Synergy between adjacent zinc fingers in sequence-specific DNA recognition

Zif268-like zinc fingers are generally regarded as independent DNA-binding modules that each specify three base pairs in adjacent, but discrete, subsites. However, crystallographic evidence suggests that a contact also can occur from the second helical position of one finger to the subsite of the preceding finger. Here we show for the three-finger DNA-binding domain of the protein Zif268, and a panel of variants, that deleting the putative contact from finger 3 can affect the binding specificity for the 5′ base in the adjoining triplet, which forms part of the binding site of finger 2. This finding demonstrates that Zif268-like zinc fingers can specify overlapping 4-bp subsites, and that sequence specificity at the boundary between subsites arises from synergy between adjacent fingers. This has important implications for the design and selection of zinc fingers with novel DNA binding specificities.     

Monomeric site-specific nucleases for genome editing

Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5'-CNNNG-3' cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5'-CNNNG-3' motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.     

Stimulation of endogenous neurogenesis by anti-EFRH immunization in a transgenic mouse model of Alzheimer's disease

Neurogenesis is a subject of intense interest and extensive research, but it stands at the center of a bitter debate over ethical and practical problems. Neurodegenerative diseases, such as Alzheimer's disease (AD), accompanied by a shifting balance between neurogenesis and neurodegeneration, are suitable for stimulation of neurogenesis for the benefit of diseased patients. We have previously shown that Abs against the EFRH sequence of beta-amyloid peptide (AbetaP) prevent aggregation and disaggregate AbetaP both in vitro and in vivo. EFRH, located in the soluble tail of the N-terminal region, acts as a regulatory site controlling both solubilization and disaggregation processes in the AbetaP molecule. Here we show that anti-EFRH immunotherapy of a platelet-derived amyloid precursor protein transgenic mouse model of AD stimulates endogenous neurogenesis, suggested by elevated numbers of BrdU-incorporated cells, most of which are colocalized with a marker of mature neurons, NeuN. These newly born neurons expressed the activity-dependent gene Zif268, indicating their functional integration and participation in response to synaptic input in the brain. These findings suggest that anti-amyloid immunotherapy may promote recovery from AD or other diseases related to AbetaP overproduction and neurotoxicity by restoring neuronal population, as well as cognitive functions in treated patients.     

Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ib alpha using canine-human chimeras.

Platelet glycoprotein Ib-IX-V (GPIb-IX-V) mediates adhesion to von Willebrand factor (vWF) in (patho)physiological thrombus formation. vWF binds the N-terminal 282 residues of GPIb alpha, consisting of an N-terminal flank (His1-Ile35), 7 leucine-rich repeats (Leu36-Ala200), a C-terminal flank (Phe201-Gly268), and a sulfated tyrosine sequence (Asp269-Glu282). By expressing canine-human chimeras of GPIb alpha on Chinese hamster ovary cells, binding sites for functional anti-GPIb alpha antibodies to individual domains were previously mapped, and it was shown that leucine-rich repeats 2 to 4 were required for optimal vWF recognition under static or flow conditions. Using novel canine-human chimeras dissecting the C-terminal flank, it is now demonstrated that (1) Phe201-Glu225 contains the epitope for AP1, an anti-GPIb alpha monoclonal antibody that inhibits both ristocetin- and botrocetin-dependent vWF binding; (2) VM16d, an antibody that preferentially inhibits botrocetin-dependent vWF binding, recognizes the sequence Val226-Gly268, surrounding Cys248, which forms a disulfide-bond with Cys209; (3) vWF binding to chimeric GPIb alpha is comparable to wild-type in 2 chimeras in which the sixth leucine-rich repeat was of the same species as the first disulfide loop (Phe201-Cys248) of the C-terminal flank, suggesting an interaction between these domains may be important for optimal vWF binding; and (4) replacing the C-terminal flank second disulfide loop (Asp249-Gly268) in human GPIb alpha with the corresponding canine sequence enhanced vWF binding under static and flow conditions, providing the first evidence for a gain-of-function phenotype associated with the second loop of the C-terminal flank.     

Epitope mapping of inhibitory antibodies against platelet glycoprotein Ibalpha reveals interaction between the leucine-rich repeat N-terminal and C-terminal flanking domains of glycoprotein Ibalpha

The interaction of von Willebrand factor (vWF) with the platelet receptor glycoprotein Ibalpha (GPIbalpha) is important for platelet adhesion at high shear stress. Two functionally important antigenic areas within GPIbalpha were identified through the characterization of 5 new inhibitory anti-GPIb monoclonal antibodies (mAbs). The binding sites of 3 of these anti-GPIb mAbs, which were intercompeting and potently inhibiting shear stress-induced binding of vWF, were mapped within the N-terminal amino acid (aa) 1-59 area by the use of canine-human chimeras. These antibodies, however, had little or no effect (approximately 40% inhibition) on the binding of vWF induced by either botrocetin or ristocetin. On the other hand, the anti-GPIb mAbs 24G10 and 6B4, which blocked GPIb-vWF binding under all conditions examined, bound to 2 different regions of GPIbalpha, aa 1-81 and aa 201-268, respectively. The epitope for 6B4 was further narrowed by phage display revealing 2 sets of peptide sequences aligning within aa 259-262 and aa 230-242. In the latter region of GPIbalpha, the gain-of-function platelet-type von Willebrand disease (PT-vWD) mutations have been identified. Alignment was partially confirmed because the binding of 6B4 to recombinant GPIbalpha fragments carrying either one of the PT-vWD mutations was considerably impaired but not completely abolished. In contrast, mAb 24G10 bound more strongly to mutant PT-vWD GPIbalpha. However, although 24G10 competed with 6B4 for binding to platelets, it bound to an epitope within aa 1-81 of GPIbalpha. In conclusion, 2 functionally important areas within GPIbalpha were identified: one localized within the leucine-rich repeat N-terminal aa 1-59 area and one composed of residues aa 1-81 in close contact with aa 201-268. Moreover, further support is provided for the existence of an intramolecular interaction between the N-terminal flanking (aa 1-81) and C-terminal flanking (aa 201-268) regions. (Blood. 2001;98:652-660)     

Sequence-specific recognition of DNA by zinc-finger peptides derived from the transcription factor Sp1.

We have overexpressed and purified two peptide fragments of Sp1 that contain the three "zinc-finger" domains necessary for specific Sp1 DNA binding. These peptides assume a stable, folded conformation in solution in the presence of Zn2+ as shown by DNA binding assays and NMR spectroscopy. Mobility-shift assays demonstrate that the Sp1 peptides recognize a number of different Sp1 DNA binding sites (GC boxes, with the core sequence GGGCGG). The dissociation constant for a 92-amino acid peptide binding to the GGGGCGGGGC sequence (Kd approximately 10 nM) and the relative affinities for several other DNA sequences definitively demonstrate Sp1-like binding properties. The thermodynamic binding site for Sp1-Zn92 has been mapped using the primer-extension/mobility-shift assay revealing that the 5' portion of the GC box DNA sequence (GGG GCG) contributes more strongly to the total binding energy than the 3' portion (GGGC). These findings are interpreted in the context of the Sp1 amino acid sequence in comparison with the structurally characterized Zif-268/DNA complex. A model is proposed that offers a structural explanation for the ability of Sp1 to recognize a diverse array of DNA sequences in terms of the individual (and different) DNA binding properties of each of the three zinc-finger domains.     

Direct effect of cocaine on epigenetic regulation of PKC? gene repression in the fetal rat heart

Maternal cocaine administration during gestation caused a down-regulation of PKC? expression in the heart of adult offspring resulting in an increased sensitivity to ischemia and reperfusion injury. The present study investigated the direct effect of cocaine in epigenetic modification of PKC? gene repression in the fetal heart. Hearts were isolated from gestational day 17 fetal rats and treated with cocaine in an ex vivo organ culture system. Cocaine treatment for 48 h resulted in significant decreases in PKC? protein and mRNA abundance and increases in CpG methylation at two SP1 binding sites in the PKC? promoter region (− 346 and − 268). Electrophoretic mobility shift assays demonstrated that CpG methylation of both SP1 sites inhibited SP1 binding. Consistently, chromatin immunoprecipitation assays showed that cocaine treatment significantly decreased binding of SP1 to the SP1 sites in the intact fetal heart. Reporter gene assays revealed that site-directed mutations of CpG methylation at both SP1 sites significantly reduced the PKC? promoter activity while methylation of a single site at either − 346 or − 268 did not have a significant effect. The causal effect of increased methylation in the cocaine-induced down-regulation of PKC? was demonstrated with the use of DNA methylation inhibitors. The presence of either 5-aza-2′-deoxycytodine or procainamide blocked the cocaine-induced increase in SP1 sites methylation and decrease in PKC? mRNA. The results demonstrate a direct effect of cocaine in epigenetic modification of DNA methylation and programming of cardiac PKC? gene repression linking prenatal cocaine exposure and pathophysiological consequences in the heart of adult offspring.     

Hereditary 1,25-dihydroxyvitamin D resistant rickets due to a mutation causing multiple defects in vitamin D receptor function

Hereditary vitamin D-resistant rickets (HVDRR) is an autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). We studied a young Saudi Arabian girl who exhibited the typical clinical features of HVDRR, but without alopecia. Analysis of her VDR gene revealed a homozygous T to C mutation in exon 7 that changed isoleucine to threonine at amino acid 268 (I268T). From crystallographic studies of the VDR ligand-binding domain, I268 directly interacts with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and is involved in the hydrophobic stabilization of helix H12. We recreated the I268T mutation and analyzed its effects on VDR function. In ligand binding assays, the I268T mutant VDR exhibited an approximately 5- to 10-fold lower affinity for [(3)H]1,25(OH)(2)D(3) compared with the wild-type (WT) VDR. The I268T mutant required approximately a 65-fold higher concentration of 1,25(OH)(2)D(3) to be equipotent in gene transactivation. Both retinoid X receptor heterodimerization and coactivator binding were reduced in the I268T mutant. Analogs of 1,25(OH)(2)D(3) have been proposed as potential therapeutics for patients with HVDRR. Interestingly, in protease sensitivity assays, treatment with the potent vitamin D analog, 20-epi-1,25(OH)(2)D(3), stabilized I268T mutant proteolytic fragments better than 1,25(OH)(2)D(3). Moreover, 20-epi-1,25(OH)(2)D(3) restored transactivation of the I268T mutant to levels exhibited by WT VDR treated with 1,25(OH)(2)D(3). In conclusion, we describe a novel mutation, I268T, in the VDR ligand-binding domain that alters ligand binding, retinoid X receptor heterodimerization, and coactivator binding. These combined defects in VDR function cause resistance to 1,25(OH)(2)D(3) action and result in the syndrome of HVDRR.     

Localization of hepatocyte nuclear factor‐4α in the nucleolus and nucleus is regulated by its C‐terminus

Aims/Introduction: Mutations in hepatocyte nuclear factor‐4α (HNF4α) lead to various diseases, among which C‐terminal deletions of HNF4α are exclusively responsible for maturity onset diabetes of the young 1 (MODY1). MODY is an autosomal dominant disease characterized by a primary defect in insulin response to glucose, suggesting that the C‐terminus of HNF4α is important for pancreatic β‐cell function. To clarify the role of the C‐terminus of HNF4α, changes in cellular localization and the binding ability to its regulator were examined, specifically in the region containing Q268, which deletion causes MODY1. Materials and Methods: Cellular localization of mutant HNF4α were examined in monkey kidney 7 (COS7), Chinese hamster ovary, rat insulinoma and mouse insulinoma cells, and their binding activity to other proteins were examined by fluorescence resonance energy transfer (FRET) in COS7 cells. Results: Although wild‐type HNF4α was localized in the nucleoplasm in transfected cultured cells, Q268X‐HNF4α was located predominantly in the nucleolus. Deletion analysis of the C‐terminus of HNF4α showed that the S337X‐HNF4α mutant, and other mutants with shorter amino acid sequences (S337‐K194), were mostly localized in the nucleolus. HNF4α mutants with amino acid sequences shorter than the W192X‐HNF4α mutant gradually spread to the nucleoplasm in accordance with their lengths. The A250X‐HNF4α mutant was capable of causing the accumulation of HNF4α or the small heterodimer partner (SHP), one of the HNF4α regulators, in the nucleolus. However, the R154X‐HNF4α mutant did not have binding ability to wild‐type HNF4α or SHP, and thus was seen in the nucleus. Conclusions: The C‐terminus sites might play a key role in facilitating the nucleolar and subnucleolar localization of HNF4α. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2012.00210.x, 2012)