Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

Zviran, Asaf; Mor, Nofar; Rais, Yoach; Gingold, Hila; Peles, Shani; Chomsky, Elad; Viukov, Sergey; Buenrostro, Jason D.; Scognamiglio, Roberta; Weinberger, Leehee; Manor, Yair S.; Krupalnik, Vladislav; Zerbib, Mirie; Hezroni, Hadas; Jaitin, Diego Adhemar; Larastiaso, David; Gilad, Shlomit; Schwessinger, Benjamin; Gafni, Ohad; Mousa, Awni; Ayyash, Muneef; Sheban, Daoud; Bayerl, Jonathan; Aguilera-Castrejon, Alejandro; Massarwa, Rada; Maza, Itay; Hanna, Suhair; Stelzer, Yonatan; Ulitsky, Igor; Greenleaf, William J.; Tanay, Amos; Trumpp, Andreas; Amit, Ido; Pilpel, Yitzhak; Novershtern, Noa; Hanna, Jacob H.

doi:10.1016/j.stem.2018.11.014

Cited by 55 publications

(61 citation statements)

References 18 publications

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“…4f). This transient induction became even more apparent when we called H3K27ac peaks in two recent, independent iPSC reprogramming studies [74,75] (Fig. 4h-i).…”

Section: Starr-seq Reveals Endogenous Retrovirus Enhancers That Are Rmentioning

confidence: 80%

STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

et al. 2020

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Background Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics. Results Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in “ground-state” (2i+LIF; 2iL) and “metastable” (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency. Conclusions In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.

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“…4f). This transient induction became even more apparent when we called H3K27ac peaks in two recent, independent iPSC reprogramming studies [74,75] (Fig. 4h-i).…”

Section: Starr-seq Reveals Endogenous Retrovirus Enhancers That Are Rmentioning

confidence: 80%

STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

et al. 2020

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“…Defined cocktails of transcription factors (TFs) are capable of interconverting cell types from different lineages and to reprogram somatic donor cells to self-renewing pluripotent cells 1–3 . The induction of pluripotent stem cells (iPSCs) from fibroblasts by Oct4, Sox2, Klf4, and c-Myc (OSKM) is a widely used model system to study the underlying molecular mechanisms of cell fate conversion 4–8 . Reprogramming-capable TFs originate from diverse structural families without any obvious evolutionary relationship, and even closely related factors can vary widely in their reprogramming competency.…”

Section: Introductionmentioning

confidence: 99%

Pluripotency reprogramming by competent and incompetent POU factors uncovers temporal dependency for Oct4 and Sox2

et al. 2019

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Oct4, along with Sox2 and Klf4 (SK), can induce pluripotency but structurally similar factors like Oct6 cannot. To decode why Oct4 has this unique ability, we compare Oct4-binding, accessibility patterns and transcriptional waves with Oct6 and an Oct4 mutant defective in the dimerization with Sox2 (Oct4 defSox2 ). We find that initial silencing of the somatic program proceeds indistinguishably with or without Oct4. Oct6 mitigates the mesenchymal-to-epithelial transition and derails reprogramming. These effects are a consequence of differences in genome-wide binding, as the early binding profile of Oct4 defSox2 resembles Oct4, whilst Oct6 does not bind pluripotency enhancers. Nevertheless, in the Oct6-SK condition many otherwise Oct4-bound locations become accessible but chromatin opening is compromised when Oct4 defSox2 occupies these sites. We find that Sox2 predominantly facilitates chromatin opening, whilst Oct4 serves an accessory role. Formation of Oct4/Sox2 heterodimers is essential for pluripotency establishment; however, reliance on Oct4/Sox2 heterodimers declines during pluripotency maintenance.

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“…We then obtained the genomic binding sites (peaks) of the 23 lncRNAs from the aforementioned experiments, and found the ratio of this overlap from published data ( Figure 2G ) is highly correlated with the corresponding PIRCh-seq signal among most of the lncRNAs ( Figure 2H ). The Spearman correlation coefficients of the ratio of the overlap ChIP-seq peaks 39 with the lncRNA’s PIRCh-seq enrichment scores in the same cell line were significantly higher than random permutations ( Figure 2I , P <0.0001). These results further confirm that PIRCh-seq reliably identifies chromatin associated lncRNAs.…”

Section: Resultsmentioning

confidence: 92%

“…We found a total of 23 expressed lncRNAs in the LnChrom database, including Xist, Firre, Rmrp, Tug1 and etc., and all of them were positively enriched in our PIRCh experiment and 14 of which were significant with P-value<0.05, suggesting the high sensitivity of the PIRCh approach in identifying chromatin associated lncRNAs. Furthermore, we obtained the genomic binding sites (peaks) of 23 lncRNAs from the aforementioned experiments, and overlapped them with the histone ChIP-seq peaks 12 and got a ratio of the overlap for each lncRNA. We then calculated the Spearman correlation coefficients of these ratios with their corresponding lncRNA’s PIRCh-seq enrichment scores in the same cell line (normalized by the total number of different ChIP-seq peaks), and found that these correlations were significantly higher than random permutations.…”

Section: Methodsmentioning

confidence: 99%

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Functional classification of noncoding RNAs associated with distinct histone modifications by PIRCh-seq

Fang

Chu

et al. 2019

Preprint

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32 33 Many long noncoding RNAs (lncRNAs) regulate gene transcription through 34 binding to histone modification complexes. Therefore, a comprehensive study of nuclear 35RNAs in a histone modification-specific manner is critical to understand their regulatory 36 mechanisms. Here we develop a method named Profiling Interacting RNAs on Chromatin 37 by deep sequencing (PIRCh-seq), in which we profile chromatin-associated transcriptome 38in 5 different cell types using antibodies recognizing histone H3 and 6 distinct histone 39 modifications associated with active or repressive chromatin states. PIRCh-seq identified 40chromatin-associated RNAs with substantially less contamination by nascent transcripts, 41as compared to existing methods. We classified chromatin-enriched lncRNAs into 6 42 functional groups based on the patterns of their association with specific histone 43 modifications. LncRNAs were enriched with different chromatin modifications in 44 different cell types, suggesting lncRNAs' regulation may also be cell type-specific. By 45 integrating profiles of RNA secondary structure and RNA m 6 A modification, we found 46 that RNA bases which bind to chromatin tend to be more single stranded. We discovered 47 hundreds of allele-specific RNA-chromatin interactions, nominating specific single 48 nucleotide variants that alter RNA association with chromatin. These results provide a 49 unique resource to globally study the functions of chromatin-associated lncRNAs and 50 elucidate the basic mechanisms of chromatin-RNA interaction.

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Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

Cited by 55 publications

References 18 publications

STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

Pluripotency reprogramming by competent and incompetent POU factors uncovers temporal dependency for Oct4 and Sox2

Functional classification of noncoding RNAs associated with distinct histone modifications by PIRCh-seq

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