Transcriptional Regulation of an Endoplasmic Reticulum Calcium Sensor
Material below summarizes the article NEUROD2 Regulates Stim1 Expression and Store-Operated Calcium Entry in Cortical Neurons, published on February 27, 2017, in eNeuro and authored by Gokhan Guner, Gizem Guzelsoy, Fatma Sadife Isleyen, Gulcan Semra Sahin, Cansu Akkaya, Efil Bayam, Eser Ilgin Kotan, Alkan Kabakcioglu, and Gulayse Ince-Dunn.
The cerebral cortex is the seat of higher cognitive functions such as learning, memory, planning, language acquisition, attention, and consciousness. In order to carry out these complex tasks, the cortex is composed of myriad types of neurons amounting to approximately 20-25 billion in humans.
A large proportion of these neurons — excitatory neurons — use the excitatory neurotransmitter glutamate and assemble into circuits that function in local communications and long-distance communications with other parts of the brain. A formidable task for the developing cerebral cortex is the production, differentiation, and assembly of excitatory cortical neurons into properly functioning networks.
A unique feature of brain development is that brain development is not only shaped by inherited genetic instructions but also by neuronal activity that is either spontaneous or driven by sensory experience. Calcium-regulated transcription factors emerge as integrators of the cellular effects of neuronal activity and transduce them into changes in gene expression programs.
Our interest in such a factor began when NEUROD2 was identified by a screen designed to clone calcium-regulated transcriptional activators in cortical neurons. NEUROD2 was then characterized as a transcription factor that is highly expressed in the differentiating excitatory neurons of the cortex.
Since its original cloning, a number of studies demonstrated NEUROD2’s essential role in various aspects of cortical development, such as the formation of neural maps that represent sensory stimuli and the maturation of dendrites and synapses. We reasoned that a potentially informative approach to understanding the cellular effects of neuronal activity-regulated gene expression would be to identify the genome-wide targets of a transcription factor such as NEUROD2.
To achieve this goal, we applied a ChIP-Seq (chromatin immunoprecipitation and sequencing) approach to identify genomic locations that were directly bound by NEUROD2 within the cerebral cortex of newly born mice pups. ChIP-Seq is a biochemical purification method that relies on immunoprecipitation of covalently crosslinked DNA binding proteins of interest to their target sequences followed by high-throughput sequencing of associated DNA.
As expected, our computational analyses of all NEUROD2 binding sites revealed that a large proportion of them mapped onto promoter regions of target genes. Unexpectedly, however, we also observed that even a larger proportion of binding events were localized to non-promoter regions located within genes or at intergenic regions.
We reasoned that at least a subset of these binding events might correspond to cis-regulatory elements. These are DNA sequences that are typically non-coding and bind multiple transcription factors to regulate gene expression in a manner that is specific to a particular cell type, developmental stage, or external stimulus. Therefore, we focused our attention to a top NEUROD2 binding site that was localized to a potential cis-regulatory element.
This top NEUROD2 binding site was localized within an intron of a gene called Stim1 (Stromal Interaction Molecule 1). Interestingly, the protein product of Stim1 gene encodes a regulator of a particular pathway of calcium entry into neurons. This pathway, named store-operated calcium entry (SOCE), is composed of calcium influx through plasma membrane calcium channels which are specifically activated by the emptying of endoplasmic reticulum calcium stores. STIM1 protein is a sensor of ER calcium content, and upon ER emptying, it induces calcium influx from the external milieu.
Initially, we questioned how Stim1 expression was impacted by NEUROD2. Upon silencing of Neurod2 in primary cortical neurons, we observed a significant increase in Stim1 expression at the mRNA and protein level, suggesting that NEUROD2 acts as a suppressor of Stim1 gene.
Our results suggested a testable physiological hypothesis, which is that NEUROD2 fine-tunes neuronal SOCE. To address this intriguing possibility, we carried out live calcium-imaging experiments following knockdown or overexpression of NEUROD2.
Indeed, our results demonstrated that while suppression of NEUROD2 expression upregulates SOCE, its overexpression augments SOCE. Thus, our results demonstrated that NEUROD2 is a critical regulator of neuronal SOCE.
Store-operated calcium entry is attracting increased interest from the neuroscience community in light of recent studies demonstrating its role in guiding the growth of axons, migration of neurons, and the pathogenesis of certain neurological diseases like epilepsy and Alzheimer’s disease.
In contrast to other sources of calcium influx, very little is known about the mechanisms by which SOCE regulates signaling and many aspects of physiological and morphological maturation of neurons. Specifically, how SOCE itself is regulated by upstream gene expression programs in neurons is essentially completely unknown.
We hypothesize that a feedback regulatory loop might exist in neurons for the regulation of SOCE. In such a scenario, calcium influx activates NEUROD2, which in turn suppresses Stim1 expression, thereby inhibiting an essential component of store-operated calcium entry.
Our future experiments will focus on directly testing this intriguing possibility.
NEUROD2 Regulates Stim1 Expression and Store-Operated Calcium Entry in Cortical Neurons. Gokhan Guner, Gizem Guzelsoy, Fatma Sadife Isleyen, Gulcan Semra Sahin, Cansu Akkaya, Efil Bayam, Eser Ilgin Kotan, Alkan Kabakcioglu, Gulayse Ince-Dunn. eNeuro Feb 2017, 4 (1) DOI: 10.1523/ENEURO.0255-16.2017