Approximately 75% of the human genome is transcribed, the majority of which does not encode protein.However, many noncoding RNAs (ncRNAs) are rapidly degraded after transcription, and relatively few have established functions, questioning the significance of this observation. Here we show that esBAF, a SWI/SNF family nucleosome remodeling factor, suppresses transcription of ncRNAs from~57,000 nucleosome-depleted regions (NDRs) throughout the genome of mouse embryonic stem cells (ESCs). We show that esBAF functions to both keep NDRs nucleosome-free and promote elevated nucleosome occupancy adjacent to NDRs. Reduction of adjacent nucleosome occupancy upon esBAF depletion is strongly correlated with ncRNA expression, suggesting that flanking nucleosomes form a barrier to pervasive transcription. Upon forcing nucleosome occupancy near two NDRs using a nucleosome-positioning sequence, we found that esBAF is no longer required to silence transcription. Therefore, esBAF's function to enforce nucleosome occupancy adjacent to NDRs, and not its function to maintain NDRs in a nucleosome-free state, is necessary for silencing transcription over ncDNA. Finally, we show that the ability of a strongly positioned nucleosome to repress ncRNA depends on its translational positioning. These data reveal a novel role for esBAF in suppressing pervasive transcription from open chromatin regions in ESCs.[Keywords: esBAF; ncRNA; chromatin remodeling; nucleosome occupancy; transcription] Supplemental material is available for this article.Received October 1, 2014; revised version accepted January 7, 2015.Transcription by RNA polymerase II (RNAPII) is widespread throughout the genome and not limited to coding sequences. In fact, nongenic regions account for the majority of sequences transcribed by RNAPII in higher eukaryotes (Djebali et al. 2012;Stamatoyannopoulos 2012). Noncoding RNA (ncRNA) is produced from many different regulatory regions in cells, including the 39 ends of protein-coding genes, divergent transcription from promoters, and other distal regulatory elements, including enhancers or other gene-distal DNase I-hypersensitive sites (DHSs) (Jacquier 2009). While ncRNAs such as ribosomal RNAs (rRNAs), microRNAa (miRNAs), Piwiinteracting RNAs (piRNAs), and some long ncRNAs (lncRNAs) have well-established functions, the functions of the vast majority of ncRNAs discovered by transcriptome sequencing, if any, are unknown.Antisense transcripts originating from promoters and aberrant transcription from intragenic regions have been well described in a diverse set of eukaryotes (Kaplan 2003;Carrozza et al. 2005;Cheung et al. 2008;Preker et al. 2008;Seila et al. 2008;Xie et al. 2011;Carvalho et al. 2013). In addition, transcription termination sites (TTSs) possess a promoter architecture sufficient to permit transcription initiation (Whitehouse et al. 2007;Murray et al. 2012;Castelnuovo et al. 2014). The mechanisms that regulate transcription start site (TSS)-associated ncRNA expression have recently been examined by several groups (Huan...