Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulators of genomic processes and cellular specification. Many lncRNAs regulate chromatin by functionally impacting the epigenetic state through direct interactions with chromatin-modifying proteins. We developed a protocol to enrich for chromatin-lncRNA interactions and used this technique to identify several candidate lncRNAs that interact with the Polycomb group (PcG) proteins. Our immunoprecipitation protocol uses a crosslinked chromatin fraction as the input and employs stringent washes and cross-validation techniques to dramatically decrease mRNA signal (as a metric of transient interactions or false positives), and increase the dynamic range of conventional RNA immunoprecipitation protocols. Applying this protocol to the PRC1 component Bmi1, we have identified 11 PcG-interacting lncRNA candidates whose expression impacts the transcription of many other chromatin factors and PcG targets. We focus on knockdown of one lncRNA candidate, CAT7, which increases expression of several homeobox-containing transcription factors as well as chromatin interacting proteins, including Trithorax group proteins, Jumanji-domain containing proteins, and PcG-like proteins in HeLa cells. Consistent with the observed increase in gene expression, knockdown of CAT7 decreases PcG binding (Suz12, H3K27me3 and Bmi1) at the promoter of the homeodomain protein Mnx1, located at the boundary of an adjacent gene desert. During early motor neuron differentiation from embryonic stem cells, knockdown of CAT7 is accompanied by changes in expression of master regulators of neuronal specification: increased upregulation Mnx1, upregulation of Isl1, and downregulation of Irx3, as well as changes in expression to several other PcG-regulated targets. Overall, this protocol is the first of its kind to efficiently identify de novo interactions between the PcG proteins and lncRNAs which impact PcG binding or PcG target gene expression.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/11744453 |
| Date | 04 February 2016 |
| Creators | Ray, Mridula Kumari |
| Contributors | Kingston, Robert Edward |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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