A dyslexia-associated variant in DCDC2 changes gene expression

Abstract

Reading disability (RD) or dyslexia is a common neurogenetic disorder. Two genes, KIAA0319 and DCDC2, have been identified by association studies of the DYX2 locus on 6p21.3. We previously identified a 2445 bp deletion, and a compound STR within the deleted region (BV677278), in intron 2 of DCDC2. The deletion and several alleles of the STR are strongly associated with RD (P = 0.00002). In this study we investigated whether BV677278 is a regulatory region for DCDC2 by electrophoretic mobility shift and luciferase reporter assays. We show that oligonucleotide probes from the STR bind nuclear protein from human brain, and that alleles of the STR have a range of DCDC2-specific enhancer activities. Five alleles displayed strong enhancer activity and increased gene expression, while allele 1 showed no enhancer activity. These studies suggest that the association of BV677278 with RD reflects a role as a modifier of DCDC2 expression.

Fig. 1

EMSA detects oligonucleotide probes from BV677278 that bind protein in human brain nuclear lysate. a Sequence of EMSA probes relative to the BV677278 repeat sequence. b EMSA detects oligonucleotide probes from BV677278 that bind protein in human brain nuclear lysate. Lanes 1–3 are the Oct2A positive control, which contains a specific, well-characterized recognition sequence present in the probe. EMSA3 (lanes 4–6) and EMSA4 (lanes 7–9) are non-overlapping double-stranded, 20-mer oligonucleotide probes with sequence extracted from BV677278 (a). Lanes 1, 4, and 7 are baseline mobilities of labeled probes without human brain nuclear lysate. Lanes 2, 5, and 8 show probes bound to a protein in human brain nuclear lysate and the consequent electrophoretic mobility shifts. Lanes 3, 6, and 9 show that binding can be competed with identical “cold” unlabeled probe

Fig. 2

The EMSA3 interaction is specific. a Summary of the labeled and unlabeled probes used in (b) and (c). Labeled EMSA3 probe was used in all lanes, in both (b) and (c). Competition assays were done with a 200-fold molar excess of unlabeled EMSA3 probe in lane 3, of unlabeled EMSA3-Scram1 probe in lane 4, of unlabeled EMSA3-Scram2 probe in lane 5, of unlabled EBNA1 probe in lane 6, and of unlabeled EMSA4 probe in lane 7 (b only). b The EMSA3 interaction is specific in human brain nuclear lysate. The shift observed in lane 2 (arrow) vanishes when a 200-fold molar excess of unlabeled EMSA3 probe is added to the binding reaction (lane 3). However, a 200-fold molar excess of either one of the two unlabeled scrambled EMSA3 probes (lanes 4 and 5) does not compete with the labeled probe, nor does a 200-fold molar excess of unlabeled EBNA1 probe (lane 6) or unlabeled EMSA4 probe (lane 7). c The EMSA3 interaction is present and specific in the Raji cell line (human Burkitt lymphoma) and in the P19 cell line (mouse embryonal carcinoma, undifferentiated). In both of these cell lines, the behavior of the EMSA3 shift (arrow) is similar to that observed in human brain nuclear lysate (b)

Fig. 3

BV277278 enhancer activity with DCDC2-specific promoter. a Schematic of pGL3-Basic vector constructs. LUC+ is the luciferase gene. BV677278(+) means the orientation of the sequence is the same as that of the DCDC2 transcript. BV677278(−) means the orientation of the sequence is reversed with respect to the DCDC2 transcript. “600” in “B/600” is the 600 bp 5-prime of the ATG start codon of DCDC2, serving as the DCDC2-specific promoter. “B” in “B/600” stands for Basic vector from Promega. “A” in “B/600/A” stands for alleles of BV677278. b BV677278 enhancer activity with DCDC2-specific promoter. The X-axis shows the names of the constructs. The Y-axis shows signal intensity, normalized to co-transfected Renilla luciferase, and averaged among three replicates. B/EA1(−) and B/EA1(+): Basic vector with BV677278 allele 1 in the enhancer position, in the + or − orientation, respectively. Basic/600: Basic vector with DCDC2-specific promoter at the 5′ position, upstream of luciferase reporter gene (conventional promoter position). B/600/A1(−) and B/600/A1(+): Basic/600 construct with allele 1 of BV677278 3-prime of the luciferase gene (conventional enhancer position). This nomenclature continues for alleles 3, 4, 5, 6, and 10. SV40 Promoter: Promoter vector from Promega with heterologous SV40 promoter. (−) and (+) indicate the orientation of the BV677278 alleles relative to DCDC2’s orientation along 6p22; (+) denotes the same orientation as the DCDC2 transcript. Bars represent standard deviation for triplicate experiments with each construct. Asterisks represent a statistically significant difference in luciferase signal intensity relative to the Basic/600 construct (P < 0.01), after correction for multiple testing