scholarly journals Encoded, click-reactive DNA-binding domains for programmable capture of specific chromatin segments

2020 ◽  
Vol 11 (46) ◽  
pp. 12506-12511
Author(s):  
Anna Witte ◽  
Álvaro Muñoz-López ◽  
Malte Metz ◽  
Michal R. Schweiger ◽  
Petra Janning ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Binding Protein ◽  
Protein Domains ◽  
Dna Binding Protein ◽  
Noncanonical Amino Acids ◽  
Dna Binding Domains ◽  
Binding Domains

We report programmable DNA-binding protein domains bearing genetically encoded noncanonical amino acids for click-biotinylation and enrichment of bound chromatin segments from cells for downstream analyses.

scholarly journals Rhodopsin targeted transcriptional silencing by DNA-binding

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Salvatore Botta ◽  
Elena Marrocco ◽  
Nicola de Prisco ◽  
Fabiola Curion ◽  
Mario Renda ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Binding Protein ◽  
Regulatory Element ◽  
Transcriptional Silencing ◽  
Dna Binding Protein ◽  
Gain Of Function ◽  
Dna Binding Domains ◽  
Binding Domains

Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.

scholarly journals REB1, a yeast DNA-binding protein with many targets, is essential for growth and bears some resemblance to the oncogene myb.

1990 ◽  
Vol 10 (10) ◽  
pp. 5226-5234 ◽  
Author(s):  
Q D Ju ◽  
B E Morrow ◽  
J R Warner
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Essential Gene ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Physiological Role ◽  
Dna Binding Protein ◽  
Reading Frame ◽  
Chromosome Ii

REB1 is a DNA-binding protein that recognizes sites within both the enhancer and the promoter of rRNA transcription as well as upstream of many genes transcribed by RNA polymerase II. We report here the cloning of the gene for REB1 by screening a yeast genomic lambda gt11 library with specific oligonucleotides containing the REB1 binding site consensus sequence. The REB1 gene was sequenced, revealing an open reading frame encoding 809 amino acids. The predicted protein was highly hydrophilic, with numerous OH-containing amino acids and glutamines, features common to many of the general DNA-binding proteins of Saccharomyces cerevisiae, such as ABF1, RAP1, GCN4, and HSF1. There was some homology between a portion of REB1 and the DNA-binding domain of the oncogene myb. REB1 is an essential gene that maps on chromosome II. However, the physiological role that it plays in the cell has yet to be established.

AND-1, a natural chimeric DNA-binding protein, combines an HMG-box with regulatory WD-repeats

1997 ◽  
Vol 110 (9) ◽  
pp. 1051-1062 ◽  
Author(s):  
A. Kohler ◽  
M.S. Schmidt-Zachmann ◽  
W.W. Franke
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Hmg Box ◽  
Amino Terminal ◽  
Wd Repeats ◽  
Carboxy Terminal

Using a specific monoclonal antibody (mAb AND-1/23-5-14) we have identified, cDNA-cloned and characterized a novel DNA-binding protein of the clawed toad, Xenopus laevis, that is accumulated in the nucleoplasm of oocytes and various other cells. This protein comprises 1,127 amino acids, with a total molecular mass of 125 kDa and a pI of 5.27. It is encoded by a mRNA of approximately 4 kb and contains, in addition to clusters of acidic amino acids, two hallmark motifs: the amino-terminal part harbours seven consecutive ‘WD-repeats’, which are sequence motifs of about 40 amino acids that are characteristic of a large group of regulatory proteins involved in diverse cellular functions, while the carboxy terminal portion possesses a 63-amino-acid-long ‘HMG-box’, which is typical of a family of DNA-binding proteins involved in regulation of chromatin assembly, transcription and replication. The DNA-binding capability of the protein was demonstrated by DNA affinity chromatography and electrophoretic mobility shift assays using four-way junction DNA. Protein AND-1 (acidic nucleoplasmic DNA-binding protein) appears as an oligomer, probably a homodimer, and has been localized throughout the entire interchromatinic space of the interphase nucleoplasm, whereas during mitosis it is transiently dispersed over the cytoplasm. We also identified a closely related, perhaps orthologous protein in mammals. The unique features of protein AND-1, which is a ‘natural chimera’ combining properties of the WD-repeat and the HMG-box families of proteins, are discussed in relation to its possible nuclear functions.

scholarly journals Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains

2000 ◽  
Vol 348 (1) ◽  
pp. 151-158 ◽  
Author(s):  
Mate TOLNAY ◽  
Lajos BARANYI ◽  
George C. TSOKOS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Rna Binding ◽  
Dna Binding Domains ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Binding Domains ◽  
Double Stranded Dna ◽  
Nuclear Ribonucleoprotein

Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiquitous protein that binds RNA and DNA sequences specifically, and has been implicated in the transcriptional regulation of the human complement receptor 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, providing direct proof that hnRNP D0 possesses a transactivator domain. We found, using truncated hnRNP D0 proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, using a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA-binding domains alone were not able to bind double-stranded DNA. Nevertheless, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the importance of the 24-amino-acid region in DNA binding, and suggested the involvement of the 19-amino-acid alternative insert which is present in isoforms B and D. The N-terminus had an inhibitory effect on binding of hnRNP D0 to single-stranded, but not to double-stranded, DNA. Although both recombinant hnRNP D0B and D0D bound DNA, only the B isoform recognized DNA in vivo. We propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA-binding transactivator and has distinct transactivator and DNA-binding domains.

REB1, a yeast DNA-binding protein with many targets, is essential for growth and bears some resemblance to the oncogene myb

1990 ◽  
Vol 10 (10) ◽  
pp. 5226-5234
Author(s):  
Q D Ju ◽  
B E Morrow ◽  
J R Warner
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Essential Gene ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Physiological Role ◽  
Dna Binding Protein ◽  
Reading Frame ◽  
Chromosome Ii

REB1 is a DNA-binding protein that recognizes sites within both the enhancer and the promoter of rRNA transcription as well as upstream of many genes transcribed by RNA polymerase II. We report here the cloning of the gene for REB1 by screening a yeast genomic lambda gt11 library with specific oligonucleotides containing the REB1 binding site consensus sequence. The REB1 gene was sequenced, revealing an open reading frame encoding 809 amino acids. The predicted protein was highly hydrophilic, with numerous OH-containing amino acids and glutamines, features common to many of the general DNA-binding proteins of Saccharomyces cerevisiae, such as ABF1, RAP1, GCN4, and HSF1. There was some homology between a portion of REB1 and the DNA-binding domain of the oncogene myb. REB1 is an essential gene that maps on chromosome II. However, the physiological role that it plays in the cell has yet to be established.

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