An experimental study of the effects of SNPs in the TATA boxes of the <i>GRIN1, ASCL3</i> and <i>NOS1</i> genes on interactions with the TATA-binding protein

Abstract

The GRIN1, ASCL3, and NOS1 genes are associated with various phenotypes of neuropsychiatric disorders. For instance, these genes contribute to the development of schizophrenia, Alzheimer’s and Parkinson’s diseases, and epilepsy. These genes are also associated with various cancers. For example, ASCL3 is overexpressed in breast cancer, and NOS1, in ovarian cancer cell lines. Based on our findings and literature data, we had previously obtained results suggesting that the single-nucleotide polymorphisms (SNPs) that disrupt erythropoiesis are highly likely to be associated with cognitive and neuropsychiatric disorders in humans. In the present work, using SNP_TATA_Z-tester, we investigated the influence of unannotated SNPs in the TATA boxes of the promoters of the GRIN1, ASCL3, and NOS1 genes (which are involved in neuropsychiatric disorders and cancers) on the interaction of the TATA boxes with the TATA-binding protein (TBP). Double-stranded oligodeoxyribonucleotides identical to the TATA-containing promoter regions of the GRIN1, ASCL3, and NOS1 genes (reference and minor alleles) and recombinant human TBP were employed to study in vitro (by an electrophoretic mobility shift assay) kinetic characteristics of the formation of TBP–TATA complexes and their affinity. It was found, for example, that allele A of rs1402667001 in the GRIN1 promoter increases TBP–TATA affinity 1.4-fold, whereas allele C in the TATA box of the ASCL3 promoter decreases the affinity 1.4-fold. The lifetime of the complexes in both cases decreased by ~20 % due to changes in the rates of association and dissociation of the complexes (ka and kd, respectively). Our experimental results are consistent with the literature showing GRIN1 underexpression in schizophrenic disorders as well as an increased risk of cervical, bladder, and kidney cancers and lymphoma during ASCL3 underexpression. The effect of allele A of the –27G&gt;A SNP (rs1195040887) in the NOS1 promoter is suggestive of an increased risk of ischemic damage to the brain in carriers. A comparison of experimental TBP–TATA affinity values (KD) of wild-type and minor alleles with predicted ones showed that the data correlate well (linear correlation coefficient r = 0.94, p &lt; 0.01).

Plant_SNP_TATA_Z-Tester: A Web Service That Unequivocally Estimates the Impact of Proximal Promoter Mutations on Plant Gene Expression

“…Within Tikhonov’s regularization [ 61 ], this correlation ( Figure 2 a) characterizes a similarity between the ill-posed inverse problem of evaluating the transcriptional outcome of mutations in plant proximal promoters ( Figure 1 ) and the analogous well-posed problem for humans, which has already been solved using our public Web service Human_SNP_TATA_Z-tester [ 28 ] (see in-depth description in the Supplementary Materials [ 18 , 28 , 29 , 30 , 31 , 32 , 33 , 35 , 36 , 37 , 38 , 39 , 41 , 42 , 62 , 63 , 64 , 65 , 66 , 67 ]). With this in mind, Figure 2 depicts how we adapted it step-by-step for comparing between wildtype and mutant variants of the plant promoter DNA sequences under study in their effects on gene expression, i.e., our new Web service Plant_SNP_TATA_Z-tester created in this work.…”

“…We processed DNA sequences by means of our public Web service Plant_SNP_TATA_Z-tester (e.g., Figure 3 a and Figure 6 ) created in this work, as depicted in Figure 2 . To this end, as its prototype, we used our previously developed Web service Human_SNP_TATA_Z-tester [ 28 ] (see description [ 28 , 29 , 30 , 31 , 32 , 33 , 35 , 36 , 37 , 38 , 39 , 41 , 42 , 62 , 63 , 64 , 65 , 66 , 67 ] in the Supplementary Materials ), and we expanded it only with Equation (2) in line with Tikhonov’s regularization [ 61 ].…”

“…( a ) The interchangeability of hsTBP ( x -axis) and atTBP ( y -axis) for transcription in vitro [ 45 ]. ( b ) Using Human_SNP_TATA_Z-tester [ 28 ] (see in-depth description in the Supplementary Materials [ 18 , 28 , 29 , 30 , 31 , 32 , 33 , 35 , 36 , 37 , 38 , 39 , 41 , 42 , 62 , 63 , 64 , 65 , 66 , 67 ]), we estimated the −ln(K D; hsTBP ) values of hsTBP affinity for the 90 bp DNA sequence of either the wildtype or the mutant variant of the Agrobacterium tumefaciens T-DNA TC7 promoter ( AtTC7 ) [ 54 ] from [ 45 ], as shown by arrow 1. ( c ) Statistically significant linear regression of the predicted K D;hsTBP values in relation to the F hsTBP values measured using hsTBP [ 45 ], as expressed by Equation (1) written above the dashed line.…”

“…The supporting information can be downloaded at . References [ 18 , 28 , 29 , 30 , 31 , 32 , 33 , 35 , 36 , 37 , 38 , 39 , 41 , 42 , 62 , 63 , 64 , 65 , 66 , 67 ] are cited in the Supplementary Materials.…”