Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors (TFs), as is exemplified by reprogramming somatic cells to pluripotent stem cells (PSCs) via expression of OCT4, KLF4, SOX2 and cMYC. How TFs orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a TF-centric view of chromatin reorganization and its association to 3D enhancer rewiring and transcriptional changes during #
Downregulation of Rpd3, a homologue of mammalian Histone Deacetylase 1 (HDAC1), extends lifespan in Drosophila melanogaster. Once revealed that long-lived fruit flies exhibit limited cardiac decline, we investigated whether Rpd3 downregulation would improve stress resistance and/or lifespan when targeted in the heart. Contested against three different stressors (oxidation, starvation and heat), heart-specific Rpd3 downregulation significantly enhanced stress resistance in flies. However, these higher levels of resistance were not observed when Rpd3 downregulation was targeted in other tissues or when other long-lived flies were tested in the heart-specific manner. Interestingly, the expressions of anti-aging genes such as sod2, foxo and Thor, were systemically increased as a consequence of heart-specific Rpd3 downregulation. Showing higher resistance to oxidative stress, the heart-specific Rpd3 downregulation concurrently exhibited improved cardiac functions, demonstrating an increased heart rate, decreased heart failure and accelerated heart recovery. Conversely, Rpd3 upregulation in cardiac tissue reduced systemic resistance against heat stress with decreased heart function, also specifying phosphorylated Rpd3 levels as a significant modulator. Continual downregulation of Rpd3 throughout aging increased lifespan, implicating that Rpd3 deacetylase in the heart plays a significant role in cardiac function and longevity to systemically modulate the fly's response to the environment.
Dysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in severe developmental defects and other diseases, but the molecular mechanisms that ensure imprint stability remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which serves as a noncanonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the DNA (de)methylation machinery..
30 2 HIGHLIGHTS 31 • The IG-DMR is a bipartite element with distinct allele-specific functions 32 • A non-canonical enhancer within the IG-DMR prevents DNA methyltransferase activity 33 • Targeted epigenome editing allows induction of specific imprinting phenotypes 34 • CRISPRi reveals a functional hierarchy between DMRs that dictates imprint stability 35 KEYWORDS 36 Genomic imprinting, Dlk1-Dio3, IG-DMR, DNA methylation, pluripotent stem cells, bipartite 37 element, enhancer, Tet enzymes, Dnmt3, epigenome editing. 38 39 SUMMARY 40Dysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in 41 severe developmental defects and other diseases, but the molecular mechanisms that ensure 42 imprint stability remain incompletely understood. Here, we dissect the functional components of 43 the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the 44 mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying 45 an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic 46 regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet 47 dioxygenases and a maternally unmethylated regulatory element, which serves as a non-48 canonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de 49 novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements 50 leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic 51 changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although 52 the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability 53 of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These 54 findings establish the IG-DMR as a novel type of bipartite control element and provide 55 mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the 56 DNA (de)methylation machinery. 57 58 Polycomb Repressive Complex II (PRC2) (Zhao et al., 2010, Das et al., 2015, Kaneko et al., 87 2014, Sanli et al., 2018). This suggests that the control of Gtl2 expression by the IG-DMR (Lin et 88 al., 2003, Kota et al., 2014, Luo et al., 2016, Das et al., 2015) is essential for maintenance of 89 imprinting at Dlk1-Dio3. However, how the IG-DMR achieves this regulation in an allele-specific 90 4 manner elusive. Targeted deletions of the IG-DMR (~4kb region) in mice have shown that 91 transmission of maternal deletion results in LOI, and specifically in paternalization of the 92 maternal allele, including loss of Gtl2 and bi-allelic expression of Dlk1 (Lin et al., 2003). 93 However, transmission of the paternal deletion results in no phenotype (Lin et al., 2003, Das et 94 al., 2015), which is surprising given that the paternal IG-DMR becomes "imprinted" by DNA 95 methylation in primordial germ cells (PGCs) (Sato et al., 2011, Nowak et al., 2011, SanMiguel...
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