Histone deacetylase inhibition rescues structural and functional brain deficits in a mouse model of Kabuki syndrome

Abstract

Kabuki syndrome is caused by haploinsufficiency for either of two genes that promote the opening of chromatin. If an imbalance between open and closed chromatin is central to the pathogenesis of Kabuki syndrome, agents that promote chromatin opening might have therapeutic potential. We have characterized a mouse model of Kabuki syndrome with a heterozygous deletion in the gene encoding the lysine-specific methyltransferase 2D (Kmt2d), leading to impairment of methyltransferase function. In vitro reporter alleles demonstrated a reduction in histone 4 acetylation and histone 3 lysine 4 trimethylation (H3K4me3) activity in mouse embryonic fibroblasts from Kmt2d(+/βGeo) mice. These activities were normalized in response to AR-42, a histone deacetylase inhibitor. In vivo, deficiency of H3K4me3 in the dentate gyrus granule cell layer of Kmt2d(+/βGeo) mice correlated with reduced neurogenesis and hippocampal memory defects. These abnormalities improved upon postnatal treatment with AR-42. Our work suggests that a reversible deficiency in postnatal neurogenesis underlies intellectual disability in Kabuki syndrome.

Fig. 1. Hippocampal memory defects in a Kmt2d^+/βGeo mouse model of Kabuki syndrome

(A) Domain organization of KMT2D in human and mouse, with the relative position of the H3K4 methyltransferase SET domain indicated in red and other domains by additional colors. The human and murine chromosomal assignment (Chr) is shown. (B) The Kmt2d^βGeo targeting event introduced a β-Geo cassette including a strong splice acceptor (SA) sequence and a 3′ cleavage and polyadenylation signal (pA) into intron 50 of Kmt2d on mouse chromosome 15 (fig. S1A). (C) Real-time PCR using primers specific for exons 20 or 52 of Kmt2d (arrows) confirmed a substantial reduction (∼50%) in mRNA corresponding to sequences distal to the β-Geo insertion site when compared to proximal sequences in Kmt2d^+/βGeo mice, in comparison to Kmt2d^+/+ littermates. Results reflected three technical replicates for each of three Kmt2d^+/+ and two Kmt2d^+/βGeo mice. (D) ChIP-seq revealed a genome-wide deficiency of H3K4me3 in cells from Kmt2d^+/βGeomice when compared to cells from Kmt2d^+/+ littermates. A positive value indicates a higher locus-specific peak in Kmt2d^+/βGeo mice. Each point corresponds to a genomic location with a peak in at least one sample. Significantly differentially bound loci are red, whereas others are gray. (E) There was no difference in positional preference between genotypes during the habituation phase [identical objects (L/R)] of the novel object recognition test. Kmt2d^+/βGeo mice spent less time with a novel object (L) and more with a habituated object (R) compared to Kmt2d^+/+ littermates. Kmt2d^+/+ littermates also demonstrated significant improvement from habituation phase [novel object (L)], whereas Kmt2d^+/βGeo mice did not. n = 13 (+/+), n = 10 (+/βGeo). (F) Kmt2d^+/βGeo mice showed a reduced frequency in platform zone crossings during the probe trial phase of Morris water maze testing. n = 48 (+/+), n = 32 (+/βGeo). *P < 0.05; ^†P < 0.005; ^††P < 0.001, t test.

Fig. 2. A global deficiency of H3K4me3in the dentate gyrus associated with reduced granule cell layer volume and neurogenesis in Kmt2d^+/βGeo mice

(A) Immunofluorescence revealed intense expression of KMT2D (red signal) in the dentate gyrus granule cell and pyramidal layers of Kmt2d^+/+ mice. (B) Immunofluorescence for H3K4me3 (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) in the granule cell layer of Kmt2d^+/βGeo mice and Kmt2d^+/+littermates. (C) Quantification revealed a reduced H3K4me3/DAPI signal intensity ratio within the granule cell layer of Kmt2d^+/βGeo mice compared to Kmt2d^+/+ littermates. n = 9 (+/+), n = 5 (+/βGeo). (D and E) Calculation of granule cell layer area (red outline) in every sixth brain slice allowed the demonstration of reduced granule cell layer volume in Kmt2d^+/βGeo mice compared to Kmt2d^+/+ littermates. n = 4 (+/+), n = 5 (+/βGeo). (F and G) Immunofluorescence revealed reduced representation of cells positive for DCX, a marker for neurogenesis, in the granule cell layer of Kmt2d^+/βGeo mice compared to Kmt2d^+/+ littermates. n = 4 (+/+), n = 4 (+/βGeo). *P < 0.05; ^††P < 0.001, t test.

Fig. 3. H3K4me3 epigenetic reporter allele demonstrated decreased activity in Kmt2d^+/βGeocells

(A) Domain organization encoded by the H4ac and H3K4me3 reporter alleles. The H4ac indicator includes H4 (lysine positions indicated), the C- and N-terminal halves of enhanced GFP (EGFP) separated by a short linker (L), the TAFII binding domain (BD), and a repetitive nuclear localization signal (NLS). The H3K4me3 indicator includes the H3 and the TAF3-PHD. (B) Recognition of the histone tail mark by the relevant histone reader leads to reconstitution of GFP structure and function (fluorescence). (C and D) The acetylation indicator demonstrated increasing fluorescence with increasing amounts of the HDACi SAHA. (E) Activity of the H4ac indicator was lost upon mutagenesis of all potential acetylation sites from lysine to arginine. (F) The H3K4me3 indicator demonstrated a dose-dependent response to the HDACi AR-42 with decreased cell numbers at higher doses (red line). (G) Activity was greatly reduced upon mutagenesis of K4 in the H3 tail and D890A/ W891A and M882A in the reader pocket. (H) The H3K4me3 indicator showed reduced activity in murine embryonic fibroblasts (MEFs) derived from Kmt2d^+/βGeomice compared to Kmt2d^+/+ littermates. Both genotypes showed a dose-dependent response to AR-42, with Kmt2d^+/βGeo MEFs achieving untreated wild-type levels of activity at a dose of 5 µM. n = 3 (+/+), n = 3 (+/βGeo), biological replicates for each dose. ^**P < 0.01; ^††P < 0.001, t test.

Fig. 4. In vivo effects of AR-42

(A to D) One- to 2-month-old mice of both genotypes showed an increase in H3K4me3 (A and B) (n = 5 to 6 per group) associated with a dose-dependent increase in neurogenesis in Kmt2d^+/βGeo mice (C and D) (monitored by normalized DCX expression) (n = 4 to 6 per group) upon treatment with the HDACi AR-42. There was no difference in either H3K4me3 or neurogenesis between Kmt2d^+/βGeo and Kmt2d^+/+ animals at a dose of 10 mg/kg per day. (E) The genome-wide deficiency of H3K4me3 seen in Kmt2d^+/βGeo mice was improved upon treatment with AR-42 (10 mg/kg per day). (F) The reduced frequency of platform crossing seen during Morris water maze testing of Kmt2d^+/βGeo mice was normalized upon treatment with AR-42 (10 mg/kg per day) [n = 48 (+/+, no treatment), n = 32 (+/βGeo, no treatment), n = 14 (+/+, 10 mg/kg per day AR-42), n = 9 (+/βGeo, 10 mg/kg per day AR-42)]. *P < 0.05; **P < 0.01; ^†P < 0.005; ^††P < 0.001, t test.