Target DNA Binding Sites Research Articles

Noncanonical binding of transcription factors: time to revisit specificity?

Transcription factors (TFs) are one of the most studied classes of DNA-binding proteins that have a direct functional impact on gene transcription and thus, on human physiology and disease. The mechanisms that TFs use for recognizing target DNA binding sites have been studied for nearly five decades, yet they remain poorly understood. It is classically assumed that a TF recognizes a specific sequence pattern, or motif, as its binding sites. However, recent studies are consistently finding examples of noncanonical binding, that is, TFs binding at sites that do not resemble their sequence motifs. Here we review the current literature on four major types of noncanonical TF binding, namely binding based on DNA shape readout, at Guanine-quadruplex structures, at repeat sequences, and bispecific binding. These examples point to a critical need for studies to unify our current observations, many of which are at odds with the "one TF, one motif" view, into a more comprehensive definition of the DNA-binding specificity of TFs.

Comprehensive In Silico Characterization of the Coding and Non-Coding SNPs in Human Dectin-1 Gene with the Potential of High-Risk Pathogenicity Associated with Fungal Infections.

The human C-type lectin domain family 7 member A (CLEC7A) gene encodes a Dectin-1 protein that recognizes beta-1,3-linked and beta-1,6-linked glucans, which form the cell walls of pathogenic bacteria and fungi. It plays a role in immunity against fungal infections through pathogen recognition and immune signaling. This study aimed to explore the impact of nsSNPs in the human CLEC7A gene through computational tools (MAPP, PhD-SNP, PolyPhen-1, PolyPhen-2, SIFT, SNAP, and PredictSNP) to identify the most deleterious and damaging nsSNPs. Further, their effect on protein stability was checked along with conservation and solvent accessibility analysis by I-Mutant 2.0, ConSurf, and Project HOPE and post-translational modification analysis using MusiteDEEP. Out of the 28 nsSNPs that were found to be deleterious, 25 nsSNPs affected protein stability. Some SNPs were finalized for structural analysis with Missense 3D. Seven nsSNPs affected protein stability. Results from this study predicted that C54R, L64P, C120G, C120S, S135C, W141R, W141S, C148G, L155P, L155V, I158M, I158T, D159G, D159R, I167T, W180R, L183F, W192R, G197E, G197V, C220S, C233Y, I240T, E242G, and Y3D were the most structurally and functionally significant nsSNPs in the human CLEC7A gene. No nsSNPs were found in the predicted sites for post-translational modifications. In the 5' untranslated region, two SNPs, rs536465890 and rs527258220, showed possible miRNA target sites and DNA binding sites. The present study identified structurally and functionally significant nsSNPs in the CLEC7A gene. These nsSNPs may potentially be used for further evaluation as diagnostic and prognostic biomarkers.

High-resolution crystal structure of the anti-CRISPR protein AcrIC5

As a result of the long-term battle of bacteria and archaea against invaders such as viruses and genetic mobile elements, they have developed CRISPR-Cas systems for self-defense, which allows them to remove the viral genetic material introduced into host cells via infection. To fight against this bacterial immune system, however, viruses have also evolved to produce multiple anti-CRISPR proteins that can inhibit the bacterial CRISPR-Cas system. In this study, we introduced a tentative inhibitory activity against a type I–C CRISPR-Cas system by determining the crystal structure of AcrIC5 from Pseudomonas delhiensis. Structural analysis revealed that AcrIC5 was composed of noble folds comprising two antiparallel sheets and three helices. Although AcrIC5 did not directly interact with either the type I–C cascade from Neisseria lactamia or the type I–F cascade from Pseudomonas aeruginosa in our analysis, a highly acidic surface feature indicated that AcrIC5 may be DNA mimic Acrs that directly binds to the target DNA binding site in type I–C cascade and inhibits the recruitment of the target DNA to this cascade.

A computational in silico approach to predict high-risk coding and non-coding SNPs of human PLCG1 gene.

PLCG1 gene is responsible for many T-cell lymphoma subtypes, including peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), adult T-cell leukemia/lymphoma along with other diseases. Missense mutations of this gene have already been found in patients of CTCL and AITL. The non-synonymous single nucleotide polymorphisms (nsSNPs) can alter the protein structure as well as its functions. In this study, probable deleterious and disease-related nsSNPs in PLCG1 were identified using SIFT, PROVEAN, PolyPhen-2, PhD-SNP, Pmut, and SNPS&GO tools. Further, their effect on protein stability was checked along with conservation and solvent accessibility analysis by I-mutant 2.0, MUpro, Consurf, and Netsurf 2.0 server. Some SNPs were finalized for structural analysis with PyMol and BIOVIA discovery studio visualizer. Out of the 16 nsSNPs which were found to be deleterious, ten nsSNPs had an effect on protein stability, and six mutations (L411P, R355C, G493D, R1158H, A401V and L455F) were predicted to be highly conserved. Among the six highly conserved mutations, four nsSNPs (R355C, A401V, L411P and L455F) were part of the catalytic domain. L411P, L455F and G493D made significant structural change in the protein structure. Two mutations-Y210C and R1158H had post-translational modification. In the 5’ and 3’ untranslated region, three SNPs, rs139043247, rs543804707, and rs62621919 showed possible miRNA target sites and DNA binding sites. This in silico analysis has provided a structured dataset of PLCG1 gene for further in vivo researches. With the limitation of computational study, it can still prove to be an asset for the identification and treatment of multiple diseases associated with the target gene.

Engineering Bioactive Dimeric Transcription Factor Analogs via Palladium Rebound Reagents.

Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression in 50% of all human cancers. Still, targeting Myc remains a challenge due to the lack of small molecule binding pockets in the tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic binding of the target DNA-binding site. We combined automated flow peptide chemistry with palladium(II) oxidative addition complexes (OACs) to engineer covalent protein dimers derived from the DNA-binding domains of Myc, Max, and Omomyc TF analogs. Palladium-mediated cross-coupling of synthesized protein monomers resulted in milligram quantities of seven different covalent homo- and heterodimers. The covalent helical dimers were found to bind DNA and exhibited improved thermal stability. Cell-based studies revealed the Max-Max covalent dimer is cell-penetrating and interfered with Myc-dependent gene transcription resulting in reduced cancer cell proliferation (EC50 of 6 μM in HeLa). RNA sequencing and gene analysis of extracted RNA from treated cancer cells confirmed that the covalent Max-Max homodimer interferes with Myc-dependent transcription. Flow chemistry, combined with palladium(II) OACs, has enabled a practical strategy to generate new bioactive compounds to inhibit tumor cell proliferation.

SWI/SNF Complex Mutations Promote Thyroid Tumor Progression and Insensitivity to Redifferentiation Therapies.

Mutations of subunits of the SWI/SNF chromatin remodeling complexes occur commonly in cancers of different lineages, including advanced thyroid cancers. Here we show that thyroid-specific loss of Arid1a, Arid2, or Smarcb1 in mouse BRAFV600E-mutant tumors promotes disease progression and decreased survival, associated with lesion-specific effects on chromatin accessibility and differentiation. As compared with normal thyrocytes, BRAFV600E-mutant mouse papillary thyroid cancers have decreased lineage transcription factor expression and accessibility to their target DNA binding sites, leading to impairment of thyroid-differentiated gene expression and radioiodine incorporation, which is rescued by MAPK inhibition. Loss of individual SWI/SNF subunits in BRAF tumors leads to a repressive chromatin state that cannot be reversed by MAPK pathway blockade, rendering them insensitive to its redifferentiation effects. Our results show that SWI/SNF complexes are central to the maintenance of differentiated function in thyroid cancers, and their loss confers radioiodine refractoriness and resistance to MAPK inhibitor-based redifferentiation therapies. SIGNIFICANCE: Reprogramming cancer differentiation confers therapeutic benefit in various disease contexts. Oncogenic BRAF silences genes required for radioiodine responsiveness in thyroid cancer. Mutations in SWI/SNF genes result in loss of chromatin accessibility at thyroid lineage specification genes in BRAF-mutant thyroid tumors, rendering them insensitive to the redifferentiation effects of MAPK blockade.This article is highlighted in the In This Issue feature, p. 995.

Structural and mechanistic insights into the CRISPR inhibition of AcrIF7.

The CRISPR–Cas system provides adaptive immunity for bacteria and archaea to combat invading phages and plasmids. Phages evolved anti-CRISPR (Acr) proteins to neutralize the host CRISPR–Cas immune system as a counter-defense mechanism. AcrIF7 in Pseudomonas aeruginosa prophages strongly inhibits the type I-F CRISPR–Cas system. Here, we determined the solution structure of AcrIF7 and identified its target, Cas8f of the Csy complex. AcrIF7 adopts a novel β1β2α1α2β3 fold and interacts with the target DNA binding site of Cas8f. Notably, AcrIF7 competes with AcrIF2 for the same binding interface on Cas8f without common structural motifs. AcrIF7 binding to Cas8f is driven mainly by electrostatic interactions that require position-specific surface charges. Our findings suggest that Acrs of divergent origin may have acquired specificity to a common target through convergent evolution of their surface charge configurations.

BET-seq: Binding energy topographies revealed by microfluidics and high-throughput sequencing.

Biophysical models of transcriptional regulation rely on energetic measurements of the binding affinities between transcription factors (TFs) and target DNA binding sites. Historically, assays capable of measuring TF-DNA binding affinities have been relatively low-throughput (measuring ~103 sequences in parallel) and have required significant specialized equipment, limiting their use to a handful of laboratories. Recently, we developed an experimental assay and analysis pipeline that allows measurement of binding energies between a single TF and up to 106 DNA species in a single experiment (Binding Energy Topography by sequencing, or BET-seq) (Le et al., 2018). BET-seq employs the Mechanically Induced Trapping of Molecular Interactions (MITOMI) platform to purify DNA bound to a TF at equilibrium followed by high coverage sequencing to reveal relative differences in binding energy for each sequence. While we have previously used BET-seq to refine the binding affinity landscapes surrounding high-affinity DNA consensus target sites, we anticipate this technique will be applied in future work toward measuring a wide variety of TF-DNA landscapes. Here, we provide detailed instructions and general considerations for DNA library design, performing BET-seq assays, and analyzing the resulting data.

Dynamic regulation of the angiotensinogen gene by DNA methylation, which is influenced by various stimuli experienced in daily life.

Angiotensinogen (AGT) has a central role in maintaining blood pressure and fluid balance. DNA methylation is an epigenomic modification maintaining a steady pattern in somatic cells. Herein we summarize the link between AGT regulation and DNA methylation. DNA methylation negatively regulates AGT expression and dynamically changes in response to continuous AGT promoter stimulation. High-salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT enhancer-binding protein-binding sites, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue. Salt-dependent hypertension may be partially affected by increased adipose AGT expression. Because angiotensin II is a well-established aldosterone-releasing hormone, stimulation of adipose AGT by aldosterone creates a positive feedback loop. This effect is pathologically associated with obesity-related hypertension, although it would be physiologically favorable for humans to efficiently retain their body fluid. The clear difference in DNA demethylation patterns between aldosterone and cortisol indicates a difference in the respective target DNA-binding sites between mineralocorticoid and glucocorticoid receptors in the AGT promoter. Stimulation-induced interactions between transcription factors and target DNA-binding sites trigger DNA demethylation. Dynamic changes in DNA methylation occur in relaxed chromatin regions both where transcription factors actively interact and where transcription is initiated. In contrast to rapid histone modifications, DNA demethylation and remethylation will progress relatively slowly over days or years. A wide variety of stimuli in daily life will continue to slowly and dynamically change DNA methylation patterns throughout life. Wise choices of beneficial stimuli will improve health.

Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors

BackgroundIn vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking.ResultsWe investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element.ConclusionsWe have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription.

Analysis of target DNA binding sites through NMR structural analysis of the MLV IN CTD and homology modeling of the CCD domains.

Analysis of target DNA binding sites through NMR structural analysis of the MLV IN CTD and homology modeling of the CCD domains.

Interactions between the R2R3-MYB transcription factor, AtMYB61, and target DNA binding sites.

Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.

A Novel Quick Method for Detecting Target DNA Binding Sites of Protein

DREB (dehydration responsive element binding protein) transcription factors play important roles in stress response and regulation of plant growth and development. In general, the traditional method of DNase I foot-printing is used to identify DNA binding of transcription factor. The DNA probes were labeled with isotope and then polyacrylamide gel electrophoresis was performed to separately digested labeled-DNA fragments, which had low resolution ratio and complexity in operation, and was not suitable for detecting interactions in large scales. To explore the transcriptional regulation mechanism of GmDREB3, the DNase I foot-printing method was improved. Combing with electrophoretic mobility shift assay (EMSA), DNA was labeled by fluorescence instead of isotope and the capillary electrophoresis instead of polyacrylamide gel electrophoresis was used to detect the digested DNA fragments and further identify DNA binding region of GmMYB1 in promoter of GmDREB3 gene. DNA binding site of GmMYB1 in GmDREB3 promoter was confirmed via the method of restriction enzyme digesting DNA. To further confirm binding element in GmDREB3 promoter, we used a putative DNA binding element of GmMYB1 to complete EMSA, indicating that GmMYB1 can bind target DNA element in vitro. In short, compared with classic DNase I foot-printing, the modified method is more rapid, accurate and reliable, and will be a high throughput method to identify interactions between proteins and target DNA sites.

A Novel Quick Method for Detecting Target DNA Binding Sites of Protein

A Novel Quick Method for Detecting Target DNA Binding Sites of Protein

Retroviral Integrases Promote Fraying of Viral DNA Ends

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.

Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

DNA linking number change induced by sequence-specific DNA-binding proteins

Sequence-specific DNA-binding proteins play a key role in many fundamental biological processes, such as transcription, DNA replication and recombination. Very often, these DNA-binding proteins introduce structural changes to the target DNA-binding sites including DNA bending, twisting or untwisting and wrapping, which in many cases induce a linking number change (ΔLk) to the DNA-binding site. Due to the lack of a feasible approach, ΔLk induced by sequence-specific DNA-binding proteins has not been fully explored. In this paper we successfully constructed a series of DNA plasmids that carry many tandem copies of a DNA-binding site for one sequence-specific DNA-binding protein, such as λ O, LacI, GalR, CRP and AraC. In this case, the protein-induced ΔLk was greatly amplified and can be measured experimentally. Indeed, not only were we able to simultaneously determine the protein-induced ΔLk and the DNA-binding constant for λ O and GalR, but also we demonstrated that the protein-induced ΔLk is an intrinsic property for these sequence-specific DNA-binding proteins. Our results also showed that protein-mediated DNA looping by AraC and LacI can induce a ΔLk to the plasmid DNA templates. Furthermore, we demonstrated that the protein-induced ΔLk does not correlate with the protein-induced DNA bending by the DNA-binding proteins.

Genome-Wide Analyses of Recombination Prone Regions Predict Role of DNA Structural Motif in Recombination

HapMap findings reveal surprisingly asymmetric distribution of recombinogenic regions. Short recombinogenic regions (hotspots) are interspersed between large relatively non-recombinogenic regions. This raises the interesting possibility of DNA sequence and/or other cis- elements as determinants of recombination. We hypothesized the involvement of non-canonical sequences that can result in local non-B DNA structures and tested this using the G-quadruplex DNA as a model. G-quadruplex or G4 DNA is a unique form of four-stranded non-B DNA structure that engages certain G-rich sequences, presence of such motifs has been noted within telomeres. In support of this hypothesis, genome-wide computational analyses presented here reveal enrichment of potential G4 (PG4) DNA forming sequences within 25618 human hotspots relative to 9290 coldspots (p<0.0001). Furthermore, co-occurrence of PG4 DNA within several short sequence elements that are associated with recombinogenic regions was found to be significantly more than randomly expected. Interestingly, analyses of more than 50 DNA binding factors revealed that co-occurrence of PG4 DNA with target DNA binding sites of transcription factors c-Rel, NF-kappa B (p50 and p65) and Evi-1 was significantly enriched in recombination-prone regions. These observations support involvement of G4 DNA in recombination, predicting a functional model that is consistent with duplex-strand separation induced by formation of G4 motifs in supercoiled DNA and/or when assisted by other cellular factors.

Camptothecin Induces Nuclear Export of Prohibitin Preferentially in Transformed Cells through a CRM-1-dependent Mechanism

Prohibitin is a growth-suppressive protein that has multiple functions in the nucleus and the mitochondria. Our earlier studies had shown that prohibitin represses the activity of E2F transcription factors while enhancing p53-mediated transcription. At the same time, prohibitin has been implicated in mediating the proper folding of mitochondrial proteins. We had found that treatment of cells with camptothecin, a topoisomerase 1 inhibitor, led to the export of prohibitin and p53 from the nucleus to the mitochondria. Here we show that the camptothecin-induced export of prohibitin occurs preferentially in transformed cell lines, but not in untransformed or primary cells. Cells that did not display the translocation of prohibitin were refractive to the apoptotic effects of camptothecin. The translocation was mediated by a putative nuclear export signal at the C-terminal region of prohibitin; fusion of the nuclear export signal (NES) of prohibitin to green fluorescence protein led to its export from the nucleus. Leptomycin B could inhibit the nuclear export of prohibitin showing that it was a CRM-1-dependent event driven by Ran GTPase. Confirming this, prohibitin was found to physically interact with CRM-1, and this interaction was significantly higher in transformed cells. Delivery of a peptide corresponding to the NES of prohibitin prevented the export of prohibitin to cytoplasm and protected cells from apoptosis. These results suggest that the regulated translocation of prohibitin from the nucleus to the mitochondria facilitates its pleiotropic functions and might contribute to its anti-proliferative and tumor suppressive properties.

Novel Target Sequences for Pax-6 in the Brain-specific Activating Regions of the Rat Aldolase C Gene

Upstream activating sequences of the rat aldolase C gene are shown here to confer brain-specific expression in transgenic mice. In addition to binding sites described previously for the brain-expressed POU proteins Brn-1 and Brn-2 (Skala, H., Porteu, A., Thomas, M., Szajnert, M. F., Okazawa, H., Kahn, A., and Phan-Dinh-Tuy, F. (1998) J. Biol. Chem. 273, 31806-31814), we have identified two novel DNA elements critical for an interaction with a brain-specific, high affinity DNA-binding protein. Characterization of this binding protein showed it to be sensitive to thiol oxidation and stable to heat at 100 degrees C. This protein was purified on the basis of its thermostability and its selective adsorption to streptavidin magnetic particles via a biotinylated multimer of its target DNA binding site. Liquid chromatography coupled to tandem mass spectrometry analysis, binding competition with consensus oligonucleotides, and antibody supershift assays led to its identification as the homeodomain paired protein Pax-6. This result suggests that the brain-specific aldolase C gene could constitute a new target for the transcription factor Pax-6, which is implicated increasingly in neurogenesis.