FIGURE 4.

Noncoding transcript AK090153 is targeted by REST. (A) Expression of AK090153 in NS5 was validated by RT-PCR. A control reverse transcription (RT) reaction was carried out without RTase enzyme (−RT). (B) The ability of RE1 mus4186 to repress reporter gene expression in a REST-dependent manner was demonstrated as in Figure 3B by luciferase assay. (C) AK090153 resides on mouse Chromosome 2 and encompasses the protein-coding gene Sptlc3 on the opposite strand. The validated REST binding site (RE1) mus4186 lies toward the 3′ intronic region of AK090153 (red rectangle). A CpG island encompasses the transcriptional start site (green rectangle). (D) Adenovirally delivered dominant-negative REST (Ad DN:REST) de-repressed transcription of AK090153 in the neural stem cell line, NS5. Transcript levels were measured by means of intron-spanning Taqman probes and normalized to the housekeeping gene β-actin. Assays were carried out on four control replicates (adenovirus only, Ad) and five dominant-negative replicates (Ad DN:REST). No expression of Sptlc3 could be detected using SYBR green qPCR with the same cDNA samples. Statistical significance was calculated using Student's t-test.