Mutation In Erythroid Specific Transcription Factor KLF1 Causes Hereditary Spherocytosis In the Nan (Neonatal Anemia) Hemolytic Anemia Mouse Model

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3217-3217
Author(s):  
Robert A White ◽  
Daniel P. Heruth ◽  
Troy Hawkins ◽  
Derek Logsdon ◽  
Margaret Gibson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hemolytic Anemia ◽  
Zinc Finger ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Hereditary Spherocytosis ◽  
Competitive Inhibition ◽  
Conflicts Of Interest ◽  
Dominant Negative ◽  
Zinc Finger Domain

Abstract Abstract 3217 The zinc finger protein Erythroid Krüuppel-like factor (EKLF, KLF1) regulates definitive erythropoiesis and terminal differentiation of red blood cells. KLF1 facilitates transcription through high affinity binding to CACCC elements within its erythroid-specific target genes which include genes encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins lead to the hemolytic anemia Hereditary Spherocytosis (HS). We have identified a new HS gene by studying the hemolytic anemia mouse mutant Nan (Neonatal Anemia). Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in Nan mice. The causative nature of the E339D mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1+/− knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild type KLF1. Competitive inhibition may help explain the apparent disconnect between the finding that Nan/+ heterozygous mice are anemic, whereas Klf1+/− heterozygous mice are normal and haplo-sufficient. This is the first direct association of a KLF1mutation with a disease in adult mammals. After examining a small population of HS patients, we also discovered one HS patient with a KLF1 mutation, which resulted in a significant amino acid substitution (T251I) in the activator/repressor domain, 28 amino acid residues upstream of the first zinc finger domain. This HS subject had no known mutations in the exons or intron/exon boundaries of EMS genes (SPTA1, SPTB, ANK1, SLC4A1) which comprise 85% of HS mutations in humans. The lack of a known genetic mutation in EMS genes leaves this patient's KLF1 mutation as the leading candidate defect. The identification of the gene causing the Nan mutation is significant because the Nan mutant has allowed discovery of a new HS gene which may also cause this disease in humans. In addition, the putative dominant/negative competitive inhibition of the Nan mutation makes the Nan mouse an excellent model system to study the function of KLF1. Disclosures: No relevant conflicts of interest to declare.

AML-1/ETO fusion protein is a dominant negative inhibitor of transcriptional repression by the promyelocytic leukemia zinc finger protein

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3939-3947 ◽  
Author(s):  
Ari Melnick ◽  
Graeme W. Carlile ◽  
Melanie J. McConnell ◽  
Adam Polinger ◽  
Scott W. Hiebert ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Zinc Finger ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Promyelocytic Leukemia Zinc Finger ◽  
Dominant Negative Inhibitor

Abstract The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RARα fusion protein and, in a similar manner, inhibits RARα target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RARα and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia.

AML-1/ETO fusion protein is a dominant negative inhibitor of transcriptional repression by the promyelocytic leukemia zinc finger protein

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3939-3947 ◽  
Author(s):  
Ari Melnick ◽  
Graeme W. Carlile ◽  
Melanie J. McConnell ◽  
Adam Polinger ◽  
Scott W. Hiebert ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Zinc Finger ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Promyelocytic Leukemia Zinc Finger ◽  
Dominant Negative Inhibitor

The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RARα fusion protein and, in a similar manner, inhibits RARα target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RARα and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia.

Human proviral mRNAs down regulated in choriocarcinoma encode a zinc finger protein related to Krüppel

1990 ◽  
Vol 10 (8) ◽  
pp. 4401-4405 ◽  
Author(s):  
N Kato ◽  
K Shimotohno ◽  
D VanLeeuwen ◽  
M Cohen
Keyword(s):  
Amino Acid ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Genomic Sequences ◽  
Cdna Clones ◽  
Leader Region ◽  
Rna Transcripts ◽  
Finger Protein

RNA transcripts of the HERV-R (ERV3) human provirus that are abundant in placenta but absent in choriocarcinoma contain nonproviral genomic sequences at their 3' ends. We report here the isolation of cDNA clones of these genomic sequences. The transcripts encode a Krüppel-related zinc finger protein consisting of a unique leader region and more than 12 28-amino-acid finger motifs.

scholarly journals Single amino acid exchanges in separate domains of the Drosophila serendipity delta zinc finger protein cause embryonic and sex biased lethality.

Genetics ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 905-916 ◽  
Author(s):  
M Crozatier ◽  
K Kongsuwan ◽  
P Ferrer ◽  
J R Merriam ◽  
J A Lengyel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Essential Gene ◽  
Larval Instar ◽  
Single Amino Acid ◽  
Isolation And Characterization ◽  
Finger Protein

Abstract The Drosophila serendipity (sry) delta (delta) zinc finger protein is a sequence-specific DNA binding protein, maternally inherited by the embryo and present in nuclei of transcriptionally active cells throughout fly development. We report here the isolation and characterization of four ethyl methanesulfate-induced zygotic lethal mutations of different strengths in the sry delta gene. For the stronger allele, all of the lethality occurs during late embryogenesis or the first larval instar. In the cases of the three weaker alleles, most of the lethality occurs during pupation; moreover, those adult escapers that emerge are sterile males lacking partially or completely in spermatozoa bundles. Genetic analysis of sry delta thus indicates that it is an essential gene, whose continued expression throughout the life cycle, notably during embryogenesis and pupal stage, is required for viability. Phenotypic analysis of sry delta hemizygote escaper males further suggests that sry delta may be involved in regulation of two different sets of genes: genes required for viability and genes involved in gonadal development. All four sry delta alleles are fully rescued by a wild-type copy of sry delta, but not by an additional copy of the sry beta gene, reinforcing the view that, although structurally related, these two genes exert distinct functions. Molecular characterization of the four sry delta mutations revealed that these mutations correspond to single amino acid replacements in the sry delta protein. Three of these replacements map to the same (third out of seven) zinc finger in the carboxy-terminal DNA binding domain; interestingly, none affects the zinc finger consensus residues. The fourth mutation is located in the NH2-proximal part of the protein, in a domain proposed to be involved in specific protein-protein interactions.

scholarly journals Residues in the Swi5 Zinc Finger Protein That Mediate Cooperative DNA Binding with the Pho2 Homeodomain Protein

1998 ◽  
Vol 18 (11) ◽  
pp. 6436-6446 ◽  
Author(s):  
Leena T. Bhoite ◽  
David J. Stillman
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Binding Proteins ◽  
Zinc Finger Protein ◽  
Interaction Analysis ◽  
Dna Binding Proteins ◽  
Two Hybrid

ABSTRACT The Swi5 zinc finger and the Pho2 homeodomain DNA-binding proteins bind cooperatively to the HO promoter.Pho2 (also known as Bas2 or Grf10) activates transcription of diverse genes, acting with multiple distinct DNA-binding proteins. We have performed a genetic screen to identify amino acid residues in Swi5 that are required for synergistic transcriptional activation of a reporter construct in vivo. Nine unique amino acid substitutions within a 24-amino-acid region of Swi5, upstream of the DNA-binding domain, reduce expression of promoters that require both Swi5 and Pho2 for activation. In vitro DNA binding experiments show that the mutant Swi5 proteins bind DNA normally, but some mutant Swi5 proteins (resulting from SWI5* mutations) show reduced cooperative DNA binding with Pho2. In vivo experiments show that these SWI5* mutations sharply reduce expression of promoters that require both SWI5 and PHO2, while expression of promoters that require SWI5 but arePHO2 independent is largely unaffected. This suggests that these SWI5* mutations do not affect the ability of Swi5 to bind DNA or activate transcription but specifically affect the region of Swi5 required for interaction with Pho2. Two-hybrid experiments show that amino acids 471 to 513 of Swi5 are necessary and sufficient for interaction with Pho2 and that the SWI5* point mutations cause a severe reduction in this two-hybrid interaction. Analysis of promoter activation by these mutants suggests that this small region of Swi5 has at least two distinct functions, conferring specificity for activation of the HO promoter and for interaction with Pho2.

scholarly journals Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso

2012 ◽  
Vol 109 (38) ◽  
pp. 15229-15234 ◽  
Author(s):  
Bethany A. Buck-Koehntop ◽  
Robyn L. Stanfield ◽  
Damian C. Ekiert ◽  
Maria A. Martinez-Yamout ◽  
H. Jane Dyson ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Zinc Finger ◽  
Dna Sequences ◽  
Genome Stability ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Epigenetic Modification ◽  
Cpg Dinucleotides ◽  
Finger Protein ◽  
Dna Motif

Methylation of CpG dinucleotides in DNA is a common epigenetic modification in eukaryotes that plays a central role in maintenance of genome stability, gene silencing, genomic imprinting, development, and disease. Kaiso, a bifunctional Cys2His2 zinc finger protein implicated in tumor-cell proliferation, binds to both methylated CpG (mCpG) sites and a specific nonmethylated DNA motif (TCCTGCNA) and represses transcription by recruiting chromatin remodeling corepression machinery to target genes. Here we report structures of the Kaiso zinc finger DNA-binding domain in complex with its nonmethylated, sequence-specific DNA target (KBS) and with a symmetrically methylated DNA sequence derived from the promoter region of E-cadherin. Recognition of specific bases in the major groove of the core KBS and mCpG sites is accomplished through both classical and methyl CH···O hydrogen-bonding interactions with residues in the first two zinc fingers, whereas residues in the C-terminal extension following the third zinc finger bind in the opposing minor groove and are required for high-affinity binding. The C-terminal region is disordered in the free protein and adopts an ordered structure upon binding to DNA. The structures of these Kaiso complexes provide insights into the mechanism by which a zinc finger protein can recognize mCpG sites as well as a specific, nonmethylated regulatory DNA sequence.

A Novel GATA1 Mutation (Ter414Arg) in a Family with the Rare X-Linked Blood Group Lu(a-b-) Phenotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1979-1979 ◽  
Author(s):  
Belinda K Singleton ◽  
David Roxby ◽  
John Stirling ◽  
Frances A Spring ◽  
Carolyn Wilson ◽  
...  
Keyword(s):  
Blood Group ◽  
Zinc Finger ◽  
Conflicts Of Interest ◽  
Large Family ◽  
Rflp Analysis ◽  
Blood Group Antigens ◽  
Zinc Finger Domain ◽  
Carboxy Terminus ◽  
Amino Terminal ◽  
Gata1 Mutation

Abstract Abstract 1979 Poster Board I-1001 The X-borne transcription factor GATA-1 is essential for erythroid and megakaryocyte development. In 1986, Norman et al. (Vox Sang 51:49) described a large family in which the rare Lu(a-b-) blood group phenotype is inherited as the result of an X-borne gene. Serologic and flow cytometric analyses confirmed the suppression of Lutheran blood group antigens on the red blood cells from the original male propositus. Analysis of DNA from the propositus revealed a mutation (1240T>C) in the termination codon of GATA1 converting TGA to a codon for arginine (CGA). The mutation predicts a translated GATA-1 protein containing an additional 41 amino acids at the carboxy terminus. DNA from an unaffected sister of the propositus had a wild-type GATA1 sequence. The GATA1 mutation was not present in 78 random blood donors as determined by restriction fragment length polymorphism (RFLP) analysis using BspHI. The propositus, who is now 64, has an Hb of 122 g/l, a low platelet count (86 × 109/l) with occasional macrothrombocytes (diameter 4-5 um) and a history of bruising easily. Several mutations in the amino terminal zinc finger domain of GATA-1 have been linked with thrombocytopenia and thalassemia or dsyerythropoietic anemia and the presence of macrothrombocytes. Our data provide evidence that mutations outside the zinc finger domain can affect GATA-1 functions in erythroid and megakaryocyte differentiation. In this case, the predicted extended carboxy terminus may interfere with GATA-1 interactions involving other DNA-binding proteins. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Epigenetic Modifier Mutations in the LL-100 Panel

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5271-5271
Author(s):  
Hilmar Quentmeier ◽  
Claudia Pommerenke ◽  
Hans G. Drexler
Keyword(s):  
Amino Acid ◽  
Drug Development ◽  
Cell Line ◽  
Cell Lines ◽  
Human Cell ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
Human Cell Line ◽  
Dominant Negative ◽  
Epigenetic Modifier

Abstract The NCI-60 human cell line panel, developed for use in drug development comprises sixty human cancer cell lines derived from nine different tissues. Only six cell lines of the NCI-60 were derived from blood cancers. Therefore, most forms and subtypes of leukemia and lymphoma are not represented in the NCI-60 panel. To respond to this apparent gap, we suggest the novel LL-100 panel, 100 leukemia and lymphoma cell lines representing the major leukemia/lymphoma entities, for basic research and drug development. Whole exome sequencing and RNA sequencing were performed to identify mutations in 100 cell lines. Here we list the 100 cell lines, ordered by subtype and show mutations in epigenetic modifier genes. We found cell lines with mutations in ASXL1, EZH2, IDH1, TET2 and in DNMT3A. Hitherto, cell line OCI-AML3 was the only human cell line described with a DNMT3A mutation. Twenty-two percent of patients with acute myeloid leukemia contain DNMT3A mutations and the median overall survival with DNMT3A mutations is shorter than without. Most DNMT3A mutations are heterozygous and alter amino acid R882, R882H being the most common DNMT3A mutation in AML. Exogenously mutant murine R878H (equivalent to human R882H) inhibits DNMT3A activity in a dominant negative manner. We describe here that the AML cell line SET-2 carries a heterozygous G to A transition at chr2_25234373 (hg38) which leads to the DNMT3A R882H amino acid substitution. Chip-based methylation analysis revealed that the described DNMT3A targets IRF8, KLF2, HOXA11 and HOXB2 are hypomethylated in cell lines OCI-AML3 (DNMT3A R882C) and in SET-2 (DNMT3A R882H). These data suggest that SET-2 is a novel model cell line for functional analysis of the DNMT3A R882 mutation and a first gain in knowledge through data mining the LL-100 panel. Disclosures No relevant conflicts of interest to declare.

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