IV. CELL CIRCUIT RECONSTRUCTION: Stem cell renewal and differentiation - transcriptional and epigenetic regulation by proteins and lincRNAs

We proposed to decipher the chromatin regulatory circuit of ES cells, focusing on the protein and RNA complexes that catalyze histone modification and/or physically interact with modified histones. We will leverage the developed technological approaches and analytical strategies of Aim 1 and 2 in the following four steps. (1) We will identify candidate components of the ES cell chromatin circuit, based on ES-specific expression of chromatin modifiers and lincRNAs, and use perturbation assays to determine their functional influence on an ES cell expression signature and phenotype (Aim 4.1). (2) We will determine the physical circuitry of these candidates by comprehensively mapping binding interactions between each candidate and genomic DNA regions, and by identifying lincRNAs associated with different chromatin complexes (Aim 4.2.1, 4.2.2). (3) We will systematically perturb each component and monitor the resulting changes in chromatin states and complex formation, using diverse assays (Aim 4.2.3). (4) We will integrate the physical and functional data, with available information on TF binding into a model of the circuit that sustains the pluripotent cell state (Aim 4.3).

Publications

Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells.

Citation: Ram O, Goren A, Amit I, Shoresh N, Yosef N, Ernst J, Kellis M, Gymrek M, Issner R, Coyne M, Durham T, Zhang X, Donaghey J, Epstein CB, Regev A, Bernstein BE. Cell. 2011 Dec 23;147(7):1628-39.
Link to journal: http://dx.doi.org/10.1016/j.cell.2011.09.057

Abstract: Hundreds of chromatin regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs colocalize in characteristic patterns at distinct chromatin environments, at genes of coherent functions, and at distal regulatory elements. When comparing between cell types, CRs redistribute to different loci but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.

lincRNAs act in the circuitry controlling pluripotency and differentiation.

Citation: Guttman M, Donaghey J, Carey BW, Garber M, Grenier JK, Munson G, Young G, Lucas AB, Ach R, Bruhn L, Yang X, Amit I, Meissner A, Regev A, Rinn JL, Root DE, Lander ES. Nature. 2011 Aug 28;477(7364):295-300.
Link to journalhttp://dx.doi.org/10.1038/nature10398

Abstract: Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.

Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. 

Citation: Guttman M, Garber M, Levin JZ, Donaghey J, Robinson J, Adiconis X, Fan L, Koziol MJ, Gnirke A, Nusbaum C, Rinn JL, Lander ES, Regev A.Nat Biotechnol. 2010 May;28(5):503-10. Epub 2010 May 2. Erratum in: Nat Biotechnol. 2010 Jul;28(7):756.
Link: http://dx.doi.org/10.1038/nbt.1633

Abstract: Massively parallel cDNA sequencing (RNA-Seq) provides an unbiased way to study a transcriptome, including both coding and noncoding genes. Until now, most RNA-Seq studies have depended crucially on existing annotations and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We applied it to mouse embryonic stem cells, neuronal precursor cells and lung fibroblasts to accurately reconstruct the full-length gene structures for most known expressed genes. We identified substantial variation in protein coding genes, including thousands of novel 5' start sites, 3' ends and internal coding exons. We then determined the gene structures of more than a thousand large intergenic noncoding RNA (lincRNA) and antisense loci. Our results open the way to direct experimental manipulation of thousands of noncoding RNAs and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes.