Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells

“Genes differentially expressed between ESCs and NECs were identified using DESeq2 (FDR < 0.05 and a log2 fold change of >1.5).”

“7 (http://homer.salk.edu/homer/) (Heinz et al., 2010).”

“Data were converted to GRCh37 using liftOver (Kent et al., 2002).”

“A/B compartments were called by computing the principal components of the distance- and coverage-corrected interaction profile correlation matrix at 250 kb resolution (Lieberman-Aiden et al., 2009).”

“TADs were identified based on directionality indices (Dixon et al., 2012) of Hi-C interactions 1 Mb upstream and downstream from a 25 kb sliding window every 5 kb steps, which were then smoothed using a running average over a ± 25 kb window.”

“The definition of chromatin signatures has enabled the genome-wide identification of enhancer elements across multiple human cell types (ENCODE Project Consortium, 2012; Heintzman et al., 2007, 2009; Pennacchio et al., 2006; Rada-Iglesias et al., 2011; Kundaje et al., 2015).”

“…activity associated with poised and active enhancers and have been used to show that widespread changes in enhancer position and activity occur upon cell fate decisions such as the lineage commitment of pluripotent cells (Creyghton et al., 2010; Rada-Iglesias et al., 2011; Zentner et al., 2011).”

“The definition of chromatin signatures has enabled the genome-wide identification of enhancer elements across multiple human cell types (ENCODE Project Consortium, 2012; Heintzman et al., 2007, 2009; Pennacchio et al., 2006; Rada-Iglesias et al., 2011; Kundaje et al., 2015).”

“We assessed their localisation with respect to higher order features of chromosome architecture including TADs and Insulated Neighborhoods (INs) (Dixon et al., 2012; Ji et al., 2016; Nora et al., 2012; Sexton et al., 2012).”

“The definition of chromatin signatures has enabled the genome-wide identification of enhancer elements across multiple human cell types (ENCODE Project Consortium, 2012; Heintzman et al., 2007, 2009; Pennacchio et al., 2006; Rada-Iglesias et al., 2011; Kundaje et al., 2015).”

“We used ChromHMM (Ernst and Kellis, 2012) to integrate these histone marks and to define four combinatorial chromatin states in both ESCs and NECs, as follows: active (characterised by H3K4me3 and/or H3K27ac); Polycomb-associated (H3K27me3); poised (H3K4 methylation and H3K27me3); and background…”

“However, a regulatory function for some ‘unmarked’ PIRs also cannot be ruled out as recent mutagenesis experiments have identified functional elements that lack ’classic’ chromatin annotations (Pradeepa et al., 2016; Rajagopal et al., 2016).”

“We assessed their localisation with respect to higher order features of chromosome architecture including TADs and Insulated Neighborhoods (INs) (Dixon et al., 2012; Ji et al., 2016; Nora et al., 2012; Sexton et al., 2012).”

“These TAD definitions were used to compute the fraction of signifi- cant PCHi-C interactions falling inside TADs, alongside TADs reported by Dixon et al., 2015.”

“…the whole dataset, we found a significant concordance between the chro- matin states at promoters and their PIRs (Figure 1D), which is in line with previous studies in other human cell types (Jin et al., 2013; Mifsud et al., 2015; Sanyal et al., 2012) and provides validation of our dataset.”

“…decisions are associated with profound changes in chromatin organisation, which underlie the activation of lineage-specific and the silencing of lineage-inappropriate genes (Buecker and Wysocka, 2012; Bulger and Groudine, 2010; Calo and Wysocka, 2013; Hallikas et al., 2006; Ong and Corces, 2012).”

“Coverage- and distance-related correction factors of the binned data were computed at 25 kb and 250 kb resolutions, based on the iterative correction algorithm (Imakaev et al., 2012).”

“It is gener- ally accepted that this long-range action is facilitated by DNA-looping interactions (Pennacchio et al., 2013; Shlyueva et al., 2014).”

“Finally, there is also a possibility that multiple enhancers are jointly engaged in ‘chromatin hubs’ with promoters, rather than acting individually (Hanscombe et al., 1991; Jiang et al., 2016; Patrinos et al., 2004; Tolhuis et al., 2002; Wijgerde et al., 1995).”

“PIRs were also significantly enriched for sites bound by the architectural protein CTCF (Figure 1—figure supplement 2B; based on ENCODE data available for ESCs only), consistent with previous observations in other cell types (Jin et al., 2013; Phillips-Cremins et al., 2013; Sanyal et al., 2012).”

“…studies provided evidence of interactions associated with transcriptional changes upon line- age commitment (Denholtz et al., 2013; Kieffer-Kwon et al., 2013; Phillips-Cremins et al., 2013; eLife digest Virtually every cell in the body contains the same set of DNA, which encodes thousands of genes.”

“…1 of 26 Cis-regulatory elements are often considerable distances away from their target gene promoters and may not control their nearest genes (Carvajal et al., 2001; Jeong et al., 2006; Marinić et al., 2013; Pennacchio et al., 2006; Ruf et al., 2011; Sagai et al., 2005; Spitz et al., 2003).”

“…15 of 26 Theoretically, the generally independent enhancer action also enables activation signals from individual elements to quantitatively ‘add up’ (at least to some extent) to promote stronger transcrip- tional outputs (Arnold et al., 2013; Bothma et al., 2015; Lam et al., 2015; Spivakov, 2014).”

“Embryo reporter assays and enhancer activity patterns are from the VISTA Enhancer Browser (Visel et al., 2007).”

“Over the last decade, chromosome conformation capture (3C) and derived methods have enabled the biochemical mapping of looping interactions to offer new insights into their architecture across different cell types (Dekker et al., 2013; de Laat and Duboule, 2013; Schmitt et al., 2016).”

“…to enrich Hi-C libraries for interactions involving the promoters of most annotated genes, providing a global view on promoter interactions that is independent of the activity of interacting regions and identity of proteins recruited to them (Mifsud et al., 2015; Schoenfelder et al., 2015a).”

“Hi-C analysis and the definition of TADs, TAD boundaries and compartments Raw sequencing reads were processed using the HiCUP pipeline (Wingett et al., 2015), which mapped sequencing read pairs against the human genome (GRCh37), filtered out experimental arti- facts such as circularized reads and…”

“The emerging studies at the single-molecule level (such as [Bartman et al., 2016; Fukaya et al., 2016]) will likely shed further light on the molecular mechanisms that underpin the principles of CRU organisation.”

“It is gener- ally accepted that this long-range action is facilitated by DNA-looping interactions (Pennacchio et al., 2013; Shlyueva et al., 2014).”

“…activity associated with poised and active enhancers and have been used to show that widespread changes in enhancer position and activity occur upon cell fate decisions such as the lineage commitment of pluripotent cells (Creyghton et al., 2010; Rada-Iglesias et al., 2011; Zentner et al., 2011).”

“…gene regulation by multiple enhancers The CRU view provides an opportunity to consider multi-modular gene regulation in early human development that has hitherto been studied on a limited number of genes, predominantly in model organisms (Barolo, 2012; Cannavò et al., 2016; Hong et al., 2008).”

“Finally, there is also a possibility that multiple enhancers are jointly engaged in ‘chromatin hubs’ with promoters, rather than acting individually (Hanscombe et al., 1991; Jiang et al., 2016; Patrinos et al., 2004; Tolhuis et al., 2002; Wijgerde et al., 1995).”

“…1 of 26 Cis-regulatory elements are often considerable distances away from their target gene promoters and may not control their nearest genes (Carvajal et al., 2001; Jeong et al., 2006; Marinić et al., 2013; Pennacchio et al., 2006; Ruf et al., 2011; Sagai et al., 2005; Spitz et al., 2003).”

“…specific determinants of chromosomal interactions are still not fully understood, which presents challenges for the high-confidence predic- tion of regulatory interactions from sequence and epigenetic information (Mora et al., 2016; Roy et al., 2015; Shlyueva et al., 2014; Whalen et al., 2016).”

“PCHi-C data normalisation and signal detection using the CHiCAGO pipeline (Cairns et al., 2016) identified 75,795 significant cis-interactions between promoters and other genomic regions in ESCs and 75,624 in NECs.”

“…1 of 26 Cis-regulatory elements are often considerable distances away from their target gene promoters and may not control their nearest genes (Carvajal et al., 2001; Jeong et al., 2006; Marinić et al., 2013; Pennacchio et al., 2006; Ruf et al., 2011; Sagai et al., 2005; Spitz et al., 2003).”

“For INs, we used the published genomic coordinates (available for ESCs only) that were defined on the basis of cohesin ChIA-PET and CTCF-binding data (Ji et al., 2016).”

“We assessed their localisation with respect to higher order features of chromosome architecture including TADs and Insulated Neighborhoods (INs) (Dixon et al., 2012; Ji et al., 2016; Nora et al., 2012; Sexton et al., 2012).”

“…been suggested that this cis-regulatory organisation ensures the robustness of gene regulation and can buffer the effects of deleterious sequence variation, as well as providing opportunities for evolutionary innovation (Barolo, 2012; Cannavò et al., 2016; Hong et al., 2008; Perry et al., 2010).”

“The emerging studies at the single-molecule level (such as [Bartman et al., 2016; Fukaya et al., 2016]) will likely shed further light on the molecular mechanisms that underpin the principles of CRU organisation.”

“Over the last decade, chromosome conformation capture (3C) and derived methods have enabled the biochemical mapping of looping interactions to offer new insights into their architecture across different cell types (Dekker et al., 2013; de Laat and Duboule, 2013; Schmitt et al., 2016).”

“These studies provided evidence of interactions associated with transcriptional changes upon line- age commitment (Denholtz et al., 2013; Kieffer-Kwon et al., 2013; Phillips-Cremins et al., 2013; eLife digest Virtually every cell in the body contains the same set of DNA, which encodes thousands of…”

“…bound by specific proteins (such as cohesin and RNA polymerase II) have made it possible to detect subsets of promoter-enhancer interactions at high resolution (Heidari et al., 2014; Li et al., 2015; Sanyal et al., 2012) and delineate their dynamics during cell differentiation and reprogramming.”

“…decisions are associated with profound changes in chromatin organisation, which underlie the activation of lineage-specific and the silencing of lineage-inappropriate genes (Buecker and Wysocka, 2012; Bulger and Groudine, 2010; Calo and Wysocka, 2013; Hallikas et al., 2006; Ong and Corces, 2012).”

“…bound by specific proteins (such as cohesin and RNA polymerase II) have made it possible to detect subsets of promoter-enhancer interactions at high resolution (Heidari et al., 2014; Li et al., 2015; Sanyal et al., 2012) and delineate their dynamics during cell differentiation and reprogramming.”

“These studies provided evidence of interactions associated with transcriptional changes upon line- age commitment (Denholtz et al., 2013; Kieffer-Kwon et al., 2013; Phillips-Cremins et al., 2013; eLife digest Virtually every cell in the body contains the same set of DNA, which encodes thousands of…”

“However, a regulatory function for some ‘unmarked’ PIRs also cannot be ruled out as recent mutagenesis experiments have identified functional elements that lack ’classic’ chromatin annotations (Pradeepa et al., 2016; Rajagopal et al., 2016).”

“…1 of 26 Cis-regulatory elements are often considerable distances away from their target gene promoters and may not control their nearest genes (Carvajal et al., 2001; Jeong et al., 2006; Marinić et al., 2013; Pennacchio et al., 2006; Ruf et al., 2011; Sagai et al., 2005; Spitz et al., 2003).”

“However, a regulatory function for some ‘unmarked’ PIRs also cannot be ruled out as recent mutagenesis experiments have identified functional elements that lack ’classic’ chromatin annotations (Pradeepa et al., 2016; Rajagopal et al., 2016).”

“…gene regulation by multiple enhancers The CRU view provides an opportunity to consider multi-modular gene regulation in early human development that has hitherto been studied on a limited number of genes, predominantly in model organisms (Barolo, 2012; Cannavò et al., 2016; Hong et al., 2008).”

“Examples of instructive and permissive interactions have been described in pluripotent cell reprogramming and differentiation (Apostolou et al., 2013; Denholtz et al., 2013; Phillips- Cremins et al., 2013; Wei et al., 2013; Zhang et al., 2013), but little was known on a global scale about which…”

“DOI: 10.7554/eLife.21926 2 of 26 Zhang et al., 2013), as well as revealed interactions formed in anticipation of changes in gene activity (Apostolou et al., 2013; Ghavi-Helm et al., 2014; Wei et al., 2013).”

“…decisions are associated with profound changes in chromatin organisation, which underlie the activation of lineage-specific and the silencing of lineage-inappropriate genes (Buecker and Wysocka, 2012; Bulger and Groudine, 2010; Calo and Wysocka, 2013; Hallikas et al., 2006; Ong and Corces, 2012).”

“However, analyses in Drosophila have identified exceptions to additive enhancer activity (Bothma et al., 2015) and have provided exam- ples of enhancers that activate more than one promoter in a coordinated fashion (Fukaya et al., 2016), which is not immediately expected from the ‘hit-and-run’…”

“Finally, there is also a possibility that multiple enhancers are jointly engaged in ‘chromatin hubs’ with promoters, rather than acting individually (Hanscombe et al., 1991; Jiang et al., 2016; Patrinos et al., 2004; Tolhuis et al., 2002; Wijgerde et al., 1995).”

“…connectivity to regions associated with Polycomb-mediated repression and poising, reinforcing the role of Polycomb-group proteins in controlling chromosomal topology at transcriptionally inactive genes (Entrevan et al., 2016; Li et al., 2015; Schoenfelder et al., 2015b; Vieux-Rochas et al., 2015).”

“…1 of 26 Cis-regulatory elements are often considerable distances away from their target gene promoters and may not control their nearest genes (Carvajal et al., 2001; Jeong et al., 2006; Marinić et al., 2013; Pennacchio et al., 2006; Ruf et al., 2011; Sagai et al., 2005; Spitz et al., 2003).”

“5 mouse embryo (EMAGE gene expression database; EMAGE:1689; [Richardson et al., 2014]) broadly matches the combined tissue-restricted pattern of its enhancers.”

“…(potentially at both single-state and dual-state CRUs) that is consistent with the ‘hit-and-run’ model of transcrip- tional regulation (Schaffner, 1988; Varala et al., 2015), and provides a flexible way to fine-tune the expression of multi-enhancer genes (Guerrero et al., 2010; Lagha et al., 2012).”

“…1 of 26 Cis-regulatory elements are often considerable distances away from their target gene promoters and may not control their nearest genes (Carvajal et al., 2001; Jeong et al., 2006; Marinić et al., 2013; Pennacchio et al., 2006; Ruf et al., 2011; Sagai et al., 2005; Spitz et al., 2003).”

“DOI: 10.7554/eLife.21926 2 of 26 Zhang et al., 2013), as well as revealed interactions formed in anticipation of changes in gene activity (Apostolou et al., 2013; Ghavi-Helm et al., 2014; Wei et al., 2013).”

“…gene regulation by multiple enhancers The CRU view provides an opportunity to consider multi-modular gene regulation in early human development that has hitherto been studied on a limited number of genes, predominantly in model organisms (Barolo, 2012; Cannavò et al., 2016; Hong et al., 2008).”

“…specific determinants of chromosomal interactions are still not fully understood, which presents challenges for the high-confidence predic- tion of regulatory interactions from sequence and epigenetic information (Mora et al., 2016; Roy et al., 2015; Shlyueva et al., 2014; Whalen et al., 2016).”

“…specific determinants of chromosomal interactions are still not fully understood, which presents challenges for the high-confidence predic- tion of regulatory interactions from sequence and epigenetic information (Mora et al., 2016; Roy et al., 2015; Shlyueva et al., 2014; Whalen et al., 2016).”

“…15 of 26 Theoretically, the generally independent enhancer action also enables activation signals from individual elements to quantitatively ‘add up’ (at least to some extent) to promote stronger transcrip- tional outputs (Arnold et al., 2013; Bothma et al., 2015; Lam et al., 2015; Spivakov, 2014).”

“Promoter – enhancer interactions are vitally important for gene regulation and their disruption may lead to pronounced developmental abnormalities (Epstein, 2009).”

“…15 of 26 Theoretically, the generally independent enhancer action also enables activation signals from individual elements to quantitatively ‘add up’ (at least to some extent) to promote stronger transcrip- tional outputs (Arnold et al., 2013; Bothma et al., 2015; Lam et al., 2015; Spivakov, 2014).”

“Over the last decade, chromosome conformation capture (3C) and derived methods have enabled the biochemical mapping of looping interactions to offer new insights into their architecture across different cell types (Dekker et al., 2013; de Laat and Duboule, 2013; Schmitt et al., 2016).”

“Finally, there is also a possibility that multiple enhancers are jointly engaged in ‘chromatin hubs’ with promoters, rather than acting individually (Hanscombe et al., 1991; Jiang et al., 2016; Patrinos et al., 2004; Tolhuis et al., 2002; Wijgerde et al., 1995).”

“Furthermore, while it is generally accepted that many genes are controlled by multiple regulatory elements (Barolo, 2012; Miguel-Escalada et al., 2015) our understanding of multi-modular gene regulation remains limited, particularly in the context of mammalian development and stem cell…”

“Here, we use PCHi-C in human embryonic stem cells (ESCs) and ESC-derived neuroectodermal cells (NECs) (Bajpai et al., 2009) to create a high-resolution resource of promoter contacts and their dynamics during early lineage commitment in the context of extensive chromatin changes that occur at the…”

“…(potentially at both single-state and dual-state CRUs) that is consistent with the ‘hit-and-run’ model of transcrip- tional regulation (Schaffner, 1988; Varala et al., 2015), and provides a flexible way to fine-tune the expression of multi-enhancer genes (Guerrero et al., 2010; Lagha et al., 2012).”