Artificial Zinc Finger DNA Binding Domains: Versatile Tools for Genome Engineering and Modulation of Gene Expression

Zinc finger proteins (ZFP) play important roles in cellular processes. The DNA binding region of ZFP consists of 3 zinc finger DNA binding domains connected by amino acid linkers, the sequence TGQKP connects ZF1 and ZF2, and TGEKP connects ZF2 with ZF3. Linkers act to tune the zinc finger protein in the right position to bind its DNA target, the type of amino acid residues and length of linkers reflect on ZF1-ZF2-ZF3 interactions and contribute to the search and recognition process of ZF protein to its DNA target. Linker mutations and the affinity of the resulting mutants to specific and nonspecific DNA targets were studied by MD simulations and MM_GB(PB)SA. The affinity of mutants to DNA varied with type and position of amino acid residue. Mutation of K in TGQKP resulted in loss in affinity due to the loss of positive K interaction with phosphates, mutation of G showed loss in affinity to DNA, WT protein and all linker mutants showed loss in affinity to a nonspecific DNA target, this finding confirms previous reports which interpreted this loss in affinity as due to ZF1 having an anchoring role, and ZF3 playing an explorer role in the binding mechanism. The change in ZFP-DNA affinity with linker mutations is discussed in view of protein structure and role of linker residues in binding.

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Control of bacterial anti-phage signaling by a WYL domain transcription factor

10.1101/2022.01.04.474952 ◽

2022 ◽

Author(s):

Chelsea L Blankenchip ◽

Justin V Nguyen ◽

Rebecca K Lau ◽

Qiaozhen Ye ◽

Yajie Gu ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Promoter Region ◽

Bound State ◽

Signaling Proteins ◽

Abortive Infection ◽

Immune Systems ◽

Dna Binding Domains ◽

Binding Domains

Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how these systems are regulated to avoid activation and cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW reveal how the protein switches from a DNA binding-competent state to a ligand-bound state that cannot bind DNA due to misalignment of dimer-related DNA binding domains. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that CapW disruption likely results in toxicity due to uncontrolled CBASS expression. Our results parallel recent findings with BrxR, a transcription factor associated with the BREX anti-phage system, and suggest that CapW and BrxR are the founding members of a family of universal anti-phage signaling proteins.

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Base sequence discrimination by zinc-finger DNA-binding domains

Nature ◽

10.1038/349175a0 ◽

1991 ◽

Vol 349 (6305) ◽

pp. 175-178 ◽

Cited By ~ 136

Author(s):

Jeannette Nardelli ◽

Toby J. Gibson ◽

Christine Vesque ◽

Patrick Charnay

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Base Sequence ◽

Dna Binding Domains ◽

Binding Domains

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Male-specific Fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains

BMC Genomics ◽

10.1186/1471-2164-14-659 ◽

2013 ◽

Vol 14 (1) ◽

pp. 659 ◽

Cited By ~ 39

Author(s):

Justin E Dalton ◽

Justin M Fear ◽

Simon Knott ◽

Bruce S Baker ◽

Lauren M McIntyre ◽

...

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Dna Binding Domains ◽

Binding Domains ◽

Male Specific

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Developmentally Regulated Mouse Gene NK10 Encodes a Zinc Finger Repressor Protein with Differential DNA-Binding Domains

DNA and Cell Biology ◽

10.1089/dna.1995.14.971 ◽

1995 ◽

Vol 14 (11) ◽

pp. 971-981 ◽

Cited By ~ 29

Author(s):

RITA LANGE ◽

ANNETTE CHRISTOPH ◽

HANS-JÜRGEN THIESEN ◽

GABY VOPPER ◽

KENNETH R. JOHNSON ◽

...

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Mouse Gene ◽

Developmentally Regulated ◽

Repressor Protein ◽

Dna Binding Domains ◽

Binding Domains

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Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1786 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1786-1795 ◽

Cited By ~ 138

Author(s):

J F Morris ◽

R Hromas ◽

F J Rauscher

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Zinc Finger ◽

Binding Sites ◽

Zinc Fingers ◽

Consensus Sequences ◽

Dna Binding Domains ◽

Binding Domains ◽

Gel Shift ◽

Myeloid Zinc Finger

The myeloid zinc finger gene 1, MZF1, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine- and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-kappa B half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNA-binding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNA-binding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a transcriptional regulator of hematopoiesis.

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Differential abilities to engage inaccessible chromatin diversify vertebrate HOX binding patterns

10.1101/2019.12.29.890335 ◽

2019 ◽

Cited By ~ 3

Author(s):

Milica Bulajić ◽

Divyanshi Srivastava ◽

Jeremy S Dasen ◽

Hynek Wichterle ◽

Shaun Mahony ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Dna Binding Domain ◽

Regulatory Activity ◽

Dna Binding Domains ◽

Binding Domains ◽

Posterior Group ◽

Differential Gene ◽

Domain Similarity

ABSTRACTWhile Hox genes encode for conserved transcription factors (TFs), they are further divided into anterior, central, and posterior groups based on their DNA-binding domain similarity. The posterior Hox group expanded in the deuterostome clade and patterns caudal and distal structures. We aim to address how similar HOX TFs diverge to induce different positional identities. We studied HOX TF DNA-binding and regulatory activity during an in vitro motor neuron differentiation system that recapitulates embryonic development. We find diversity in the genomic binding profiles of different HOX TFs, even among the posterior group paralogs that share similar DNA binding domains. These differences in genomic binding are explained by differing abilities to bind to previously inaccessible sites. For example, the posterior group HOXC9 has a greater ability to bind occluded sites than the posterior HOXC10, producing different binding patterns and driving differential gene expression programs. From these results, we propose that the differential abilities of posterior HOX TFs to bind to previously inaccessible chromatin drive patterning diversification.

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