ 102

Author(s):

Mario Renda ◽

Ilaria Baglivo ◽

Bonnie Burgess-Beusse ◽

Sabrina Esposito ◽

Roberto Fattorusso ◽

...

Keyword(s):

Dna Binding ◽

Large Scale ◽

Specific Binding ◽

Zinc Fingers ◽

Dna Interaction ◽

Ctcf Binding ◽

Specific Expression ◽

Binding Factor ◽

Allele Specific ◽

Enhancer Blocking

The DNA-binding protein CTCF (CCCTC binding factor) mediates enhancer blocking insulation at sites throughout the genome and plays an important role in regulating allele-specific expression at the Igf2/H19 locus and at other imprinted loci. Evidence is also accumulating that CTCF is involved in large scale organization of genomic chromatin. Although CTCF has 11 zinc fingers, we show here that only 4 of these are essential to strong binding and that they recognize a core 12-bp DNA sequence common to most CTCF sites. By deleting individual fingers and mutating individual sites, we determined the orientation of binding. Furthermore, we were able to identify the specific finger and its point of DNA interaction that are responsible for the loss of CTCF binding when CpG residues are methylated in the imprinted Igf2/H19 locus. This single interaction appears to be critical for allele-specific binding and insulation by CTCF.

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Aberrant Expression of CD19 as a Marker of Monocytic Lineage in Acute Myelogenous Leukemia

American Journal of Clinical Pathology ◽

10.1093/ajcp/107.3.283 ◽

1997 ◽

Vol 107 (3) ◽

pp. 283-291 ◽

Cited By ~ 8

Author(s):

John T. Brandt ◽

Joseph A. Tisone ◽

Jo Ellen Bohman ◽

Karl S. Theil

Keyword(s):

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Aberrant Expression ◽

Acute Myelogenous ◽

Monocytic Lineage

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Positive transcriptional control of the pyridoxal phosphate biosynthesis genes pdxST by the MocR-type regulator PdxR of Corynebacterium glutamicum ATCC 13032

Microbiology ◽

10.1099/mic.0.044818-0 ◽

2011 ◽

Vol 157 (1) ◽

pp. 77-88 ◽

Cited By ~ 29

Author(s):

Nina Jochmann ◽

Susanne Götker ◽

Andreas Tauch

Keyword(s):

Dna Binding ◽

Corynebacterium Glutamicum ◽

Transcriptional Control ◽

Pyridoxal Phosphate ◽

Specific Binding ◽

Consensus Sequence ◽

Regulatory Protein ◽

Band Shift ◽

Promoter Sequences ◽

Amino Terminal

The pdxR (cg0897) gene of Corynebacterium glutamicum ATCC 13032 encodes a regulatory protein belonging to the MocR subfamily of GntR-type transcription regulators and consisting of an amino-terminal winged helix–turn–helix DNA-binding domain and a carboxy-terminal aminotransferase-like domain. A defined deletion in the pdxR gene resulted in the decreased expression of the divergently orientated pdxST genes coding for the subunits of pyridoxal 5′-phosphate synthase. The pdxST mutant C. glutamicum NJ0898 and the pdxR mutant C. glutamicum AMH17 showed vitamin B6 auxotrophy that was restored by supplementing the growth medium with either pyridoxal, pyridoxal 5′-phosphate or pyridoxamine. The genetic organization of the 89 bp pdxR–pdxST intergenic region was elucidated by mapping the 5′ ends of the respective transcripts, followed by detection of typical promoter sequences. Bioinformatic pattern searches and comparative genomics revealed three DNA motifs with the consensus sequence AAAGTGGW(−/T)CTA, overlapping the deduced promoter sequences and serving as candidate DNA-binding sites for PdxR. DNA band shift assays with the purified PdxR protein demonstrated the specific binding of the transcription regulator to double-stranded 40-mer sequences containing the detected motifs, thereby confirming the direct regulatory role of PdxR in activating the expression of the pdxST genes.

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Functional divergence between the half-sites of the DNA-binding sequence for the yeast transcriptional regulator Rap1p

Biochemical Journal ◽

10.1042/bj3410477 ◽

1999 ◽

Vol 341 (3) ◽

pp. 477-482 ◽

Cited By ~ 10

Author(s):

Fátima-Zahra IDRISSI ◽

Benjamin PIÑA

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Functional Divergence ◽

Consensus Sequence ◽

Transcriptional Regulator ◽

Dna Complexes ◽

Dna Binding Sites ◽

Binding Sequence ◽

Half Site

The yeast transcriptional regulator Rap1p binds to the DNA consensus sequence ACACCCAYACAYYY. We have previously shown that DNA-binding sites in which all four Y (Y = T or C) positions were Ts (UASrpg sequences) synergized more efficiently to activate transcription than sequences in which all Ys were Cs (telomere sequences) [F.-Z. Idrissi, J. Fernández-Larrea and B. Piña (1998) J. Mol. Biol. 284, 925-935]. Here we provide evidence that the DNA consensus sequence for Rap1p behaves as a combination of two ACAYYY half-sites with different functionality, the presence of Ts in the second half-site being the determinant for the transcriptional behaviour of the UASrpg sequences. DNA structure in the different complexes with Rap1p varied from being relatively uniform to appear rather distorted, this also being dependent on the presence of Ts in the second half-site. These distortions did not cause sharp bends or kinks in the DNA molecule. Computer analysis suggests that high-affinity binding of Rap1p to UASrpg sequences requires a rearrangement of the C-terminal Myb domain of the protein. We propose that the structural alterations in Rap1p-DNA complexes, both in the DNA and in the protein, affect the transcription potential of the complex in an allosteric manner. We also propose that the dimeric nature of the Rap1 DNA-binding domain is a key structural feature that explains the disparate functions of its DNA-binding sites in vivo.

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