Link for full list of publications
(2019). Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs. Molecular Cell. 75:(5)905-+. Abstract
Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.
(2019). Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules. Cell Stem Cell. 24:(2)328-341.e9. Abstract
The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.
(2017). High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency. BioRxiv. Abstract
The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM) has provoked interest to define the molecular characteristics of the process. Despite important progress, the dynamics of epigenetic reprogramming at high resolution in correctly reprogrammed iPSCs and throughout the entire process remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using a highly efficient deterministic murine reprogramming system engineered through optimized inhibition of Gatad2a-Mbd3/NuRD repressive sub-complex. This comprehensive characterization provides single-day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. CAPGs are predominantly regulated by Myc, and are enriched for cell biosynthetic regulatory functions. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis demonstrated that de novo DNA methylation is not required for highly efficient conducive iPSC reprogramming, and identified a group of super-enhancers targeted by OSK, whose early demethylation marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are distinctively regulated by multiple synergetic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naive pluripotency.
Stelzer, Y*., Bar, S*., Bartok, O., Afik, S., Ronen, D., Kadener, S., and Benvenisty, N. Differentiation of human parthenogenetic pluripotent stem cells reveals multiple tissue- and isoform-specific imprinted transcripts. Cell Reports 11, 308-320.
Stelzer, Y., Sagi, I., Yanuka, O., Eiges, R., and Benvenisty, N. The noncoding RNA IPW regulates the imprinted DLK1-DIO3 locus in an induced pluripotent stem cell model of Prader-Willi syndrome. Nature Genetics 46, 551-557.
Stelzer, Y*., Ronen, D*., Bock, C., Boyle, P., Meissner, A., and Benvenisty, N. Identification of novel imprinted differentially methylated regions by global analysis of human-parthenogenetic-induced pluripotent stem cells. Stem Cell Reports 1, 79-89.
Pick, M*., Stelzer, Y*., Bar-Nur, O., Mayshar, Y., Eden, A., and Benvenisty, N. Clone- and gene-specific aberrations of parental imprinting in human induced pluripotent stem cells. Stem Cells 27, 2686-2690.
* Equally Contributing Authors