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. 2017 Dec;42(13):2516-2526.
doi: 10.1038/npp.2017.91. Epub 2017 May 4.

Associations of the Intellectual Disability Gene MYT1L With Helix-Loop-Helix Gene Expression, Hippocampus Volume and Hippocampus Activation During Memory Retrieval

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Associations of the Intellectual Disability Gene MYT1L With Helix-Loop-Helix Gene Expression, Hippocampus Volume and Hippocampus Activation During Memory Retrieval

Agnieszka Kepa et al. Neuropsychopharmacology. .
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Abstract

The fundamental role of the brain-specific myelin transcription factor 1-like (MYT1L) gene in cases of intellectual disability and in the etiology of neurodevelopmental disorders is increasingly recognized. Yet, its function remains under-investigated. Here, we identify a network of helix-loop-helix (HLH) transcriptional regulators controlled by MYT1L, as indicated by our analyses in human neural stem cells and in the human brain. Using cell-based knockdown approaches and microarray analyses we found that (1) MYT1L is required for neuronal differentiation and identified ID1, a HLH inhibitor of premature neurogenesis, as a target. (2) Although MYT1L prevented expression of ID1, it induced expression of a large number of terminal differentiation genes. (3) Consistently, expression of MYT1L in the human brain coincided with neuronal maturation and inversely correlated with that of ID1 and ID3 throughout the lifespan. (4) Genetic polymorphisms that reduced expression of MYT1L in the hippocampus resulted in increased expression of ID1 and ID3, decreased levels of the proneural basic HLH (bHLH) transcriptional regulators TCF4 and NEUROD6 and decreased expression of genes involved in long-term potentiation and synaptic transmission, cancer and neurodegeneration. Furthermore, our neuroimaging analyses indicated that MYT1L expression associated with hippocampal volume and activation during episodic memory recall, as measured by blood-oxygen-level-dependent (BOLD) signals. Overall, our findings suggest that MYT1L influences memory-related processes by controlling a neuronal proliferation/differentiation switch of ID-bHLH factors.

Figures

Figure 1
Figure 1
Changes in gene expression patterns during differentiation of the human neural stem cell line SPC04. Changes in HLH (ID1 and ID3) and bHLH (NEUROG1 and NEUROG2) mRNA levels (a), and induction of a subset of terminal differentiation genes co-expressed with MYT1L (b) were accessed by microarray analyses. Results show variations in expression in various stages of differentiation (pre-differentiation, and 3 or 7 days after induction of differentiation), relative to that of undifferentiated cells and represent mean±SEM of n=3 independent experiments.
Figure 2
Figure 2
Effects of MYT1L knockdown on neural differentiation and global gene expression in SP04 cells. Cells were transduced with lentiviral vectors encoding shMYT1L or a non-silencing shRNA control and induced to differentiate for 7 or 14 days. (a) Efficiency of MYT1L knockdown accessed by PCR 7 and 14 days after induction of differentiation. Effects of treatment with shMYT1L on MYT1L and mRNA levels, split according to the length of differentiation. Results represent mean±SEM of n=3 independent experiments; **p<0.01. (b) Effects of shMYT1L on morphology of SPC04 cells, 7 days after induction of differentiation. Changes in morphology are evident when comparing phase contrast pictures of cells transduced with control, non-silencing lentiviruses with that of cells transduced with shMYT1L-encoding lentiviruses: control cells showed enhanced neuronal features such as increased neurite elongation and retracted cell bodies. In contrast, shMYT1L prevented these morphological changes, resulting in a flat, more adherent morphology. The fluorescence pictures reveal the high efficiency of transduction by the lentiviruses that expressed the enhanced green fluorescent protein as marker. Scale bar=100 μm. (c) Quantification of the morphological changes described in (b) were obtained using the neurite outgrowth plugin in MetaMorph; total neurite length and average length of neurite per cell and cell soma area were recorded in a total of 473 cells (number of cells per group: control MYT1L, n=298; shMYT1L, n=175). Unpaired t-tests were used to compare means in each group: ****p<0.0001. (d) Microarray mRNA profiling analysis reveals target genes differentially affected by MYT1L. Probe sets differentially expressed following shMYT1L treatment were clustered together based on euclidean distance, and their expression levels (each line represents a single probe set) are displayed as fold change compared to control treatment. (e) Functional clustering of differently expressed genes showing enrichment for genes involved in neuronal maturation and the extracellular matrix composition.
Figure 3
Figure 3
Analyses of gene expression in the human brain across the lifetime reveals patterns of MYT1L mRNA expression that correlates highly with that of NEUROD6, notably in the neocortex, the hippocampus and the amygdala. Conversely, MYT1L expression correlates inversely with that of ID1 and ID3 throughout the life span.
Figure 4
Figure 4
Effects of a MYT1L-associated eQTL on gene expression in the hippocampus. (a) Genomic view of the MYT1L locus, indicating SNPs acting in cis to influence MYT1L expression (MYT1L eQTLs, red bars). These SNPs are all in high linkage disequilibrium (r2>0.8), surrounding a conserved distant-acting and tissue-specific transcriptional enhancer (ie, human element hs1385, labeled as element_1385) from the VISTA Enhancer database, (Pennacchio et al, 2006). Genomic co-ordinates are based on the hg19 genome assembly. (b) The minor alleles at these eQTLs decrease mRNA levels of MYT1L (cis-effects) in the hippocampus, as illustrated for the rs55800610 gene variant. The minor A-allele at this SNP also associates with reduced NEUROD6 and TCF4 mRNA levels and increased mRNA levels of ID1 and ID3 in the hippocampus (trans-effects). Because of the low minor allele frequency (MAF=0.07), heterozygotes and individuals homozygotes for the minor allele were grouped for analyses. Genotypes count: A-carriers (AA+AT), n=20; TT, n=114. (c and d) Gene set enrichment analyses of all genes downregulated in individuals carriers of the rs55800610 A-allele indicating their clustering into specific biological functions (c) and belonging to a known biological pathway (d). The dashed vertical line represents the significance threshold (Padj 0.05). (e) Genes whose expression is affected by MYT1L decrease both in SPC04 cells and in the human hippocampus, and the measured effect sizes (negative=down-regulation, positive=up-regulation).
Figure 5
Figure 5
Associations between MYT1L expression and hippocampus volume and hippocampus activation during episodic memory recall. (a) Association of rs17338519 with hippocampal volume in a sample of N=1,398 adolescents. Linear regression analyses indicated that the number of minor T-alleles associated with lower volume of the right hippocampus (P=4.40 × 10−4). (b) Expression of MYT1L stratified by genotypes at rs17338519, in the hippocampus and cerebral cortex of 134 post-mortem human brains. For the cortex, expressions of MYT1L in the occipital, temporal, and frontal cortices were averaged. Regression analyses indicated that the presence of the T-allele at rs17338519 associated with decreased MYT1L mRNA levels in the hippocampus (P=0.034) and the cortex (P=0.0035). Genotypes count: GG=110; T-carriers=24. (c) Association of rs17338519 with hippocampal activation during an fMRI task of episodic memory recall in 285 healthy adults. The left panel shows the ROI-derived brain activation map within the hippocampus, with increased activation of the right hippocampus (x=16, y=−6, z=−12) during episodic memory recall in carriers of the T-allele (N=285; p=0.02 FWE corrected for multiple testing across ROI). The right panel shows a quantification of rs17338519 genotypes effects on the activation of the right hippocampus. Each dot represents size of effect in one subject.

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