Zinc-finger protein transcription factors (ZFP TFs)D efects in transcriptional regulation underlie numerous disease states, most notably cancer (1). A major goal of current strategies for correcting such defects is to achieve sufficient specificity of action (2). Designed zinc-finger protein transcription factors (ZFP TFs) emulate natural transcriptional control mechanisms and therefore provide an attractive tool for precisely regulating gene expression. Accurate control of gene expression is important for understanding gene function (target validation) and for developing therapeutics to treat disease (3). We and others have used engineered ZFP TFs to either activate or repress a variety of endogenous gene targets (4-11). For these proteins, or any other gene-regulation technology, to succeed as tools in drug discovery or direct agents in the clinic, their specificity of action within the genome must be precise, a challenging criterion to meet given the size and complexity of the human genome. Recent studies with small interfering RNA (12, 13) and antisense DNA͞RNA (14) have illuminated the magnitude of the task of achieving single-gene specificity in regulating the human genome.We focus here on the use of ZFP TFs in the area of oncology and specifically on the emerging role of checkpoint kinase 2 (CHK2). CHK2 acts as a key integrator of DNA-damage signals regulating cell-cycle progression, DNA repair, and cell death by phosphorylating a variety of substrates, including the p53 tumor suppressor protein (15, 16). Here we show that a designed ZFP TF targeted to a unique 18-bp recognition sequence in the promoter of the CHK2 gene binds the intended site within chromatin and represses CHK2 transcription in vivo. Moreover, repression of CHK2 by this engineered ZFP TF occurs with remarkable specificity, while simultaneously reducing CHK2 protein to levels that functionally ablate the action of this kinase. Finally, we show that constitutive expression of the ZFP TF in telomerase-immortalized, untransformed human fibroblasts provides stable repression of the CHK2 gene and results in loss of DNA-damage-induced CHK2-dependent phosphorylation of p53 on Ser-20. These data demonstrate that ZFP TFs can be exquisitely specific, yet potent repressors of gene expression and, therefore, are potentially powerful reagents for target validation and therapeutic interventions in vivo.
DNA methylation is now seen as a primary signal in the cell for mediating transcriptional repression through chromatin formation. The construction and evaluation of enzymes capable of influencing this process in vivo is therefore of significant interest. We have fused the C5-cytosine DNA methyltransferases, M.HhaI and M.HpaII, which both methylate 4 bp sequences containing a CpG dinucleotide, to a three zinc finger protein recognising a 9 bp DNA sequence. DNA methylation analyses demonstrate specific DNA methylation by both enzymes at target sites comprising adjacent methyltransferase and zinc finger subsites, targeted M.HpaII being the most specific. Binding analysis of the targeted M.HpaII enzyme reveals an 8-fold preference for binding to its target site, compared to binding to a zinc finger site alone, and an 18-fold preference over binding to a methyltransferase site alone, thereby demonstrating enhanced binding by the fusion protein, compared to its component proteins. Both DNA binding and methylation are specific for the target site up to separations of approximately 40 bp between the zinc finger and methyltransferase subsites. Ex vivo plasmid methylation experiments are also described that demonstrate targeted methylation. These targeted enzymes, however, are shown to be not fully mono-functional, retaining a significant non-targeted activity most evident at elevated protein concentrations.
A three zinc-finger protein that binds specifically to the cDNA representing the unique fusion gene BCR:Abl, associated with acute lymphoblastic leukaemia, has previously been characterised. At this breakpoint, a sequence homology of 8/9 bp exists between the BCR:Abl (fusion) and c-ABL: (parental) target sequences. We show that the three zinc-finger protein discriminates poorly between the fusion (BCR:Abl) and parental (ABL:) sequence (K:(d)s of 42.8 and 65.1 nM, respectively). In order to improve the discriminatory properties of this protein, and to demonstrate the utility of current zinc-finger databases, we have added a fourth zinc-finger to the original three zinc-finger protein. This fourth finger recognises a 3 bp subsite derived from the BCR: portion of the breakpoint and is not present in c-ABL: This novel four finger protein, which now recognises a 12 bp sequence, demonstrates improved specific binding to BcrAbl (K:(d )= 17 nM). More significantly we have shown that there is now enhanced discrimination between BcrAbl and ABL: sequences by the four finger protein than the original three finger protein.
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