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

• Published in: Journal of molecular neuroscience Vol. 67; no. 2; pp. 282 - 294
• Main Authors: Corney, B.P.A., Widnall, C.L., Rees, D.J., Davies, J.S., Crunelli, V., Carter, D.A.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-02-2019; Springer Nature B.V
• Subjects: 3' Untranslated regions; Animals; Antisense RNA; Biomedical and Life Sciences; Biomedicine; Brain; CACNG2 protein; Calcium; Calcium channels (voltage-gated); Calcium Channels - genetics; Cell Biology; Cell lines; Disease; Disease control; Domains; Epilepsy; Epilepsy - genetics; Functional morphology; Gene expression; Gene polymorphism; Gene silencing; Mice; Microsatellite Repeats; Molecular modelling; Neurochemistry; Neurology; Neurosciences; Neurotransmission; PC12 Cells; Phenotypes; Polymorphism; Polymorphism, Genetic; Potassium channels; Potassium Channels, Tandem Pore Domain - genetics; Promoter Regions, Genetic; Proteins; Proteomics; Rats; Rats, Sprague-Dawley; Regulatory sequences; Rest; RNA, Long Noncoding - genetics; RNA, Long Noncoding - metabolism; Synapsin; Synapsins - genetics; Transfection; REST; Synaptic; AMPA; lncRNA; Bidirectional; Absence epilepsy; TARP; CaRF
• ISSN: 0895-8696; 1559-1166; 1559-1166
• DOI: 10.1007/s12031-018-1208-x

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.