Abstract
Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.
Author summary
The 17q21.31 deletion syndrome, also named Koolen-de Vries syndrome (KdVS), is a rare copy number variants associated in humans with intellectual disability, friendly behavior, congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 region or by variants in the KANSL1 gene in human. The reciprocal 17q21.31 microduplication syndrome is not so well characterized. To investigate the pathophysiology of the syndromes, we studied the deletion, the duplication of the syntenic region and a heterozygous Kansl1 mutant in the mouse. We found affected morphology and cognition, similar to human condition, with genes controlling chromatin organization, synaptic transmission and neurogenesis dysregulated in the hippocampus of KdVS models. In addition we found that synaptic transmission was altered in KdVS mice. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS and extend substantially our knowledge about altered biological processes. Nevertheless, phenotypic differences between deletion and Kansl1+/- models suggested roles of other genes affected by the 17q21.31 deletion.
Introduction
The Koolen-de Vries syndrome (KdVS) has a prevalence estimated at 1/55,000 based upon CNV studies[1–3] and was primary described as a consequence of the 17q21.31 microdeletion. Patients with KdVS present characteristic facial dysmorphisms[4] and clinical features including intellectual disability, friendly behavior, hypotonia, and several brain anomalies[5–7]. Microdeletions and microduplications of genomic fragments in the 17q21.3 region ranging from 400 to 800kb have been found in individuals with intellectual disability[6, 8]; these genomic fragments include five protein-coding genes: CRHR1, SPPL2C, MAPT, STH, and KANSL1. The reciprocal duplication is much more rare than the deletion. To our knowledge, only eight patients have been described in the literature[8–12]. The symptoms are heterogeneous and include craniofacial malformations, microcephaly, psychomotor delay, poor verbal and motor skills, and reduced social interaction[8]. Two cases out of eight have been diagnosed with autism spectrum disorder (ASD).
Loss-of-function mutations and atypical deletions restricted to the KANSL1 gene, encoding the KAT8 Regulatory NSL Complex Subunit 1, have been found in several KdVS patients. Interestingly, phenotypic comparison of both the 17q21.31 microdeletion and KANSL1 heterozygous mutation patients show similar clinical severity, implicating that haploinsufficiency of KANSL1 is sufficient to cause the full manifestation of KdVS phenotype[3, 13–15]. KANSL1 is a member of the evolutionarily conserved nonspecific lethal (NSL) complex that controls various cellular functions, including transcription regulation and stem cell identity maintenance and reprogramming[16, 17]. The NSL complex contains the histone acetyltransferase MOF (males absent on the first) encoded by KAT8 which acetylates histone H4 on lysine 16 (H4K16) and with lower efficiency on lysines 5 and 8 (H4K5 and H4K8, respectively) to facilitate transcriptional activation[18, 19]. Recent studies in flies have shown that KANSL1 acts as a scaffold protein interacting with four NSL subunits including WDR5 which plays a critical role in assembling distinct histone-modifying complexes with different epigenetic regulatory functions[20].
Genes within the human 17q21.31 region are highly conserved on mouse chromosome 11E1. Crhr1, Sppl2c, Mapt and Kansl1 orthologs have all been found in the same orientation as in the human H1 haplotype. To investigate the pathophysiology of KdVS and microduplication syndrome, we generated first a mutant mice bearing deletion (Del/+), and duplication (Dup/+) of the 17q21.31-homologous Arf2-Kansl1 genetic interval and looked for phenotypes related to the human condition. We studied behavior, cognition, craniofacial and brain morphology of single Deletion carried compared to wild type and pseudo-disomic (Del/Dup) controls. Then we compared these data to results obtained with mutant mice for Kansl1 and extended our analysis to gene expression. We found a large phenotypic overlap with altered molecular mechanisms controlling the hippocampus synaptic response.
Results
Deletion of the Arf2–Kansl1 genetic interval impairs viability and alters post-natal development with reduced size and body weight
Del/+ and Dup/+ mice were generated on the C57BL/6N (B6N) genetic background (see supplementary information; Fig 1). In comparison with wild-type (wt) littermates, he Del allele frequency was reduced significantly while the transmission of the Dup allele and the Del/Dup carriers were not affected (Table 1), thus demonstrating that lethality is associated with the deletion on the B6N background.
Table 1. Transmission rates of the Arf2-Kansl1 deletion (Del) and duplication (Dup) alleles recorded at weaning.
Crossing | Genotype | Observed number | Observed ratio | χ2 | p |
---|---|---|---|---|---|
Del/+ × wt | wt | 102 | 63.4% | 5.74 | 0.02 |
Del/+ | 59 | 36.6% | |||
Dup/+ × wt | 2n | 186 | 55.0% | 1.71 | 0.20 |
Dup/+ | 152 | 45.0% | |||
Del/+ × Dup/+ | wt | 71 | 37.6% | 11.9 | 5.5x10−04 |
Del/+ | 14 | 7.4% | 23.4 | 1.3x10−06 | |
Dup/+ | 48 | 25.4% | 0.01 | 0.91 | |
Del/Dup | 56 | 29.6% | 1.62 | 0.20 |
We generated and characterized a compound Del-Dup cohort with littermates carrying four genotypes: Del/+, wt, Del/Dup, and Dup/+. First, we followed general parameters. Compared to wt mice, Dup/+ mice were underweight, whereas Del/+ and Del/Dup mice were not (Fig 1d). At 20 weeks of age. Del/+, Dup/+, and Del/Dup animals showed significantly reduced body length compared to wt littermates (Fig 1e). In comparison with wt, Del/+ littermates showed lower adiposity levels (Fig 1f) at the same age. Nevertheless, we did not detect any notable differences in feeding behavior between mutant and wt animals during a circadian activity test (S1 Fig).
Deletion of the Arf2–Kansl1 region decreases circadian activity and alters recognition and associative memory
Patients with 17q21.31 CNVs have impaired intellectual and adaptive functioning[4, 5]. As a primary experiment, we looked at the activity and the Del/+ and Dup/+ mice displayed a normal circadian pattern (S1 Fig). However, in comparison with wt, Del/+ mice showed reduced spontaneous locomotor activity (Fig 2a) as well as reduced rearing behavior during the light phase (Fig 2a). In the open field, no differences in exploration, locomotor activity, rearing behavior, or time spent in the center of the area were observed between mutant mice and wt littermates (Fig 2b; S1 Table; S2a–S2d Fig). In the elevated plus maze, Del/+ and Dup/+ mice explored the same number of arms and spent similar periods of time in open arms as those observed for wt (S1 Table). We evaluated motor coordination and learning using the rotarod test but no differences were observed between mutant Del/+ and Dup/+ mice, and wt mice (S2a–S2c Fig). Similarly, for the grip test, no differences in the muscular strength were observed between mutant mice and wt littermates (S2d Fig).
Spatial learning and memory was assessed in the Morris water maze, but similar acquisition and retention were observed in wt and mutant animals (S3 Fig). In the Y maze test, we found similar working memory parameters in wt and mutant animals (S2 Table). Next, we evaluated our models in the novel object recognition test. During the acquisition session, mice of all genotypes spent an equal amount of time exploring the sample object (S2 Table). After 3h of retention delay, Del/+ mice are able to differentiate the novel versus the familiar object but show object discrimination deficits compared to wt whereas Dup/+ and Del/Dup mice showed similar memory capacities (Fig 2c) compared to control.
Associative memory was evaluated with the fear conditioning test. No significant differences in the baseline and post-shock freezing levels were observed between mice of all genotypes (S2 Table). In the context session, Dup/+ mice displayed a higher level of freezing with significant differences in the last 2 min of the test (F(3,56) = 6.399, P < 0.001; Dup/+ vs wt: P = 0.027; Fig 2d). The cued session was performed 5 h after the context session. During the presentation of the conditioning cue, all genotypes demonstrated higher freezing incidence (Fig 2d). During the second 2-min long cue, Del/+ and Dup/+ animals showed lower and higher levels of freezing, respectively, in comparison with wt littermates (H(3, 56) = 20.609, P < 0.001; Del/+ vs wt: P = 0.002, Dup/+ vs wt: P = 0.036). To challenge cued fear extinction in those animals, we used another fear conditioning/extinction protocol with reinforced conditioned stimuli (CS) and long-term follow-up; we separated cohorts for the Del/+ and for Dup/+ with their own littermate controls. This revealed opposite effects on the capacity of Del/+ and Dup/+ animals to extinguish the fear response (Fig 2e). Dup/+ animals presented with higher levels of freezing as compared to their wt littermates suggesting that the fear trace persists in those animals. In contrast, the freezing levels of Del/+ animals were globally lower compared to those of their wt littermates, indicating that the fear memory trace is less stable in those animals.
Deletion of the Arf2–Kansl1 region increases sociability
To determine whether sociability traits were evident in our mouse models, we first used the three-chamber sociability test. Familiar and unfamiliar animals were of similar sex and genetic background than experimental animals but were of younger age in order to avoid aggressiveness. In the first phase of the test, the social interest session, Del/+ mice spent relatively more time than wt littermates exploring the unfamiliar mouse (F(3,51) = 3.447, P = 0.023; Del/+ vs wt: P = 0.017; Fig 2f, Session 1; S4c Fig). In the second phase, the social discrimination session, Del/+ mice interacted with familiar and novel congeners for longer periods compared to wt littermates as observed in the first session (F(3,51) = 6.034, P = 0.001; Del/+ vs wt: P = 0.002; Fig 2f, Session 2; S4d Fig). Nevertheless no differences in the percentage of time spent to explore the novel congener versus the familiar congener were observed between mutant mice and wt littermates (Fig 2e).
Rearrangements of the Arf2–Kansl1 region induce changes in craniofacial features and brain architecture
We studied the influence of 17q21.31-homologous CNVs on the mouse craniofacial structure. We analysed computed tomography (CT) cranial scans of animal heads combined with 3D reconstruction of skull images using 39 cranial landmarks (S5a Fig). Separate cohorts of Del/+ and Dup/+ females were used for the Euclidean distance matrix analysis[21] and MorphoJ analysis[22]. Skull size in Del/+ (T = 0.115; S5b Fig) and Dup/+ animals (T = 0.115; S5d Fig) was similar to that of wt littermates. The skull shape measurements were nominally altered in Del/+ (Z = 0.077; S5c Fig) and Dup/+ animals (Z = 0.052; S5e Fig) compared to those in wt littermates. Principal component analysis helped to identify change in the skull shape in Del/+ and the Dup/+ versus control littermates with the three main components (PC1 and PC2; S5f Fig) accounting for 59.2% of the total variance. Differences are more pronounced in the skull shape of the Del/+ mice than in wt controls with a predominantly shorter nasal bone and a broadening of the face at the level of the zygomatic spine and squamosal junction.
In addition to neuropsychiatric features, over 50% of patients with 17q21.31 microdeletion also present with various brain structure changes[4, 5, 15]. Furthermore, 50% of patients with the 17q21.31 microdeletion present with microcephaly[8]. To identify potential morphological alterations of brain regions, we analyzed the brain structure of 8 Del/+, 10 wt, 11 Del/Dup, and 8 Dup/+ mice using magnetic resonance imaging (MRI). Overall, we found significant differences in total brain volume between the genotypes (F(3, 33) = 14.14, p < 0.001; Del/+: 458±23 mm3, wt: 448±10 mm3, Del/Dup: 446±12 mm3, Dup/+: 412±13 mm3, brain volumes given as mean±sd). Dup/+ animals showed a globally reduced brain volume in comparison with that of the other genotypes. Using a segmented atlas that divides the brain into 159 separate brain regions[23–25], we examined the 83 structures of at least 1 mm3 in size. A reduction of the whole brain volume was noticed for Dup/+ animals (Fig 3a). Brain structures significantly affected after a correction for multiple testing included the hippocampus, amygdala, nucleus accumbens, cingulate complex, entorhinal cortex, frontal region, and perirhinal cortex. Notably, for the majority of these structures, we observed opposite absolute volume changes in Del/+ and Dup/+ animals in comparison with values determined in wt littermates (Fig 3b). Relative volumes of the discussed regions are represented in the supplementary information (S3 Table).
Effects of the Arf2-Kansl1 dosage on synaptic transmission and synaptic plasticity in hippocampal slices
To explore if genes from the Arf2-Kansl1 region regulate electrophysiological parameters in mouse neurons as suggested by changes in ChIP-seq profiles, we assessed basal synaptic transmission and synaptic plasticity by measuring field excitatory postsynaptic potentials (fEPSPs) in acute hippocampal slices from Del/+ and Dup/+ mice. In Del/+ mutants, we observed decreased fEPSP slopes in mutant slices, especially in response to higher stimulus strengths (S6a Fig). Mean slopes of fEPSPs invoked by the maximum stimulus strength (4.2 V) were significantly smaller in slices from Del/+ mice (1.46 ± 0.09 mV/ms) than wt littermates (1.87 ± 0.09 mV; F(1,13.34) = 8.31; P = 0.025; two-way nested ANOVA, genotype effect). The mean paired-pulse ratio of slopes of fEPSPs evoked at a 50 ms interpulse interval was also significantly lower in mutant slices (S6c Fig; F(1, 11.04) = 6.506; P = 0.027. No significant changes in LTP elicited by theta-burst stimulation were noted in slices from Del/+ mice (S6 Fig).
Basal synaptic strength was slightly enhanced in slices from Dup/+ mice, as fEPSPmax mean slope was nominally higher in slices from Dup/+ mice (2.12 ± 0.09 mV/ms) than in slices from wt littermates (1.89 ± 0.09 mV/ms; S6b Fig). However, the effect did not reach statistical significance (F(1,8.67) = 3.09; P = 0.114; two-way nested ANOVA, genotype effect). Likewise, paired-pulse facilitation (S6d Fig) and LTP were not significantly different in slices from Dupl/+ and litter-matched wt mice (S6f Fig).
Mice carrying a heterozygous deletion of Kansl1 recapitulate the majority of phenotypes observed in animals carrying the Arf2-Kansl1 deletion
We performed similar examinations of mice with heterozygous ablation of Kansl1 (Kansl1+/- mice) in the same B6N genetic background and compared the outcome with the Del/+ phenotypes.). In comparison with wt mice, Kansl1+/- adult animals were underweight (two-way ANOVA genotype effect F(1,30) = 11.729, P = 0.004; Fig 4a) Kansl1+/- mice had a significantly smaller body size (F(1,15) = 11.516, P = 0.004) and lower adiposity level (F(1,15) = 6.813, P = 0.020; Fig 4a) than wt littermates in 20 week old animals. In aggregate, these data indicate many similarities in basic traits between the Kansl1+/- and the Del/+ carriers.
Next, we examined the behavior of Kansl1+/- mice. In a circadian activity test, Kansl1+/- mice displayed normal patterns of activity (S7 Fig). However their baseline locomotor activity levels differed from those of wt littermates during the dark phase (F(1,16) = 8.482, P = 0.010) and the light phase (F(1,16) = 8.573, P = 0.010; Fig 4b). In the novel open field arena, Kansl1+/- mice demonstrated an increased level of rearing behavior (F(1,15) = 4.846, P = 0.044: Fig 4c). To investigate further this hyperactivity, we performed visual observations of animals in odorless home-cages (Fig 4d). Mutant mice displayed more intensive rearing behavior (F(1,15) = 7.207, p = 0.017) and also showed a decreased level of digging behavior (F(1,15) = 12.268, p = 0.003) in comparison with wt littermates. These results indicate a global alteration of Kansl1+/- activity characterized, in particular, by locomotor hypoactivity and vertical hyperactivity. During the learning phase of the rotarod test, Kansl1+/- mice displayed higher levels of motor coordination and learning than wt mice (two-way ANOVA genotype effect F(1,30) = 115.867, P < 0.001; Fig 4e). In the test phase, Kansl1+/- mice showed improvements for speed higher than 10rpm (Fig 4f), a phenotype not observed in the deletion due to lower power of the tests. Recognition memory was assessed in mice by using the novel object recognition task with a retention delay of 3 h. While no difference was observed in the acquisition session (S4 Table), Kansl1+/- mice displayed a significant memory impairment compared to wt during the choice session (F(1,15) = 22.566, P < 0.001; Fig 5a). Then, we evaluated associative memory with the fear conditioning test. No differences in the baseline and post-shock freezing levels were detected between Kansl1+/- mice and wt littermates in the conditioning session (Fig 5b; S4 Table). In the context session, Kansl1+/- mice displayed a lower incidence of freezing than wt littermates (H(1,16) = 6.419, P = 0.011). In the cue session, a decreased freezing level was detected in Kansl1+/- mice during the second 2-min cue period (F(1,16) = 16.748, P < 0.001). Finally, we evaluated animal social behaviors with the three-chamber sociability test and the social interaction test (Fig 5c and 5d; S8 Fig). In both tests, no differences were observed between Kansl1+/- mice and wt littermates.
Epigenetic profiling in KdVS models identifies changes in regulation of neuronal processes and synaptic activity
To identify potential gene expression differences in KdVS models, we carried out epigenetic profiling in the hippocampus, a brain region implicated in learning and memory processes, isolated from 3 Del/+, 3 Kansl1+/- and 6 wt (3+3 matched littermates). We performed ChIP-Seq of H3K4me3 which is a histone mark located in actively expressed genes. As expected, H3K4me3 marks were lower in the Arf2-Kansl1 region with half peak height values for Arf2, Crhr1, Mapt and Kansl1 in the Del/+ samples compared to those observed in wt samples and for the neighboring genes (Fig 6a). Analysis of H3K4me3 tracks (DESeq2, p<0.01) revealed 788 and 751 misregulated promoters in Del/+ and Kansl1+/-, respectively (Fig 6b). Clustering of Pearson correlations, an unbiased method to measure the degree of similarity between large data sets, showed clear segregation between conditions and high concordance of biological replicates (Fig 6c; data are available in S5 to S10 Tables).
Cell-type marker analysis (Fig 6d; see Methods)[26] revealed that up-regulated promoters observed in Del/+ mostly corresponded to genes expressed in neuronal populations (pyramidal neurons and interneurons). Furthermore, Gene Ontology (GO) analysis revealed that they present significant enrichments for the synapse (p<7.9e-20), and dendrite compartments (p<3e-14) as well as synaptic transmission (p<9.1e-24) or neurogenesis processes (p<1.3e-22; S12 and S20 Tables). In contrast, the term oxidoreductase and mitochondrion were found enriched respectively in Del/+ and Kansl1/+ down-regulated promoters using DAVID [27] but no other GO significant enrichments were found for Del/+ (S11 and S19 Tables).
Of the 470 promoters up-regulated in Del/+, 36% (172) were also up-regulated in Kansl1+/-, whereas the two genotypes shared 67% (211) of genes that were down-regulated (Fig 6e). Among all the promoters up-regulated in either of the genetic conditions, we observed 4 distinct clusters of genes (Fig 6f). For cluster 1, 160 genes enriched in non-neuronal populations (astrocytes, ependymocytes, choroid plexus, mural cells, Fig 6g) are up-regulated in the hippocampus of Kansl1 heterozygotes but not of Del/+ mice. Cluster 2 encompassed 214 genes up-regulated in both genetic conditions and enriched in markers of CA1/CA2 pyramidal neurons and astrocytes, while Cluster 3 contained 212 genes enriched in neuronal markers and whose expression was up-regulated to a greater extent in Del/+. Finally, we noted that cluster 4 comprised 162 genes upregulated specifically in Del/+ and expressed in CA1/CA2 pyramidal neurons and astrocytes. Each cluster showed a specific GO enrichment profile (Fig 6h). Several neuronal processes, including synaptic transmission and neurogenesis were overrepresented GO terms in genes from clusters 2, 3 and 4. Cluster 2 genes (up in both models) were also enriched in DNA-packaging and nucleosomes (Fig 6h). In the Del/+ hippocampi, 8 genes involved in social behavior were up-regulated and only 2 of these, Tbx1 and Nr2e1, were also dysregulated in Kansl1+/- mice (Fig 6i). These results suggest a dominance of Del/+ with respect to Kansl1 for determining social behavior.
Effects of the Arf2-Kansl1 dosage on synaptic transmission and synaptic plasticity in hippocampal slices
To explore if genes from the Arf2-Kansl1 region regulate electrophysiological parameters in mouse neurons as suggested by changes in ChIP-seq profiles, we assessed basal synaptic transmission and synaptic plasticity by measuring field excitatory postsynaptic potentials (fEPSPs) in acute hippocampal slices from Del/+ and Dup/+ mice. In Del/+ mutants, we observed decreased fEPSP slopes in mutant slices, especially in response to higher stimulus strengths (S6 Fig). Mean slopes of fEPSPs invoked by the maximum stimulus strength (4.2 V) were significantly smaller in slices from Del/+ mice (1.46 ± 0.09 mV/ms) than wt littermates (1.87 ± 0.09 mV; F(1,13.34) = 8.31; P = 0.025; two-way nested ANOVA, genotype effect). The mean paired-pulse ratio of slopes of fEPSPs evoked at a 50 ms interpulse interval was also significantly lower in mutant slices (S6 Fig; F(1, 11.04) = 6.506; P = 0.027. No significant changes in LTP elicited by theta-burst stimulation were noted in slices from Del/+ mice (S6c–S6e Fig).
Basal synaptic strength was slightly enhanced in slices from Dup/+ mice, as fEPSPmax mean slope was nominally higher in slices from Dup/+ mice (2.12 ± 0.09 mV/ms) than in slices from wt littermates (1.89 ± 0.09 mV/ms). However, the effect did not reach statistical significance (F(1,8.67) = 3.09; P = 0.114; two-way nested ANOVA, genotype effect). Likewise, paired-pulse facilitation (S6 Fig) and LTP were not significantly different in slices from Dupl/+ and litter-matched wt mice (S6 Fig).
Discussion
In this study, we described the first mouse models of Koolen-de Vries syndrome (KdVS) and 17q21.31 microduplication syndrome. Del/+ mice showed similar phenotypes observed in KdVS patients: higher level of social interaction, lower level of recognition memory, associative learning and memory and brain malformations [3, 5, 15, 28]. We found a single phenotypic similarity between patients carrying the 17q21.31 microduplication and Dup/+ mice, which is microcephaly that has been reported in 50% of the human individuals with this microduplication [8]. Several SNPs associated with risk for Alzheimer’s disease (AD) were identified near MAPT and KANSL1 in humans and they appeared to be correlated with an overexpression of both genes in different brain regions [29]. This observation was further supported by the description of a familial form of late onset AD due to the microduplication of the 17q21.31 region [30]. Thus it would be important now to follow cognition in ageing cohorts carrying the Dup/+ allele generated here as young individuals analysed in the present study do not shown any cognitive impairment, but rather more some improvement in associative memory.
Overall, the phenotypic comparison observed in the Del/+ and Kansl1+/- models confirms the critical importance of KANSL1 in KdVS[3, 15]. Nevertheless, increased social interaction was not found to be affected in Kansl1 haploinsufficient mice whereas it is predominant in humans with KANSL1 mutations [3, 15]. This discrepancy could reflect either different dosage threshold levels in the mouse and human that governs proper social interaction, with the mice needing more change to induce such friendly phenotype. Indeed we have found more genes associated with social behavior misregulated in the Del/+ brain compared to the Kansl1 heterozygotes. An educated guess would be that the haploinsufficiency of another gene(s) from the Arf2-Kansl1 region would contribute to this phenotype in mouse.
The hippocampal epigenetic analysis of 17q21.31 models unraveled several features. Only the mitochondrion term was enriched in the Kansl1/+ down-regulated genes, a situation partially similar to a recent study where KANSL1 and its partner MOF were observed in mitochondria regulating expression of genes involved in oxidative phosphorylation [31]. Interestingly the oxidoreductase term was enriched in Del/+ down-regulated genes. Thus it would be interested to follow mitochondrial activity in the mouse models. Another common set of dysregulated promoters, were largely affecting CA1 neuronal populations and neuronal functioning and includes many genes with long introns. Common up-regulated genes also appear to be implicated in DNA-packaging and nucleosomes, possibly reflecting the outcome of the misregulated KANSL1 activity. Interestingly, several genes (Adcyap1, Cntnap2, Grid1, Nrxn1, Nrxn3, Ucn, Tbx1, and Nr2e1), that are associated with disorders [32, 33], stress response [34], social behaviors [35–37] or autism spectrum disorders [38–42], are up-regulated specifically in the hippocampus of Del/+ mice. Deregulation of these genes may be a molecular underpinning of the friendly/amiable affect of 17q21.31 deletion patients. Expression of the majority of those genes (except for Txb1 and Nr2e1) was not altered in Kansl1+/- mice. We also emphasize that two overexpressed genes, Ucn and Adcyap1, and one underexpressed gene, Chd1l, found deregulated in Del/+ mice are linked to corticotropin release and are associated with stress response. Those genes may be relevant for the overly friendly social phenotypes observed in 17q21.31 deletion carriers.
Electrophysiological experiments confirmed that dosage of one or several genes within the 17q21.31 syntenic region affects basal synaptic transmission and short-term plasticity of excitatory synapses in the hippocampus. Noted disturbances in the expression level of several genes could contribute to this impairment. For example, dysregulation of Cntnap2 could affect migration of interneurons [37] and inhibitory synaptic function [43], which could, in turn, alter excitatory synaptic responses. Other gene affected by 17q21.31 mutations is Nrxn1 that shapes the balance between excitatory and inhibitory synaptic activity [44, 45]. Such a defect at the expression level may account for the change in synaptic strength and impaired learning and memory.
In conclusion, this study confirms a previously hypothesized role of KANSL1 in the manifestation of KdVS phenotypes and extends substantially our knowledge about biological processes affected by these mutations. With these new genetic tools, we can explore the function of these genes and dissect further the pathophysiological mechanisms to eventually inform potential therapeutic avenues.
Methods
Ethical statement and mouse lines
The 17p21.31 mutant mice carrying the deletion of the Arf2–Kansl1 region (noted Del/+), or the reciproqual duplication (noted Dup/+), were generated on the C57BL/6N genetic background (see Supplementary information). The Kansl1+/- mutant mice were derived in a C57BL/6N genetic background from the unique IKMP ES cell clone HEPD0766_8_G02. Kansl1tm1b(EUCOMM)Hmgu[46] animals were obtained by breeding Kansl1tm1a(EUCOMM)Hmgu/+ mice with animals expressing the Cre recombinase[47] to generate the Kansl1tm1b(EUCOMM)Hmgu/+ (noted here Kansl1+/-). The local ethics committee, Com’Eth (n°17), approved all mouse experimental procedures, under the accreditation number 2012–069.
Behavioral analysis
Behavioral studies were conducted in 12-20-week old animals. All assessments were scored blind to the genotype as recommended by the ARRIVE guidelines[48, 49]. All the experimental procedures for behavioral assessments have been described[50, 51] and are detailed in the supplementary information.
Craniofacial and image acquisition
Craniofacial phenotyping is described in the supplementary data. Magnetic resonance imaging (MRI) was used to identify alterations of brain regions in 17q21.31-homologous CNV mice (8 Del/+, 10 wt, 11 Del/Dup, and 8 Dup/+ mice). MRI scans were acquired from 41 male mice at 43 weeks of age with specimens prepared as described[52] and detailed with the image processing in the supplementary information.
Hippocampal slice electrophysiology
Acute hippocampal slices were used to record field excitatory post synaptic potentials (fEPSPs), by using an electrophysiological suite of 8 MEA60 set-ups consisting of a MEA1060-BC pre-amplifier and a filter amplifier (gain 550×) (Multi Channel Systems, Reutlingen, Germany) as described[50, 53]. All experiments were performed using two-pathway stimulation of the Schaffer collateral/commissural fibers in the CA1 area of 350-μm thick hippocampal slices (see supplementary information).
ChIP-seq
Adult hippocampi from Del/+, Kansl1+/- and wt mice were dissected and snap-frozen in liquid nitrogen. Tissue samples were ground in a liquid-nitrogen chilled mortar and the resulting powder was used for ChIP. Chipping for H3K4me3 (Diagenode A5051-001P) was performed as in (www.blueprint-epigenome.eu/UserFiles/file/Protocols/Histone_ChIP_July2014.pdf). Libraries were synthetized with KAPA Hyper prep kit (KK8504) following the manufacturer’s instructions. The libraries were pooled (4/lane) and sequenced on the illumina HiSeq. Libraries were mapped with BWA (0.6.2). Peaks were called with a custom C++ script and DEseq2 (R+) was used to perform statistical comparisons. Data are deposited in GEO under accession GSE80311. All enrichment analyses are made from standard hypergeometric tests with Benjamini or Bonferroni correction. GO annotations are updated to 25/6/2015.
Cell-types enrichments
ChIP-seq data and ChIP-seq supplementary tables were deposited in GEO and available at the link https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cnavacaedzgztgz&acc=GSE80311. Cell-types enrichments are based on the single-cell RNAseq data from Amit Zeisel et al., 2015, “cell types in the mouse cortex and hippocampus revealed by single-cell rna-seq” [26]. In this work, single-cell RNAseq was used to measure trascriptomes of >3000 single cells, allowing to define the markers of 11 different cell types in adult (P21-P30) hippocampus and somatosensory cortex. Given that also our ChIP-seq is done on adult (P30) hippocampus, single-cell RNAseq data from Zeisel et al. becomes a highly valuable resource to gain insight at the cell type level. Here we performed standard hypergeometric tests with bonferroni correction against the cell-type markers derived from data of Zeisel et al. in order to evaluate the abundance of specific markers in deregulated gene sets. A significant enrichment (p<0.01) means that a high amount of markers of a specific cell type is found in Kansl1+/- or Del/+ deregulated genes, suggesting that the latter cell type should be particularly affected. The complete statistical data and the lists of markers found in de-regulated genes are fully available in S5 to S20 Tables.
Statistical analyses for behavior and electrophysiology
All acquired behavioral data were analyzed using a one-way ANOVA analysis with a post-hoc Tukey’s test, when applicable, non-parametric Kruskal-Wallis test or Mann-Whitney U test using the Sigma Plot software (Ritme, France). The Pearson’s chi-squared test was used for mutant allele transmission. Data are represented as the mean ± s.e.m. and differences were considered to be significant if P < 0.05. When comparing freezing levels between wt, Del/+, and Dup/+ animals over different time points during extinction we used the two-Way ANOVA Repeated Measures statistical test followed by Holm-Sidak post-hoc tests to evaluate for interactions between the groups. Otherwise, when comparing wt data with those obtained from respective Del/+ or Dup/+ animals for a single time point, we used Student’s t-test. When data did not follow a normal distribution, we used the Mann-Whitney rank-based statistical test.
In electrophysiological experiments input-output relationships were compared initially by a mixed model repeated-measures ANOVA and Bonferroni post hoc test implemented in Prism 5 (GraphPad Software, San Diego, USA) using individual slice data as independent observations. Since several slices were routinely recorded from every mouse, fEPSPmax, PPF and LTP values between wt and mutant mice were compared using two-way nested ANOVA design with genotype (group) and mice (sub-group) as fixed and random factors respectively (STATISTICA v.10, StatSoft, USA). DF error was computed using the Satterthwaite’s method and main genotype effect was considered significant if P < 0.05. Graph plots and normalization were performed using OriginPro 8.5 (OriginLab, Northampton, USA). Electrophysiological data are presented as the mean ± s.e.m. with n and N indicating number of slices and mice respectively.
Supporting information
Acknowledgments
We are grateful to the animal care-takers for their services at the PHENOMIN-ICS and Babraham Research Campus. We also wish to thank members of the research groups involved and staff of the IGBMC laboratory for their helpful suggestions and discussions. We are thankful to Erica E. Davis for critical reading of the manuscript. The Kansl1 knock-out was obtained from the International Mouse Phenotyping Consortium (www.mousephenotype.org) at the French PHENOMIN node. Mouse models are available through the Infrafrontier infrastructure.
Data Availability
Expression data are available from the GEO database under the accession number GSE80311. All other data are within the paper and its Supporting Information files.
Funding Statement
This work has been supported by the National Centre for Scientific Research (CNRS), the French National Institute of Health and Medical Research (INSERM), the University of Strasbourg (UDS), the “Centre Européen de Recherche en Biologie et en Médecine”, the European commission (AnEUploidy project to YHe, LSHG-CT-2006-037627) with a fellowship from the “Fondation pour la Recherche Médicale” to TA (FDT20130928080). This study also received support from French state funds through the “Agence Nationale de la Recherche” under the frame programme Investissements d’Avenir labelled ANR-10-IDEX-0002-02, ANR-10-LABX-0030-INRT, ANR-10-INBS-07 PHENOMIN, from the EU FP7 large-scale integrated project GENCODYS (grant 241995) to YHe, MK and HGS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Expression data are available from the GEO database under the accession number GSE80311. All other data are within the paper and its Supporting Information files.