Analysis of Noncoding RNA Function at Specific Genomic Loci

Abstract

Long noncoding RNAs (lncRNAs) have emerged as important effectors of gene expression, due to their interactions with chromatin regulatory complexes and their implication in developmental processes such as genomic imprinting, dosage compensation, and maintenance of pluripotency. However, despite numerous studies suggesting regulatory roles for lncRNAs in gene expression, there is limited mechanistic insight into how lncRNAs can target and regulate specific sets of genes. To better understand the regulatory effects of lncRNAs, I explored factors contributing to interactions between lncRNAs and specific genomic loci. To this end, I analyzed genomic binding sites for two highly abundant RNAs, NEAT1 and MALAT1. NEAT1 and MALAT1 bind hundreds of genomic sites in trans, primarily localizing to active genes. NEAT1 and MALAT1 colocalize to many of these loci, but display distinct gene body binding patterns, suggesting independent but complementary functions for these RNAs. Mass spectrometry analysis of lncRNA-associated proteins identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function. Transcriptional inhibition or stimulation alters genome-wide localization of NEAT1, implying that underlying DNA sequence does not target NEAT1 to chromatin and that localization is instead responsive to the process of transcription. In addition to examining lncRNAs associated with active genes, I characterized the impact of lncRNAs on gene repression at specific loci. I focused on a human lncRNA expressed during neural differentiation, CAT7, whose locus is composed primarily of tandem repeats and is proximal to the Polycomb Repressive Complex 1 (PRC1) target genes SHH and MNX1. RNA immunoprecipitation experiments indicate CAT7 directly interacts with PRC1 in vivo. Loss of the CAT7 tandem repeats leads to derepression of MNX1 and reduced PRC1 binding to the locus. Coincident with loss of gene repression, perturbation of CAT7 leads to diminished long-range genomic contacts between MNX1 and other cis-localized PRC1-bound loci. These results suggest a role for CAT7 in maintenance of a localized region of gene repression near its site of transcription. Overall, these studies support a model wherein lncRNAs aid in the organization of subnuclear domains that coordinate the activation or repression of specific sets of genomic loci.