Allelic chromatin structure precedes imprinted expression of Kcnk9 during neurogenesis.

Differences in chromatin state inherited from the parental gametes influence the regulation of maternal and paternal alleles in offspring. This phenomenon, known as genomic imprinting, results in genes preferentially transcribed from one parental allele. While local epigenetic factors such as DNA methylation are known to be important for the establishment of imprinted gene expression, less is known about the mechanisms by which differentially methylated regions (DMRs) lead to differences in allelic expression across broad stretches of chromatin. Allele-specific higher-order chromatin structure has been observed at multiple imprinted loci, consistent with the observation of allelic binding of the chromatin-organizing factor CTCF at multiple DMRs. However, whether allelic chromatin structure impacts allelic gene expression is not known for most imprinted loci. Here we characterize the mechanisms underlying brain-specific imprinted expression of the Peg13-Kcnk9 locus, an imprinted region associated with intellectual disability. We performed region capture Hi-C on mouse brains from reciprocal hybrid crosses and found imprinted higher-order chromatin structure caused by the allelic binding of CTCF to the Peg13 DMR. Using an in vitro neuron differentiation system, we showed that imprinted chromatin structure precedes imprinted expression at the locus. Additionally, activation of a distal enhancer induced imprinted expression of Kcnk9 in an allelic chromatin structure-dependent manner. This work provides a high-resolution map of imprinted chromatin structure and demonstrates that chromatin state established in early development can promote imprinted expression upon differentiation.

Elimination of Calm1 long 3'-UTR mRNA isoform by CRISPR-Cas9 gene editing impairs dorsal root ganglion development and hippocampal neuron activation in mice.

The majority of mouse and human genes are subject to alternative cleavage and polyadenylation (APA), which most often leads to the expression of two or more alternative length 3' untranslated region (3'-UTR) mRNA isoforms. In neural tissues, there is enhanced expression of APA isoforms with longer 3'-UTRs on a global scale, but the physiological relevance of these alternative 3'-UTR isoforms is poorly understood. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that generates short (Calm1-S) and long (Calm1-L) 3'-UTR mRNA isoforms via APA. We found Calm1-L expression to be largely restricted to neural tissues in mice including the dorsal root ganglion (DRG) and hippocampus, whereas Calm1-S was more broadly expressed. smFISH revealed that both Calm1-S and Calm1-L were subcellularly localized to neural processes of primary hippocampal neurons. In contrast, cultured DRG showed restriction of Calm1-L to soma. To investigate the in vivo functions of Calm1-L, we implemented a CRISPR-Cas9 gene editing strategy to delete a small region encompassing the Calm1 distal poly(A) site. This eliminated Calm1-L expression while maintaining expression of Calm1-S Mice lacking Calm1-L (Calm1ΔL/ΔL ) exhibited disorganized DRG migration in embryos, and reduced experience-induced neuronal activation in the adult hippocampus. These data indicate that Calm1-L plays functional roles in the central and peripheral nervous systems.

Emerging Roles for 3' UTRs in Neurons.

The 3' untranslated regions (3' UTRs) of mRNAs serve as hubs for post-transcriptional control as the targets of microRNAs (miRNAs) and RNA-binding proteins (RBPs). Sequences in 3' UTRs confer alterations in mRNA stability, direct mRNA localization to subcellular regions, and impart translational control. Thousands of mRNAs are localized to subcellular compartments in neurons-including axons, dendrites, and synapses-where they are thought to undergo local translation. Despite an established role for 3' UTR sequences in imparting mRNA localization in neurons, the specific RNA sequences and structural features at play remain poorly understood. The nervous system selectively expresses longer 3' UTR isoforms via alternative polyadenylation (APA). The regulation of APA in neurons and the neuronal functions of longer 3' UTR mRNA isoforms are starting to be uncovered. Surprising roles for 3' UTRs are emerging beyond the regulation of protein synthesis and include roles as RBP delivery scaffolds and regulators of alternative splicing. Evidence is also emerging that 3' UTRs can be cleaved, leading to stable, isolated 3' UTR fragments which are of unknown function. Mutations in 3' UTRs are implicated in several neurological disorders-more studies are needed to uncover how these mutations impact gene regulation and what is their relationship to disease severity.

Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing.

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to quantify inclusion of alternative exons in connection with alternative 3' ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.

Coordination of Alternative Splicing and Alternative Polyadenylation revealed by Targeted Long-Read Sequencing.

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to resolve the connectivity of alternative exons to alternative 3' ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.

Allelic chromatin structure primes imprinted expression of Kcnk9 during neurogenesis.

Differences in chromatin state inherited from the parental gametes influence the regulation of maternal and paternal alleles in offspring. This phenomenon, known as genomic imprinting, results in genes preferentially transcribed from one parental allele. While local epigenetic factors such as DNA methylation are known to be important for the establishment of imprinted gene expression, less is known about the mechanisms by which differentially methylated regions (DMRs) lead to differences in allelic expression across broad stretches of chromatin. Allele-specific higher-order chromatin structure has been observed at multiple imprinted loci, consistent with the observation of allelic binding of the chromatin-organizing factor CTCF at multiple DMRs. However, whether allelic chromatin structure impacts allelic gene expression is not known for most imprinted loci. Here we characterize the mechanisms underlying brain-specific imprinted expression of the Peg13-Kcnk9 locus, an imprinted region associated with intellectual disability. We performed region capture Hi-C on mouse brain from reciprocal hybrid crosses and found imprinted higher-order chromatin structure caused by the allelic binding of CTCF to the Peg13 DMR. Using an in vitro neuron differentiation system, we show that on the maternal allele enhancer-promoter contacts formed early in development prime the brain-specific potassium leak channel Kcnk9 for maternal expression prior to neurogenesis. In contrast, these enhancer-promoter contacts are blocked by CTCF on the paternal allele, preventing paternal Kcnk9 activation. This work provides a high-resolution map of imprinted chromatin structure and demonstrates that chromatin state established in early development can promote imprinted expression upon differentiation.

CRISPR-Mediated Knockout of Long 3' UTR mRNA Isoforms in mESC-Derived Neurons.

Alternative cleavage and polyadenylation (APA) is pervasive, occurring for more than 70% of human and mouse genes. Distal poly(A) site selection to generate longer 3' UTR mRNA isoforms is prevalent in the nervous system, affecting thousands of genes. Here, we establish mouse embryonic stem cell (mESC)-derived neurons (mES-neurons) as a suitable system to study long 3' UTR isoforms. RNA-seq analysis revealed that mES-neurons show widespread 3' UTR lengthening that closely resembles APA patterns found in mouse cortex. mESCs are highly amenable to genetic manipulation. We present a method to eliminate long 3' UTR isoform expression using CRISPR/Cas9 editing. This approach can lead to clones with the desired deletion within several weeks. We demonstrate this strategy on the Mprip gene as a proof-of-principle. To confirm loss of long 3' UTR expression and the absence of cryptic poly(A) site usage stemming from the CRISPR deletion, we present a simple and cost-efficient targeted long-read RNA-sequencing strategy using the Oxford Nanopore Technologies platform. Using this method, we confirmed specific loss of the Mprip long 3' UTR isoform. CRISPR gene editing of mESCs thus serves as a highly relevant platform for studying the molecular and cellular functions of long 3' UTR mRNA isoforms.