Search all library resources

Genomic analysis of neuron-restrictive silencer factor activity in neuronal and non-neuronal human cell lines [electronic resource]

The neuron-restrictive silencer factor/RE-1 silencing transcription factor (NRSF/REST) is thought to be a negative regulator of neuronal genes in non-neuronal cells. However, evidence of its continued expression and activity in neurons suggests that NRSF may play other roles. A complete knowledge of NRSF target genes in neuronal and non-neuronal cells is the first step to understanding its functions. Using chromatin immunoprecipitation and quantitative PCR, I experimentally tested the occupancy of NRSF in living non-neuronal cells on 113 candidate binding sites predicted on the basis of conservation across the human, mouse, and dog genomes. These tests helped to further refine the prediction algorithm and identified a number of NRSF-bound regulatory microRNAs that may work in a feedforward loop to downregulate NRSF and its co-repressor, CoREST. I next focused on understanding NRSF recruitment in neuron-derived versus non-neuronal cell lines, using chromatin immunoprecipitation paired with ultrahigh-throughput sequencing (ChIP-seq) to get a direct, genome-wide picture of NRSF binding in human neuron-derived and non-neuronal cell lines. I found a large overlap in the NRSF binding pattern between the two cell types, particularly in binding sites found to be strongly or commonly bound. There is a subset of strong sites bound in all cell types, and weaker sites that are more likely to be cell-type specific. These common sites contain the canonical NRSE while the cell line unique sites do not. Finally, I used another ultrahigh-throughput sequencing based method to catalog and quantify all mRNA transcripts in each of the cell lines (RNA-seq) to add target gene expression to the analysis of NRSF function. Common target genes were more likely to be highly expressed in the neuron-derived cell line than in non-neuronal cell lines despite NRSF binding in both. I also found that the neuron-specific binding sites were primarily located in exons and promoters, while common or non-neuronal specific binding sites were primarily located in introns and intergenic regions. Differences in binding strength and target gene expression levels suggest that NRSF has different binding mechanisms and functions in neuron-derived and non-neuronal human cell lines.