scholarly journals Mutations in the Second Linker of KLF1 Cause Congenital Non-Spherocytic Hemolytic Anemia Due to Global Reduction of In Vivo DNA-Binding Affinity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1246-1246
Author(s):  
Stephen Huang ◽  
Kevin R. Gillinder ◽  
Annabel Sorolla ◽  
Emma Whitelaw ◽  
Andrew C Perkins
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Hemolytic Anemia ◽  
Zinc Finger ◽  
Fetal Liver ◽  
Zinc Fingers ◽  
Globin Gene ◽  
Flow Cytometric Analysis ◽  
Global Reduction

Abstract The mommeD45 mutation generates an amino acid transversion (H350R) within a conserved linker peptide between zinc fingers two and three of Klf1 (linker 2). Klf1H350R/H350R mice have mild compensated microcytic anaemia 1. Mice Carrying the H350R mutation were interbred with Klf1+/- mice. Klf1H350R/-mice have severe perinatal haemolytic anaemia, jaundice and marked splenomegaly. Haematological evaluation of these mice shows similar phenotypes to human patients who are compound heterozygotes for null and linker 2 mutations in KLF12. Analysis of Klf1H350R/- fetal liver by flow cytometry showed an increase in circulating immature CD71+ Ter119+ erythroblasts. In the bone marrow, a marked reduction in mature (Cd71- Ter119+) red blood cells was observed. Flow cytometric analysis of the spleen from Klf1H350R/- animals revealed an expansion of erythroid cells consistent with extramedullary erythropoiesis. ChIP-seq for Klf1 in 14.5DPC fetal liver from Klf1H350R/H350R mice revealed no loss in specificity when compared to wildtype Klf1, but a global reduction in affinity. Affinity measurements of recombinant zinc finger domains in vitro will be presented. By RNA-seq, we observed significantly lower expression of Klf1 target genes in mice homozygous for the H350R mutation compared to mice carrying a wildtype allele. And this correlates with reduced DNA binding observed in ChIP-seq and in vitro assays. Previous studies of the linkers in C2H2 zinc finger transcription factors have revealed their necessity as structural and regulatory components for the C2H2 class of transcription factors. Our results show the second linker of Klf1 plays an indirect role in DNA-binding and does not act just as a spacer for the zinc fingers. References: 1. Sorolla A, Tallack MR, Oey H, et al. Identification of novel hypomorphic and null mutations in Klf1 derived from a genetic screen for modifiers of alpha-globin transgene variegation. Genomics. 2015;105(2):116-122. 2. Viprakasit V, Ekwattanakit S, Riolueang S, et al. Mutations in Kruppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression. Blood. 2014;123(10):1586-1595. Disclosures Perkins: Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria.

Characterisation of Novel Hypomorphic and Null Mutations in Klf1 Derived from a Genetic Screen for Modifiers of a-Globin Transgene Variegation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3329-3329
Author(s):  
Stephen Huang ◽  
Kevin R. Gillinder ◽  
Melissa Ilsley ◽  
Graham Magor ◽  
Michael Tallack ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Red Blood Cells ◽  
Zinc Finger ◽  
Blood Cells ◽  
Zinc Fingers ◽  
Globin Gene ◽  
Position Effect ◽  
Flow Cytometric Analysis ◽  
Genetic Screen ◽  
Relative Reduction

Abstract Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated a-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for Modifiers of murine metastable epi-alleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation; i.e. an increased percentage of GFP+ circulating red blood cells. Both strains harbour point mutations in the erythroid specific transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain. D11 homozygous mice die in utero of anaemia at 14.5DPC. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have mild compensated microcytic anaemia which models the phenotype in a recently described human family. Mice Carrying the H350R mutation were interbred with Klf1+/- mice. Klf1H350R/- mice have severe perinatal haemolytic anaemia and marked splenomegaly. Furthermore blood haemoglobin content, haematocrit and red blood cell size (MCV) were significantly reduced in Klf1H350R/- mice compared to wildtype and D45 homozygous offspring of the same age. Analysis of Klf1H350R/- by flow cytometry showed an increase in circulating immature red blood cells. In the bone marrow, a lack of mature red blood cells was observed. Flow cytometric analysis of the spleen from Klf1H350R/- animals revealed an expansion of erythroid cells and a relative reduction in B and T Cells. Gel shifts assays of a recombinant Klf1 zinc finger protein with the H350R mutation showed normal binding to the b -globin promoter sequence but weak binding to the Alas2 intronic enhancer site. Furthermore b -globin gene expression was near normal whereas expression of other known Klf1 target genes was decreased. We will discuss how H350R disrupts function from ChIP-seq and RNA-seq in primary fetal liver tissue. Previous studies of the linkers in C2H2 zinc finger transcription factors have revealed their necessity as structural and regulatory components for the C2H2 class of transcription factors. Our results thus far show that the second linker of Klf1 has a role in maintaining the integrity of Klf1 function (at a subset of Klf1-occupied sites,) and does not act just as a spacer for the zinc fingers. Disclosures Perkins: Novartis Oncology: Honoraria.

scholarly journals Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN

Science ◽  
2018 ◽  
Vol 362 (6414) ◽  
pp. eaat0572 ◽  
Author(s):  
Quinlan L. Sievers ◽  
Georg Petzold ◽  
Richard D. Bunker ◽  
Aline Renneville ◽  
Mikołaj Słabicki ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Small Molecules ◽  
Zinc Finger ◽  
Ubiquitin Ligase ◽  
Biochemical Analysis ◽  
Zinc Fingers ◽  
Functional Characterization ◽  
C2h2 Zinc Finger

The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a Cys2-His2 (C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase. We screened the human C2H2 zinc finger proteome for degradation in the presence of thalidomide analogs, identifying 11 zinc finger degrons. Structural and functional characterization of the C2H2 zinc finger degrons demonstrates how diverse zinc finger domains bind the permissive drug-CRBN interface. Computational zinc finger docking and biochemical analysis predict that more than 150 zinc fingers bind the drug-CRBN complex in vitro, and we show that selective zinc finger degradation can be achieved through compound modifications. Our results provide a rationale for therapeutically targeting transcription factors that were previously considered undruggable.

scholarly journals Physical and functional sensitivity of zinc finger transcription factors to redox change.

1996 ◽  
Vol 16 (3) ◽  
pp. 1035-1046 ◽  
Author(s):  
X Wu ◽  
N H Bishopric ◽  
D J Discher ◽  
B J Murphy ◽  
K A Webster
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Sites ◽  
Redox Regulation ◽  
Dna Binding Proteins ◽  
Thiol Groups ◽  
Protein Thiol

Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites.

scholarly journals The Zinc-Sensing Mechanism of Mouse MTF-1 Involves Linker Peptides between the Zinc Fingers

2006 ◽  
Vol 26 (15) ◽  
pp. 5580-5587 ◽  
Author(s):  
Yong Li ◽  
Tomoki Kimura ◽  
John H. Laity ◽  
Glen K. Andrews
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Zinc Fingers ◽  
Gene Promoter ◽  
Zinc Sensing ◽  
Cellular Zinc

ABSTRACT Mouse metal response element-binding transcription factor-1 (MTF-1) regulates the transcription of genes in response to a variety of stimuli, including exposure to zinc or cadmium, hypoxia, and oxidative stress. Each of these stresses may increase labile cellular zinc, leading to nuclear translocation, DNA binding, and transcriptional activation of metallothionein genes (MT genes) by MTF-1. Several lines of evidence suggest that the highly conserved six-zinc finger DNA-binding domain of MTF-1 also functions as a zinc-sensing domain. In this study, we investigated the potential role of the peptide linkers connecting the four N-terminal zinc fingers of MTF-1 in their zinc-sensing function. Each of these three linkers is unique, completely conserved among all known vertebrate MTF-1 orthologs, and different from the canonical Cys2His2 zinc finger TGEKP linker sequence. Replacing the RGEYT linker between zinc fingers 1 and 2 with TGEKP abolished the zinc-sensing function of MTF-1, resulting in constitutive DNA binding, nuclear translocation, and transcriptional activation of the MT-I gene. In contrast, swapping the TKEKP linker between fingers 2 and 3 with TGEKP had little effect on the metal-sensing functions of MTF-1, whereas swapping the canonical linker for the shorter TGKT linker between fingers 3 and 4 rendered MTF-1 less sensitive to zinc-dependent activation both in vivo and in vitro. These observations suggest a mechanism by which physiological concentrations of accessible cellular zinc affect MTF-1 activity. Zinc may modulate highly specific, linker-mediated zinc finger interactions in MTF-1, thus affecting its zinc- and DNA-binding activities, resulting in translocation to the nucleus and binding to the MT-I gene promoter.

scholarly journals Multicomponent differentiation-regulated transcription factors in F9 embryonal carcinoma stem cells.

1991 ◽  
Vol 11 (3) ◽  
pp. 1686-1695 ◽  
Author(s):  
M K Shivji ◽  
N B La Thangue
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Dna Binding ◽  
Embryonal Carcinoma ◽  
Regulatory Sequence ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Dna Sequence Specificity ◽  
Cell Extracts

Murine F9 embryonal carcinoma (F9 EC) stem cells have an E1a-like transcription activity that is down-regulated as these cells differentiate to parietal endoderm. For the adenovirus E2A promoter, this activity requires at least two sequence-specific transcription factors, one that binds the cyclic AMP-responsive element (CRE) and the other, DRTF1, the DNA-binding activity of which is down-regulated as F9 EC cells differentiate. Here we report the characterization of several binding activities in F9 EC cell extracts, referred to as DRTF 1a, 1b and 1c, that recognize the DRTF1 cis-regulatory sequence (-70 to -50 region). These activities can be chromatographically separated but are not distinguishable by DNA sequence specificity. Activity 1a is a detergent-sensitive complex in which DNA binding is regulated by phosphorylation. In contrast, activities 1b and 1c are unaffected by these treatments but exist as multicomponent protein complexes even before DNA binding. Two sets of DNA-binding polypeptides, p50DR and p30DR, affinity purified from F9 EC cell extracts produce complexes 1b and 1c. Both polypeptides appear to be present in the same DNA-bound protein complex and both directly contact DNA. These affinity-purified polypeptides activate transcription in vitro in a binding-site-dependent manner. These data indicate the in F9 EC stem cells, multicomponent differentiation-regulated transcription factors contribute to the cellular E1a-like activity.

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

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.

scholarly journals On the prediction of DNA-binding preferences of C2H2-ZF domains using structural models: application on human CTCF

2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Alberto Meseguer ◽  
Filip Årman ◽  
Oriol Fornes ◽  
Ruben Molina-Fernández ◽  
Jaume Bonet ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Site ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Computational Method ◽  
Chromatin Binding ◽  
Input Structure ◽  
Binding Factor ◽  
Potential Binding

Abstract Cis2-His2 zinc finger (C2H2-ZF) proteins are the largest family of transcription factors in human and higher metazoans. To date, the DNA-binding preferences of many members of this family remain unknown. We have developed a computational method to predict their DNA-binding preferences. We have computed theoretical position weight matrices (PWMs) of proteins composed by C2H2-ZF domains, with the only requirement of an input structure. We have predicted more than two-third of a single zinc-finger domain binding site for about 70% variants of Zif268, a classical member of this family. We have successfully matched between 60 and 90% of the binding-site motif of examples of proteins composed by three C2H2-ZF domains in JASPAR, a standard database of PWMs. The tests are used as a proof of the capacity to scan a DNA fragment and find the potential binding sites of transcription-factors formed by C2H2-ZF domains. As an example, we have tested the approach to predict the DNA-binding preferences of the human chromatin binding factor CTCF. We offer a server to model the structure of a zinc-finger protein and predict its PWM.

scholarly journals Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity

2020 ◽  
Vol 21 (24) ◽  
pp. 9401
Author(s):  
Antonio Bouthelier ◽  
Florinda Meléndez-Rodríguez ◽  
Andrés A. Urrutia ◽  
Julián Aragonés
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Cellular Response ◽  
Target Gene ◽  
Target Genes ◽  
Transactivation Domain ◽  
Transactivation Domains ◽  
Relative Contribution

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.

scholarly journals Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

1995 ◽  
Vol 15 (3) ◽  
pp. 1405-1421 ◽  
Author(s):  
C C Adams ◽  
J L Workman
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
General Mechanism ◽  
Nf Kappa B ◽  
Nucleosomal Dna ◽  
Nucleosome Binding

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

scholarly journals Identification of two factors required for transcription of the ovalbumin gene.

1986 ◽  
Vol 6 (12) ◽  
pp. 4259-4267 ◽  
Author(s):  
I Sagami ◽  
S Y Tsai ◽  
H Wang ◽  
M J Tsai ◽  
B W O'Malley
Keyword(s):  
Molecular Weight ◽  
Transcription Factors ◽  
Gradient Centrifugation ◽  
Globin Gene ◽  
Sodium Dodecyl ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Gene Promoters ◽  
Early Genes

Two transcription factors, COUP and S300-II, were isolated and partially purified from HeLa cell nuclear extracts. Both factors are required for the efficient transcription in vitro of the ovalbumin gene but not the simian virus 40 early genes. COUP factor binds to the chicken ovalbumin upstream promoter (COUP) sequence which lies between -70 to -90 base pairs upstream from the cap site. A series of competition experiments with a band-shifting assay was carried out to determine the relative affinity of COUP box transcription factor for various promoters. We found that a promoter DNA fragment isolated from the ovalbumin gene competes better than those isolated from the ovomucoid, Y, and alpha-genes. In contrast, the the simian virus 40 early genes, the beta-globin gene, and the adenosine deaminase gene promoters do not compete well in this assay. The molecular weight of the COUP factor was estimated by S-300 column chromatography, glycerol gradient centrifugation to be 90,000. However, two bands were observed in sodium dodecyl sulfate gel electrophoresis of cross-linked COUP factor to a 32P-labeled oligonucleotide containing the COUP sequence. The protein moieties of the major and minor bands were estimated to be 85,000 to 90,000 and 40,000 to 45,000, respectively. The S300-II factor with an apparent molecular weight of 45,000 in an S-300 column is required for function in an in vitro reconstituted transcription system. In contrast to the COUP factor, the S300-II factor does not have apparent specificity for binding to the ovalbumin gene promoter. The S300-II factor may function by interacting with RNA polymerase or other DNA-binding transcription factors.

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