Deletion of the Chd6 exon 12 affects motor coordination

Abstract

Members of the CHD protein family play key roles in gene regulation through ATP-dependent chromatin remodeling. This is facilitated by chromodomains that bind histone tails, and by the SWI2/SNF2-like ATPase/helicase domain that remodels chromatin by moving histones. Chd6 is ubiquitously expressed in both mouse and human, with the highest levels of expression in the brain. The Chd6 gene contains 37 exons, of which exons 12-19 encode the highly conserved ATPase domain. To determine the biological role of Chd6, we generated mouse lines with a deletion of exon 12. Chd6 without exon 12 is expressed at normal levels in mice, and Chd6 Exon 12 -/- mice are viable, fertile, and exhibit no obvious morphological or pathological phenotype. Chd6 Exon 12 -/- mice lack coordination as revealed by sensorimotor analysis. Further behavioral testing revealed that the coordination impairment was not due to muscle weakness or bradykinesia. Histological analysis of brain morphology revealed no differences between Chd6 Exon 12 -/- mice and wild-type (WT) controls. The location of CHD6 on human chromosome 20q12 is overlapped by the linkage map regions of several human ataxias, including autosomal recessive infantile cerebellar ataxia (SCAR6), a nonprogressive cerebrospinal ataxia. The genomic location, expression pattern, and ataxic phenotype of Chd6 Exon 12 -/- mice indicate that mutations within CHD6 may be responsible for one of these ataxias.

Fig. 1

Generation of a Chd6 Exon 12 −/− mouse line. a Domain architecture of the Chd6 protein. The SWI2/SNF2 helicase domain contains seven conserved motifs, including motif I (ATP-binding region) and Ia (DNA binding), which are encoded by exon 12 of mChd6. b Gene targeting strategy: A portion of the mChd6 gene structure is shown, including exons 11-14 (boxes), and the HpaI restriction enzyme sites used for Southern blot analysis. The targeting vector “lox-frt-Chd6-ATPase” was designed to enable the deletion of mChd6 exon 12 by loxP site-specific recombination. The loxP sites (white triangles) and direction of transcription of the Neomycin-resistance gene (Neo) are indicated (arrow). The Frt-flanked Neo-cassette was removed by flp-mediated, site-specific recombination (Frt recombination elements are black triangles). The structure of the desired allele (Δ-exon 12) was obtained after Cre-mediated excision of the floxed exon 12. The Southern probe and the sizes of HpaI restriction fragments detected by this probe in wild-type (WT) and targeted DNA are shown. c Southern analysis of tail clip genomic DNA: The WT Chd6 allele (3.5 kb band) is distinguishable from the floxed conditional Chd6 allele (3.7 kb) and the Chd6 Exon 12 −/− allele (2.9 kb). d Western analysis: Detection of WT Chd6 (305 kDa) and mutant Δ479-558Chd6 (296 kDa) in lung extracts from WT and Chd6 Exon 12 −/− animals, respectively. The small difference in molecular weights between WT and mutant Chd6 could not be detected by this assay

Fig. 2

Exon 12 −/− mice are impaired in the ledge test compared to WT mice. Mice were placed on the 0.75 cm-wide ledge of a mouse cage and observed for 3 min. Mice were scored as having performed one of four behaviors: remaining stationary on the ledge, falling off the ledge, climbing into the cage in a controlled manner, or progressing along the ledge. a Average distance progressed by mixed lineage WT and Exon 12 −/− mice. b Percent of mixed lineage mice to perform each behavior. c Average distance crossed by 129 Sv lineage Exon 12 −/− and WT mice. d Percent of 129 Sv lineage mice to perform each behavior. Data are represented as a percentage of the total number of mice tested. Error bars are ±SEM, significant difference is indicated by * (P < 0.05 for the same behavior compared between WT and Exon 12 −/− of the same lineage)

Fig. 3

Exon 12 −/− mice have impaired rotorod performance. Mice were tested for 3 days, with three trials per day for a total of nine trials. a Average rpm reached for individual trials with mixed lineage mice. b Average rpm reached for individual trials with 129 Sv lineage mice. For both lineages, in each trial the WT results are significantly different from the Exon 12 −/− results (P < 0.01). c Average rpm for all trials with mixed lineage mice. d Average rpm for all trials with 129 Sv lineage mice. e Average rpm for all trials with 129 Sv lineage mice tested at week 20. Error bars are ±SEM, Significance of P < 0.01 is indicated by *

Fig. 4

Behavior analysis of Exon 12 −/− mice demonstrates further evidence that the sensorimotor defect is not due to muscle weakness or bradykinesis. The average for all trials is indicated for mixed lineage and 129 Sv lineage mice were tested on the wire hang (a, b), pole test (c-f), beam walking (g, h), and olfaction (i). Black bars indicate WT mice values; gray bars indicate Exon 12 −/− mice values. Error bars are ±SEM. For beam-walking and olfaction test data, the number of mice to accomplish the task is represented as a percent of the total number tested. Significance of P < 0.01 is indicated by *

Fig. 5

Behavioral analysis of mixed-lineage Exon 12 −/− mice indicates normal spatial learning and mild anxiety. Mixed-lineage mice were tested for anxiety in the open-field maze and spatial learning and anxiety in the Barnes maze. Open-field measurements included (a) center-to-wall time ratio, (b) total distance traveled in centimeters, (c) the amount of ambulatory time. The Barnes maze measurements included (d) the percentage of mice that entered the goal box, (e) the percentage of mice finding and investigating the goal, (f) the average time to find and investigate the goal for all trials, and (g) the average time to find the goal for each trial. For Barnes maze data, the number of mice to accomplish the task is represented as a percent of the total number tested. Error bars are ±SEM. Significance of P < 0.05 is indicated by *

Fig. 6

Stimulation of the Nrf2 stress response pathway does not require the Chd6 exon 12. a Treatment of MEF cells with Nrf2 pathway stimulants CDDO or Sulforaphane increased the expression of Nqo1 in Exon 12 −/− MEF cells and WT MEFs to similar levels. qPCR analysis of the level of Nqo1 normalized to β-actin expression levels. b Increased expression of Nrf2 target genes results in protection from ROS challenge at similar levels in Exon 12 −/− MEF cells and WT MEF cells. MEFs pretreated with control or CDDO were labeled with DCFDA and challenged with tBHQ. The level of DCFDA fluorescence was measured by FACScan, and the change in mean fluorescence intensity was measured as treated over untreated levels. The average of six experiments is shown