Abstract
Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment.
Highlights
Cocaine- and amphetamine-regulated transcript (CART) plays a pivotal role in neuroprotection against stroke
Electrophoretic mobility shift assay (EMSA) showed that a clear protein-intron NRSE (iNRSE) complex formed in a dose-dependent manner when an increasing amount of HeLa nuclear extracts was incubated with the CART iNRSE probe (Fig. 1B, lanes 2– 4)
The affinity of neuron-restrictive silencer factor (NRSF) with iNRSE was similar to that with promoter NRSE (pNRSE). These results indicate that the NRSF protein can bind to the iNRSE sequence in the CART gene in vitro
Summary
Cocaine- and amphetamine-regulated transcript (CART) plays a pivotal role in neuroprotection against stroke. Results: Transcriptional repressor neuron-restrictive silencer factor (NRSF) represses CART through binding to two NRSEs in the CART promoter and intron. Conclusion: NRSF represses CART transcription and antagonizes cAMP-response element-binding protein signaling through a dual NRSE mechanism. We show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment. The dual NRSF-NRSE mechanism exhibits an antagonizing effect on CREB-CRE signaling under ischemia insult These findings provide novel insights on the negative regulation of CART transcription
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