Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation

Nishiyama, Atsuya; Mulholland, Christopher B.; Bultmann, Sebastian; Kori, Satomi; Endo, Akinori; Saeki, Yasushi; Qin, Weihua; Trummer, Carina; Chiba, Yu; Yokoyama, Haruhiko; Kumamoto, Soichiro; Kawakami, Toru; Hojo, Hironobu; Nagae, Genta; Aburatani, Hiroyuki; Tanaka, Keiji; Arita, Kyohei; Leonhardt, Heinrich; Nakanishi, Makoto

doi:10.1038/s41467-020-15006-4

Cited by 97 publications

(149 citation statements)

References 70 publications

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“…S14D), which favors DNA methylation. It was previously shown that IFC-305 is able to stimulate proliferating cell nuclear antigen (PCNA) 31 , which together with a complex machinery, guides DNMT1 to hemi-methylated regions for regenerating 5mC on complementary DNA strand during replication [52][53][54][55][56][57] . As such, maintaining DNA methylation through increasing DNMT1-guiding proteins, as well as stimulating cell cycle components such as CDK4, CDK6 and cyclin D1 31 , could be an alternative IFC-305 mechanism of action which explains the retention of a proliferating phenotype.…”

Section: Discussionmentioning

confidence: 99%

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor

Rodríguez‐Aguilera

Ecsedi

Goldsmith

et al. 2020

Sci Rep

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A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies. Dynamic changes in chromatin organization and DNA modifications participate in the establishment of cell identity in normal human biology and disease 1. Epigenetic marks such as DNA methylation are characteristic of a particular cell type as they help define the associated transcriptome 2,3. DNA methyl-transferases (DNMTs) establish 5-methylcytosine (5mC) from S-adenosylmethionine (SAM), the principal methylating agent in the body derived from the methionine cycle 4. In humans, the liver is the organ with the highest turnover of SAM and

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Section: Discussionmentioning

confidence: 99%

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor

Rodríguez‐Aguilera

Ecsedi

Goldsmith

et al. 2020

Sci Rep

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“…Encouraged by the results with the antiGFP nanobody, we proceeded to test two existing nanobodies against endogenous chromatin regulators, antiHP1 33 and antiDNMT 34 , for their capacity to silence and induce epigenetic memory. The antiHP1 nanobody was shown to bind to all three isoforms of HP1 33 , while the antiDNMT1 nanobody has previously been used to immunoprecipitate endogenous DNMT1 from whole cell lysate 34 . Using a similar system as the antiGFP nanobody, we cloned fusions between rTetR and either antiHP1 or antiDNMT1, and stably integrated them into HEK293T cells containing the TagRFP reporter with TetO sites at the AAVS1 locus ( Fig.…”

Section: Nanobodies Against Dnmt1 and Hp1 Can Silence A Reporter Genementioning

confidence: 99%

Nanobody-mediated control of gene expression and epigenetic memory

Van

Fujimori

Bintu

2020

Preprint

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Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we developed a new strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

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“…The cytoplasmic extracts were aliquoted and stored at -80˚C. Chromatin purification after incubation in egg extracts was performed as previously described with modifications (39). Sperm nuclei were incubated in egg extracts supplemented with an ATP regeneration system (20 mM phosphocreatine, 4 mM ATP, 5 μg /ml creatine phosphokinase) at 3000-4000 nuclei/μL at 22˚C.…”

Section: Xenopus Interphase Egg Extracts and Purification Of Chromatinmentioning

confidence: 99%

HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation

Kumamoto

Nishiyama

Chiba

et al. 2020

Preprint

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DNA Ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1 which was recruited onto chromatins. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signalling promotes the recruitment of LIG3 on chromatins and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.

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Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation

Cited by 97 publications

References 70 publications

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor

Nanobody-mediated control of gene expression and epigenetic memory

HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation

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