Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

Patel, Anamika; Hashimoto, Hideharu; Zhang, X.; Cheng, Xiaodong

doi:10.1016/bs.mie.2016.01.019

Cited by 33 publications

(43 citation statements)

References 61 publications

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“…The function of IKZF1 N159S and IKZF1 N159T proteins was assessed by their ability to dimerize, migrate to the nucleus, and form foci by binding to PC-HC, as previously reported (14,21). Mutant proteins IKZF1 N159S and IKZF1 N159T were able to dimerize with WT IKZF1 ( Figure 5E) and migrate to the nucleus ( Figure 6A and Supplemental Figure 6).…”

Section: Discussionsupporting

confidence: 52%

“…All these positions are known to be involved in DNA binding (20) and are highly conserved across species, which highlights their importance for the function of IKZF1 ( Figure 1D). Supporting a key role of the N159 residue, p.N159A mutation was previously tested and shown to behave as a dominant-negative mutation in vitro, while its effect in vivo has not been described in animal models or extensively evaluated in humans (18,21).…”

Section: Introductionmentioning

confidence: 99%

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Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency

Boutboul¹,

Kuehn²,

Wyngaert³

et al. 2018

Journal of Clinical Investigation

137

129

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Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

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Section: Discussionsupporting

confidence: 52%

Section: Introductionmentioning

confidence: 99%

Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency

Boutboul¹,

Kuehn²,

Wyngaert³

et al. 2018

Journal of Clinical Investigation

137

129

View full text Add to dashboard Cite

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“…Cells were harvested by centrifugation, resuspended in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 500 mM NaCl, 5% (v/v) glycerol, 0.5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 25 μM ZnCl 2 , and then lysed by sonication. Lysates were mixed with polyethylenimine (Sigma) to a final concentration of 0.4% (w/v) and clarified by centrifugation at 18 000 rpm (35). Cleared extracts were loaded onto a glutathione-Sepharose 4B column (GE Healthcare) pre-equilibrated with the lysis buffer.…”

Section: Methodsmentioning

confidence: 99%

“…The GST tag was removed using PreScission protease (purified in-house), leaving five additional N-terminal residues (GPLGS). The proteins were diluted two-fold with 20 mM Tris–HCl (pH 7.5), 5% (v/v) glycerol, 25 μM ZnCl 2 , and 0.5 mM TCEP and loaded onto tandem HiTrap-Q/HiTrap-SP columns (GE Healthcare) (35). Most proteins flowed through the Q column onto the SP column from which they were eluted using a linear gradient of NaCl from 250 mM to 1 M. Finally, the pooled protein was concentrated and loaded onto a size exclusion column and eluted as a single peak in 500 mM NaCl, 20 mM Tris–HCl (pH 7.5), 5% (v/v) glycerol, and 25 μM ZnCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications

Hashimoto

Zhang

Zheng³

et al. 2016

Nucleic Acids Res

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Mutations in human zinc-finger transcription factor WT1 result in abnormal development of the kidneys and genitalia and an array of pediatric problems including nephropathy, blastoma, gonadal dysgenesis and genital discordance. Several overlapping phenotypes are associated with WT1 mutations, including Wilms tumors, Denys-Drash syndrome (DDS), Frasier syndrome (FS) and WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). These conditions vary in severity from individual to individual; they can be fatal in early childhood, or relatively benign into adulthood. DDS mutations cluster predominantly in zinc fingers (ZF) 2 and 3 at the C-terminus of WT1, which together with ZF4 determine the sequence-specificity of DNA binding. We examined three DDS associated mutations in ZF2 of human WT1 where the normal glutamine at position 369 is replaced by arginine (Q369R), lysine (Q369K) or histidine (Q369H). These mutations alter the sequence-specificity of ZF2, we find, changing its affinity for certain bases and certain epigenetic forms of cytosine. X-ray crystallography of the DNA binding domains of normal WT1, Q369R and Q369H in complex with preferred sequences revealed the molecular interactions responsible for these affinity changes. DDS is inherited in an autosomal dominant fashion, implying a gain of function by mutant WT1 proteins. This gain, we speculate, might derive from the ability of the mutant proteins to sequester WT1 into unproductive oligomers, or to erroneously bind to variant target sequences.

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“…For purification of recombinant CLAMP by FLAG-tag affinity chromatography, the coding sequence of CLAMP (CG1832-RA) was fused to a C-terminal coding sequence of FLAG affinity tag and cloned into pFastBac1, using the following forward and reverse primer: (Patel et al, 2016). Cell debris were spun down at 4°C for 30 min at 50,000 g. To the resulting supernatant 20 U Benzonase was added per 10 mL of culture and incubated with end-over-end rotation for 1 h at 4°C.…”

Section: Protein Purificationmentioning

confidence: 99%

Factor cooperation for chromosome discrimination in Drosophila

Albig

Tikhonova

Krause

et al. 2018

Preprint

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Transcription regulators select their genomic binding sites from a large pool of similar, non-functional sequences. Although general principles that allow such discrimination are known, the complexity of DNA elements often precludes a prediction of functional sites.The process of dosage compensation in Drosophila allows exploring the rules underlying binding site selectivity. The male-specific-lethal (MSL) Dosage Compensation Complex selectively binds to some 300 X-chromosomal 'High Affinity Sites' (HAS) containing GA-rich 'MSL recognition elements' (MREs), but disregards thousands of other MRE sequences in the genome. The DNA-binding subunit MSL2 alone identifies a subset of MREs, but fails to recognize most MREs within HAS. The 'Chromatin-linked adaptor for MSL proteins' (CLAMP) also interacts with many MREs genome-wide and promotes DCC binding to HAS. Using genome-wide DNA-immunoprecipitation we describe extensive cooperativity between both factors, depending on the nature of the binding sites. These are explained by physical interaction between MSL2 and CLAMP. In vivo, both factors cooperate to compete with nucleosome formation at HAS. The male-specific MSL2 thus synergises with a ubiquitous GA-repeat binding protein for refined X/autosome discrimination. KeywordsATAC-seq/ CLAMP/ DNA-immunoprecipitation/ Dosage compensation/ MSL2

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Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

Cited by 33 publications

References 61 publications

Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency

Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency

Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications

Factor cooperation for chromosome discrimination in Drosophila

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