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52


Juvenile hormones direct primordial germ cell migration to the embryonic gonad

Barton, Lacy J; Sanny, Justina; Packard Dawson, Emily; Nouzova, Marcela; Noriega, Fernando Gabriel; Stadtfeld, Matthias; Lehmann, Ruth
Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular signaling isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in invertebrates, facilitate multiple processes in reproduction. Here we investigated the role of these potent signaling molecules in embryonic germ cell development, using JHs in Drosophila melanogaster as a model system. In contrast to their established endocrine roles during larval and adult germline development, we found that JH signaling acts locally during embryonic development. Using an in vivo biosensor, we observed active JH signaling first within and near primordial germ cells (PGCs) as they migrate to the developing gonad. Through in vivo and in vitro assays, we determined that JHs are both necessary and sufficient for PGC migration. Analysis into the mechanisms of this newly uncovered paracrine JH function revealed that PGC migration was compromised when JHs were decreased or increased, suggesting that specific titers or spatiotemporal JH dynamics are required for robust PGC colonization of the gonad. Compromised PGC migration can impair fertility and cause germ cell tumors in many species, including humans. In mammals, retinoids have many roles in development and reproduction. We found that like JHs in Drosophila, RA was sufficient to impact mouse PGC migration in vitro. Together, our study reveals a previously unanticipated role of isoprenoids as local effectors of pre-gonadal PGC development and suggests a broadly shared mechanism in PGC migration.
PMID: 38215744
ISSN: 1879-0445
CID: 5633172

3D Enhancer-promoter networks provide predictive features for gene expression and coregulation in early embryonic lineages

Murphy, Dylan; Salataj, Eralda; Di Giammartino, Dafne Campigli; Rodriguez-Hernaez, Javier; Kloetgen, Andreas; Garg, Vidur; Char, Erin; Uyehara, Christopher M; Ee, Ly-Sha; Lee, UkJin; Stadtfeld, Matthias; Hadjantonakis, Anna-Katerina; Tsirigos, Aristotelis; Polyzos, Alexander; Apostolou, Effie
Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or 'hubness', positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors.
PMID: 38053013
ISSN: 1545-9985
CID: 5595532

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus

Aronson, Boaz E; Scourzic, Laurianne; Shah, Veevek; Swanzey, Emily; Kloetgen, Andreas; Polyzos, Alexander; Sinha, Abhishek; Azziz, Annabel; Caspi, Inbal; Li, Jiexi; Pelham-Webb, Bobbie; Glenn, Rachel A; Vierbuchen, Thomas; Wichterle, Hynek; Tsirigos, Aristotelis; Dawlaty, Meelad M; Stadtfeld, Matthias; Apostolou, Effie
Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.
PMID: 34710357
ISSN: 1878-1551
CID: 5042672

Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency

Vidal, Simon E; Polyzos, Alexander; Chatterjee, Kaushiki; Ee, Ly-Sha; Swanzey, Emily; Morales-Valencia, Jorge; Wang, Hongsu; Parikh, Chaitanya N; Amlani, Bhishma; Tu, Shengjiang; Gong, Yixiao; Snetkova, Valentina; Skok, Jane A; Tsirigos, Aristotelis; Kim, Sangyong; Apostolou, Effie; Stadtfeld, Matthias
Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.
PMID: 32976761
ISSN: 2213-6711
CID: 4606132

A Susceptibility Locus on Chromosome 13 Profoundly Impacts the Stability of Genomic Imprinting in Mouse Pluripotent Stem Cells

Swanzey, Emily; McNamara, Thomas F; Apostolou, Effie; Tahiliani, Mamta; Stadtfeld, Matthias
Cultured pluripotent cells accumulate detrimental chromatin alterations, including DNA methylation changes at imprinted genes known as loss of imprinting (LOI). Although the occurrence of LOI is considered a stochastic phenomenon, here we document a genetic determinant that segregates mouse pluripotent cells into stable and unstable cell lines. Unstable lines exhibit hypermethylation at Dlk1-Dio3 and other imprinted loci, in addition to impaired developmental potential. Stimulation of demethylases by ascorbic acid prevents LOI and loss of developmental potential. Susceptibility to LOI greatly differs between commonly used mouse strains, which we use to map a causal region on chromosome 13 with quantitative trait locus (QTL) analysis. Our observations identify a strong genetic determinant of locus-specific chromatin abnormalities in pluripotent cells and provide a non-invasive way to suppress them. This highlights the importance of considering genetics in conjunction with culture conditions for assuring the quality of pluripotent cells for biomedical applications.
PMID: 32187532
ISSN: 2211-1247
CID: 4352772

EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation

Lhoumaud, Priscillia; Sethia, Gunjan; Izzo, Franco; Sakellaropoulos, Theodore; Snetkova, Valentina; Vidal, Simon; Badri, Sana; Cornwell, Macintosh; Di Giammartino, Dafne Campigli; Kim, Kyu-Tae; Apostolou, Effie; Stadtfeld, Matthias; Landau, Dan Avi; Skok, Jane
Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.
PMID: 31752933
ISSN: 1474-760x
CID: 4209262

KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks

Di Giammartino, Dafne Campigli; Kloetgen, Andreas; Polyzos, Alexander; Liu, Yiyuan; Kim, Daleum; Murphy, Dylan; Abuhashem, Abderhman; Cavaliere, Paola; Aronson, Boaz; Shah, Veevek; Dephoure, Noah; Stadtfeld, Matthias; Tsirigos, Aristotelis; Apostolou, Effie
Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.
PMID: 31548608
ISSN: 1476-4679
CID: 4105382

Evaluation of Stuart et al.: Distinct Molecular Trajectories Converge to Induce Naive Pluripotency

Stadtfeld, Matthias
An example of the peer review process for "Distinct molecular trajectories converge to induce naive pluripotency" (Stuart et al., 2019) is presented here.
PMID: 31491390
ISSN: 1875-9777
CID: 4067952

Cellular trajectories and molecular mechanisms of iPSC reprogramming

Apostolou, Effie; Stadtfeld, Matthias
The discovery of induced pluripotent stem cells (iPSCs) has solidified the concept of transcription factors as major players in controlling cell identity and provided a tractable tool to study how somatic cell identity can be dismantled and pluripotency established. A number of landmark studies have established hallmarks and roadmaps of iPSC formation by describing relative kinetics of transcriptional, protein and epigenetic changes, including alterations in DNA methylation and histone modifications. Recently, technological advancements such as single-cell analyses, high-resolution genome-wide chromatin assays and more efficient reprogramming systems have been used to challenge and refine our understanding of the reprogramming process. Here, we will outline novel insights into the molecular mechanisms underlying iPSC formation, focusing on how the core reprogramming factors OCT4, KLF4, SOX2 and MYC (OKSM) drive changes in gene expression, chromatin state and 3D genome topology. In addition, we will discuss unexpected consequences of reprogramming factor expression in in vitro and in vivo systems that may point towards new applications of iPSC technology.
PMID: 29925040
ISSN: 1879-0380
CID: 3158222

Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes

Amlani, Bhishma; Liu, Yiyuan; Chen, Taotao; Ee, Ly-Sha; Lopez, Peter; Heguy, Adriana; Apostolou, Effie; Kim, Sang Yong; Stadtfeld, Matthias
The ability of induced pluripotent stem cells (iPSCs) to differentiate into all adult cell types makes them attractive for research and regenerative medicine; however, it remains unknown when and how this capacity is established. We characterized the acquisition of developmental pluripotency in a suitable reprogramming system to show that iPSCs prior to passaging become capable of generating all tissues upon injection into preimplantation embryos. The developmental potential of nascent iPSCs is comparable to or even surpasses that of established pluripotent cells. Further functional assays and genome-wide molecular analyses suggest that cells acquiring developmental pluripotency exhibit a unique combination of properties that distinguish them from canonical naive and primed pluripotency states. These include reduced clonal self-renewal potential and the elevated expression of differentiation-associated transcriptional regulators. Our observations close a gap in the understanding of induced pluripotency and provide an improved roadmap of cellular reprogramming with ramifications for the use of iPSCs.
PMID: 29420174
ISSN: 2211-1247
CID: 2947822