Michael J. Moore

Toronto Metropolitan University

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Aaron Klug

MRC Laboratory of Molecular Biology

Yen Choo

Nanyang Technological University

Mark Isalan

Imperial College London

Lindsey Reynolds

University of Louisville

Mao‐Chang Su

Kaohsiung Chang Gung Memorial Hospital

Yann Jouvenot

Bio-Rad (United States)

Katherine Howes

Lawrence Berkeley National Laboratory

Chris Ullman

Point Richmond Tech Center

Hai‐Kun Liu

Heidelberg University

Andrew R. McNamara

Roche (Switzerland)

Cooperative Institutions

Sangamo BioSciences (United States)

Point Richmond Tech Center

Lawrence Berkeley National Laboratory

European Molecular Biology Laboratory

Washington University in St. Louis

Medical Research Council

Harvard University

MRC Laboratory of Molecular Biology

University of Sussex

Centre National de la Recherche Scientifique

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Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

Proceedings of the National Academy of Sciences (2003)

Siyuan Tan Dmitry Guschin Albert R. Davalos Ya-Li Lee Andrew Snowden

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene ( CHK2 ) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics.

10.1073/pnas.2035056100

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Citations (150)

Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors

Proceedings of the National Academy of Sciences (2003)

Monika Papworth Michael J. Moore Mark Isalan Michal Minczuk Yen Choo

The herpes simplex virus 1 (HSV-1) replicative cycle begins by binding of the viral activator, VP16, to a set of sequences in the immediate-early (IE) gene promoters. With the aim of inhibiting this cycle, we have constructed a number of synthetic zinc-finger DNA-binding peptides by using recently reported methods. Peptides containing either three or six fingers, targeted to a viral promoter, were engineered as fusions with a KOX-1 transcription repression domain. These proteins bound to the HSV-1 IE175k (ICP4) promoter, in vitro , with nanomolar or subnanomolar binding affinity. However, in a chloramphenicol acetyltransferase reporter system, only the six-finger protein was found to repress VP16-activated transcription significantly. Thus the longer array of zinc fingers is required to compete successfully against VP16, one of the most powerful natural activators known. We found that the HSV-1 replication cycle can be partially repressed by the six-finger peptide with the viral titer reduced by 90%.

Chloramphenicol acetyltransferase

Transcription

RING finger domain

10.1073/pnas.252773399

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Citations (73)

Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units

Proceedings of the National Academy of Sciences (2001)

Michael J. Moore Aaron Klug Yen Choo

Multizinc finger peptides are likely to reach increased prominence in the search for the “ideal” designer transcription factor for in vivo applications such as gene therapy. However, for these treatments to be effective and safe, the peptides must bind with high affinity and, more importantly, with great specificity. Our previous research has shown that zinc finger arrays can be made to bind 18 bp of DNA with picomolar affinity, but also has suggested that arrays of fingers also may bind tightly to related sequences. This work addresses the question of zinc finger DNA binding specificity. We show that by changing the way in which zinc finger arrays are constructed—by linking three two-finger domains rather than two three-finger units—far greater target specificity can be achieved through increased discrimination against mutated or closely related sequences. These new peptides have the added capability of being able to span two short gaps of unbound DNA, although still binding with picomolar affinity to their target sites. We believe that this new method of constructing zinc finger arrays will offer greater efficacy in the fields of gene therapy and in the production of transgenic organisms than previously reported zinc finger arrays.

Zinc finger nuclease

RING finger domain

Sp1 transcription factor

LIM domain

Binding selectivity

10.1073/pnas.98.4.1437

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Citations (160)

Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors

Proceedings of the National Academy of Sciences (2003)

Lindsey Reynolds Christopher G. Ullman Michael J. Moore Mark Isalan Michelle J. West

Zinc finger domains are small DNA-binding modules that can be engineered to bind desired target sequences. Functional transcription factors can be made from these DNA-binding modules, by fusion with an appropriate effector domain. In this study, eight three-zinc-finger proteins (ZFPs) that bound HIV-1 sequences in vitro were engineered into transcription repressors by linking them to the Krüppel-associated box (KRAB) repressor domain (KOX1). One protein, ZFP HIVB-KOX, which bound to a 9-bp region overlapping two Sp1 sites, was found to repress a Tat-activated 5' LTR cellular HIV-reporter assay to almost basal levels. A related six-finger protein, HIVBA'-KOX, was made to target all three Sp1 sites in the 5' LTR promoter and efficiently inhibited both basal and Tat-activated transcription in unstimulated and mitogen-stimulated T cells. In contrast, a combination of two unlinked three-finger ZFPs, HIVA'-KOX and HIVB-KOX, which bind over the same region of DNA, resulted in less effective repression. Finally, HIVBA'-KOX was tested for its capacity to block viral replication in a cellular infection assay using the HIV-1 HXB2 strain. This ZFP was found to inhibit HIV-1 replication by 75% compared with control constructs, thus demonstrating the potential of this approach for antiviral therapy.

Transcription

Zinc finger nuclease

10.1073/pnas.252770699

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Citations (128)

CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

Nature Communications (2021)

Naveen C. Reddy Shahriyar P. Majidi Lingchun Kong Mati Nemera Cole Ferguson

Abstract Regulation of chromatin plays fundamental roles in the development of the brain. Haploinsufficiency of the chromatin remodeling enzyme CHD7 causes CHARGE syndrome, a genetic disorder that affects the development of the cerebellum. However, how CHD7 controls chromatin states in the cerebellum remains incompletely understood. Using conditional knockout of CHD7 in granule cell precursors in the mouse cerebellum, we find that CHD7 robustly promotes chromatin accessibility, active histone modifications, and RNA polymerase recruitment at enhancers. In vivo profiling of genome architecture reveals that CHD7 concordantly regulates epigenomic modifications associated with enhancer activation and gene expression of topologically-interacting genes. Genome and gene ontology studies show that CHD7-regulated enhancers are associated with genes that control brain tissue morphogenesis. Accordingly, conditional knockout of CHD7 triggers a striking phenotype of cerebellar polymicrogyria, which we have also found in a case of CHARGE syndrome. Finally, we uncover a CHD7-dependent switch in the preferred orientation of granule cell precursor division in the developing cerebellum, providing a potential cellular basis for the cerebellar polymicrogyria phenotype upon loss of CHD7. Collectively, our findings define epigenomic regulation by CHD7 in granule cell precursors and identify abnormal cerebellar patterning upon CHD7 depletion, with potential implications for our understanding of CHARGE syndrome.

Epigenomics

Haploinsufficiency

Conditional gene knockout

10.1038/s41467-021-25846-3

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Citations (28)

Tailor-Made Zinc-Finger Transcription Factors Activate FLO11 Gene Expression with Phenotypic Consequences in the Yeast Saccharomyces cerevisiae

PLoS ONE (2007)

Jia‐Ching Shieh Yu‐Che Cheng Mao‐Chang Su Michael J. Moore Yen Choo

Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity.

Transcription

10.1371/journal.pone.0000746

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Citations (14)

Design of polyzinc finger peptides with structured linkers

Proceedings of the National Academy of Sciences (2001)

Michael J. Moore Yen Choo Aaron Klug

Zinc finger domains are perhaps the most versatile of all known DNA binding domains. By fusing up to six zinc finger modules, which normally recognize up to 18 bp of DNA, designer transcription factors can be produced to target unique sequences within large genomes. However, not all continuous DNA sequences make good zinc finger binding sites. To avoid having to target unfavorable DNA sequences, we designed multizinc finger peptides with linkers capable of spanning long stretches of nonbound DNA. Two three-finger domains were fused by using either transcription factor IIIA for the Xenopus 5S RNA gene (TFIIIA) finger 4 or a non-sequence-specific zinc finger as a “structured” linker. Our gel-shift results demonstrate that these peptides are able to bind with picomolar affinities to target sequences containing 0–10 bp of nonbound DNA. Furthermore, these peptides display greater sequence selectivity and bind with higher affinity than similar six-finger peptides containing long, flexible linkers. These peptides are likely to be of use in understanding the behavior of polydactyl proteins in nature and in the targeting of human, animal, or plant genomes for numerous applications. We also suggest that in certain polydactyl peptides an individual finger can “flip” out of the major groove to allow its neighbors to bind shorter, nontarget DNA sequences.

Zinc finger nuclease

LIM domain

Linker

RING finger domain

Protein–DNA interaction

PHD finger

Transcription

Sp1 transcription factor

10.1073/pnas.98.4.1432

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Citations (68)