The role of zinc finger linkers in zinc finger protein binding to DNA

Hamed, Mazen Y.; Siam, Reema; Zaid, Roza

doi:10.1007/s10822-021-00413-6

Cited by 6 publications

(2 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…And with ZNF407 an “S to W” amino acid exchange in its “HTGEKS linker” has been set into context with an autosomal recessive intellectual impairment . Other single amino acid exchanges in zinc finger linkers of transcription regulator proteins cause loss of affinity to DNA . With the H to Y amino acid exchange of the HTGEKP linker, which has been studied in this project, a functional alteration of the ZNF331 protein with a severe impact on transcriptional dysregulation has been pointed out .…”

Section: Discussionmentioning

confidence: 99%

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C₂H₂ Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis

Scherf

Koy

Röwer

et al. 2023

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Weak-force Order (ITEM-TWO) analysis in combination with molecular modeling, the phosphorylation-dependent molecular recognition motif of the anti-HpTGEKP antibody has been investigated with binary and ternary component mixtures consisting of antibody and (phospho-) peptides. Amino acid sequences have been selected to match either the antibody's recognition motif or the cancer-related zinc finger protein mutations and phosphorylations of the respective amino acid residues. Upon electrospraying of all the components of the mixtures, that is, hexapeptides, antibody, and intact immune complexes, the produced ions were subjected to mass spectrometric mass filtering. The antibody ions as well as the immune complex ions traversed into the mass spectrometer's collision chamber, whereas paths of unbound peptide ions were blocked prior to entering the collision cell. After dissociation of the multiply charged immune complexes in the gas phase, the complex-released peptide ions were recorded after having traversed the second mass filter. Complex-released peptides were unambiguously identified by their masses using mass analysis with isotope resolution. From the results of our studies with seven (phospho-) peptides with distinct amino acid sequences, which resembled either the antibody's binding motif or mutations, we conclude the following: (i) A negatively charged phospho group, located near the peptide's N-terminus is mandatory for antibody binding when placed on the peptide surface at a precise distance to the C-terminally located positively charged ε-amino group of a lysinyl residue. (ii) A bulky amino acid residue, such as the tyrosinyl residue at the N-terminal position of the (phospho-) threoninyl residue, abolishes antibody binding. (iii) Two closely spaced phospho groups negatively interfere with the surface polarity pattern and abolish antibody binding as well. (iv) Non-phosphorylated peptides are not binding partners of the anti-HpTGEKP antibody.

show abstract

Section: Discussionmentioning

confidence: 99%

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C₂H₂ Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis

Scherf

Koy

Röwer

et al. 2023

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…1) For a certain ZnF, its preferably bound DNA triplets in live cells cannot be predicted accurately [ 24 ]. It has been found that besides those “fingerprint” amino acids, other nearby amino acids including the linker sequences also contribute to the DNA binding activity [ 17 , 18 , 25 ]. 2) It is commonly observed that not all the ZnF individuals within a tandem array are actually involved in the target recognition while the intrinsic rule of this selective usage is largely unknown [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9

Zhang

et al. 2022

Mobile DNA

View full text Add to dashboard Cite

Background Krüppel Associated Box-containing Zinc Finger Proteins (KRAB-ZFPs), representing the largest superfamily of transcription factors in mammals, are predicted to primarily target and repress transposable elements (TEs). It is challenging to dissect the distinct functions of these transcription regulators due to their sequence similarity and diversity, and also the complicated repetitiveness of their targeting TE sequences. Results Mouse KRAB-Zfps are mainly organized into clusters genomewide. In this study, we revealed that the intra-cluster members had a close evolutionary relationship, and a similar preference for zinc finger (ZnF) usage. KRAB-Zfps were expressed in a cell type- or tissue type specific manner and they tended to be actively transcribed together with other cluster members. Further sequence analyses pointed out the linker sequences in between ZnFs were conserved, and meanwhile had distinct cluster specificity. Based on these unique characteristics of KRAB-Zfp clusters, sgRNAs were designed to edit cluster-specific linkers to abolish the functions of the targeted cluster(s). Using mouse embryonic stem cells (mESC) as a model, we screened and obtained a series of sgRNAs targeting various highly expressed KRAB-Zfp clusters. The effectiveness of sgRNAs were verified in a reporter assay exclusively developed for multi-target sgRNAs and further confirmed by PCR-based analyses. Using mESC cell lines inducibly expressing Cas9 and these sgRNAs, we found that editing different KRAB-Zfp clusters resulted in the transcriptional changes of distinct categories of TEs. Conclusions Collectively, the intrinsic sequence correlations of intra-cluster KRAB-Zfp members discovered in this study suggest that the conserved cluster specific linkers played crucial roles in diversifying the tandem ZnF array and the related target specificity of KRAB-Zfps during clusters’ evolution. On this basis, an effective CRISPR-Cas9 based approach against the linker sequences is developed and verified for rapidly editing KRAB-Zfp clusters to identify the regulatory correlation between the cluster members and their potential TE targets.

show abstract

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti‐HpTGEKP Antibody and Zinc Finger Protein Linker Phospho‐Hexapeptides

et al. 2022

View full text Add to dashboard Cite

Accurate formation of antibody-antigen complexes has been relied on in both, multitudes of scientific projects and ample therapeutic and diagnostic applications. Mass spectrometrically determined dissociation behavior of immune complexes with the anti-HpTGEKP antibody revealed that the ten most frequently occurring phospho-hexapeptide linker sequences from C2H2 zinc finger proteins could be divided into two classes: orthodox binders, where strong noncovalent interactions developed as anticipated, and unorthodox binders with deviating structures and weaker binding. Phosphorylation of threonine was compulsory for antibody binding in an orthodox manner. Gas phase dissociation energy determinations of seven C2H2 zinc finger protein linker phospho-hexapeptides with orthodox binding properties revealed a bipolar binding motif of the antibody paratope. Epitope peptides, which in addition to the negatively charged phospho-threonine residue were Cterminally flanked by positively charged residues provided stronger binding, i. e. dissociation was endothermic, than peptides with acidic amino acid residues at these positions, for which dissociation was exothermic.

show abstract

The role of zinc finger linkers in zinc finger protein binding to DNA

Cited by 6 publications

References 54 publications

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C₂H₂ Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C₂H₂ Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis

Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti‐HpTGEKP Antibody and Zinc Finger Protein Linker Phospho‐Hexapeptides

Contact Info

Product

Resources

About

The role of zinc finger linkers in zinc finger protein binding to DNA

Cited by 6 publications

References 54 publications

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C2H2 Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C2H2 Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis

Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti‐HpTGEKP Antibody and Zinc Finger Protein Linker Phospho‐Hexapeptides

Contact Info

Product

Resources

About

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C₂H₂ Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C₂H₂ Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis