Functional Characterization of LncRNAs Involved in Pluripotency or Differentiation

Cold Spring Harbor Laboratory, Cold Spg Hbr NY

Investigators

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Abstract

Project Summary The over-arching theme of my research program is focused on elucidating innovative ways that the genome is expressed and regulated. Our focus is on long non-coding RNAs (lncRNAs) as they represent a major class of regulatory molecules that tend to be expressed in a cell and/or tissue specific expression pattern. The latest GENCODE annotation indicates as many lncRNA genes as protein coding genes, yet we know far less in regard to their in-depth molecular function. Since many lncRNAs are developmentally regulated, increasing evidence has suggested that they may be among the key regulatory factors in stem cells, lineage commitment, and differentiation. We have identified a significant number of lncRNAs that are upregulated in mouse embryonic stem cells (mESCs) vs neural progenitor cells. The long-term goal of this project is to begin to define functional classes of lncRNAs that have roles in pluripotency and/or differentiation. Over the next 5 years we propose to determine the function of several prioritized lncRNAs in pluripotency and/or lineage commitment and identify the specific pathways that they regulate. We prioritized 4 lncRNAs for study as: (1) all 4 are both >2-fold upregulated, and are highly expressed in mESCs vs NPCs, and (2) in addition, they have potential human orthologs based on synteny and/or partial sequence homology. In depth analysis will define how these lncRNAs regulate differentiation through their impacts on gene expression in cis or trans and/or in other aspects of cellular function. We will examine the impact of their loss or over-expression on global gene expression in mESCs as well as in early developing mouse embryos. Of particular relevance will be identifying alterations in pathways involved in pluripotency or in regulating differentiation to a specific germ layer. In addition, control and KO mESCs for each lncRNA will be examined by ChIP-seq (histone modifications, etc) and ATAC-seq to identify potential epigenetic differences and/or changes in chromatin structure. For those lncRNAs that are chromatin associated we will carry out Chromatin Isolation by RNA Purification-seq (ChIRP-seq) 54,55 to determine their association with specific genes. For those that are in the cytoplasm we will focus on whether they are competitive endogenous RNAs, have a role in regulating translation, regulating protein stability, and pursue interactions with their associated cytoplasmic compartment. Since RNAs reside in the cell as RNP complexes, and the proteins in these complexes are critical for targeting and/or function, we will use an RNA pulldown approach adapted from RNA Antisense Pulldown-Mass Spectrometry (RAP-MS) 57 that we modified and optimized during the current funding period 48 to identify specific lncRNA associated proteins. Together, the proposed studies will unravel the role of up to 4 lncRNAs and critical functional interactions between each of them and their interacting proteins/genes on a genomic, cellular, molecular, and embryonic level. The proposed studies will shed light on the functional role of these lncRNAs in pluripotency, lineage commitment and/or differentiation, and will provide new insights as to how regulatory signals instructed by lncRNAs can impact differentiation.