Regulatory Architecture of the Neuronal Cacng2/Tarpγ2 Gene Promoter: Multiple Repressive Domains, a Polymorphic Regulatory Short Tandem Repeat, and Bidirectional Organization with Co-regulated lncRNAs

CACNG2 (TARPγ2, Stargazin) is a multi-functional regulator of excitatory neurotransmission and has been implicated in the pathological processes of several brain diseases. Cacng2 function is dependent upon expression level, but currently, little is known about the molecular mechanisms that control expression of this gene. To address this deficit and investigate disease-related gene variants, we have cloned and characterized the rat Cacng2 promoter and have defined three major features: (i) multiple repressive domains that include an array of RE-1 silencing transcription factor (REST) elements, and a calcium regulatory element-binding factor (CaRF) element, (ii) a (poly-GA) short tandem repeat (STR), and (iii) bidirectional organization with expressed lncRNAs. Functional activity of the promoter was demonstrated in transfected neuronal cell lines (HT22 and PC12), but although selective removal of REST and CaRF domains was shown to enhance promoter-driven transcription, the enhanced Cacng2 promoter constructs were still about fivefold weaker than a comparable rat Synapsin-1 promoter sequence. Direct evidence of REST activity at the Cacng2 promoter was obtained through co-transfection with an established dominant-negative REST (DNR) construct. Investigation of the GA-repeat STR revealed polymorphism across both animal strains and species, and size variation was also observed in absence epilepsy disease model cohorts (Genetic Absence Epilepsy Rats, Strasbourg [GAERS] and non-epileptic control [NEC] rats). These data provide evidence of a genotype (STR)-phenotype correlation that may be unique with respect to proximal gene regulatory sequence in the demonstrated absence of other promoter, or 3′ UTR variants in GAERS rats. However, although transcriptional regulatory activity of the STR was demonstrated in further transfection studies, we did not find a GAERS vs. NEC difference, indicating that this specific STR length variation may only be relevant in the context of other (Cacna1h and Kcnk9) gene variants in this disease model. Additional studies revealed further (bidirectional) complexity at the Cacng2 promoter, and we identified novel, co-regulated, antisense rat lncRNAs that are paired with Cacng2 mRNA. These studies have provided novel insights into the organization of a synaptic protein gene promoter, describing multiple repressive and modulatory domains that can mediate diverse regulatory inputs

Unraveling genetic modifiers in the gria4 mouse model of absence epilepsy.

Absence epilepsy (AE) is a common type of genetic generalized epilepsy (GGE), particularly in children. AE and GGE are complex genetic diseases with few causal variants identified to date. Gria4 deficient mice provide a model of AE, one for which the common laboratory inbred strain C3H/HeJ (HeJ) harbors a natural IAP retrotransposon insertion in Gria4 that reduces its expression 8-fold. Between C3H and non-seizing strains such as C57BL/6, genetic modifiers alter disease severity. Even C3H substrains have surprising variation in the duration and incidence of spike-wave discharges (SWD), the characteristic electroencephalographic feature of absence seizures. Here we discovered extensive IAP retrotransposition in the C3H substrain, and identified a HeJ-private IAP in the Pcnxl2 gene, which encodes a putative multi-transmembrane protein of unknown function, resulting in decreased expression. By creating new Pcnxl2 frameshift alleles using TALEN mutagenesis, we show that Pcnxl2 deficiency is responsible for mitigating the seizure phenotype - making Pcnxl2 the first known modifier gene for absence seizures in any species. This finding gave us a handle on genetic complexity between strains, directing us to use another C3H substrain to map additional modifiers including validation of a Chr 15 locus that profoundly affects the severity of SWD episodes. Together these new findings expand our knowledge of how natural variation modulates seizures, and highlights the feasibility of characterizing and validating modifiers in mouse strains and substrains in the post-genome sequence era. PLoS Genet 2014 Jul 10; 10(7):e1004454

Genetic approaches to studying mouse models of human seizure disorders.

In conclusion, we have discussed a reverse genetics approach to studying seizure disorders in mice (Fig. 1), employing a targeted mutagenesis method to exploit the genetic defects identified in human epilepsy families. After detailed characterization of the nature of the human mutation and the mouse counterpart gene, a targeting vector containing the human disease allele is created. The endogenous mouse gene is replaced by the human disease allele through homologous recombination in ES cells, leading to the generation of chimeric animals. Mice carrying one copy or both copies of the human mutation can be bred to study the phenotypic effect of heterozygous and homozygous mutations. At this stage, one may want to split the newly created mice into two groups. One group will go through seizure phenotyping tests, while the other group will be used to generate disease allele-carrying mice on a different genetic background. Phenotypic characterization of mice on different inbred strains includes behavioral monitoring and EEG analysis looking for the occurrence of spontaneous seizures, as well as routine cage examination looking for handling-provoked seizure and ECT- and PTZ- induced seizure paradigms looking for sensitivity to these stimuli. A complete evaluation of the seizure phenotype at the whole-animal level establishes the relevance of the mouse model to the human condition. Further investigation including imaging, electrophysiology and AED response in these mouse models will shed light on the mechanistic basis of the convulsive disorder. Current epilepsy research in mouse genetics offers promise for understanding the molecular mechanisms that underlie epileptogenesis in humans. A large-scale forward genetic effort to create novel mouse mutants with seizure phenotypes by in vivo chemical mutagenesis with ethyl-nitroso urea (ENU) is underway at the Jackson Laboratory (http://www.jax.org/nmf/). Genetic mapping and isolation of the affected genes in these seizure-prone models will provide additional molecular pathways involved in seizures. The mutant mice generated through both forward and reverse genetic approaches will be a valuable resource for the biomedical community to study epilepsy at the molecular level and to characterize the pathological consequences of seizures in the whole organism