Abstract:SummaryIn mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein fro… Show more
“…To visualize the DNA methylation and centromeric regions, we co-transfected mCherry-MBD-NLS [35] and EGFP-CENPC. Accumulation of MBD signals was observed in DAPI-dense heterochromatin in TALMaj-SssI (WT)-expressing Dnmt TKO ES cells, whereas cells expressing the mutant (T313D) or mock control exhibited a uniform distribution of MBD signals in the nuclei except for the nucleoli.…”
To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. “Epigenome editing” techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10–20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50–60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.
“…To visualize the DNA methylation and centromeric regions, we co-transfected mCherry-MBD-NLS [35] and EGFP-CENPC. Accumulation of MBD signals was observed in DAPI-dense heterochromatin in TALMaj-SssI (WT)-expressing Dnmt TKO ES cells, whereas cells expressing the mutant (T313D) or mock control exhibited a uniform distribution of MBD signals in the nuclei except for the nucleoli.…”
To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. “Epigenome editing” techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10–20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50–60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.
“…1a), and here the human MBD-based probe was used to generate a zebrafish reporter line. It was predicted that the probe may cause a dosage-dependent lethal effect, and therefore it was critical to identify a proper ubiquitous promoter to generate a stable transgenic line 17 . A total of three available ubiquitous promoters ( bactin2 , ef1a and h2afx ) in zebrafish were selected to induce expression of the probe throughout the body, and the recombinant vectors were constructed via Tol2kit 24, 25 .Figure 1Generation of a zebrafish reporter ubiquitously expressing mCherry-MBD.…”
As one of the major epigenetic modifications, DNA methylation is constantly regulated during embryonic development, cell lineage commitment, and pathological processes. To facilitate real-time observation of DNA methylation, we generated a transgenic zebrafish reporter of DNA methylation (zebraRDM) via knockin of an mCherry-fused methyl-CpG binding domain (MBD) probe driven by the bactin2 promoter. The probe colocalized with heterochromatin, and its intensity was positively correlated with 5 mC immunostaining at a subcellular resolution in early embryos. Biochemical assays indicated that cells with stronger fluorescence maintained a higher level of DNA methylation, and time-lapse imaging at the blastula stage showed that the level of DNA methylation was transiently strengthened during mitosis. By crossing zebraRDM with other fluorescent transgenic lines, we demonstrate that the reporter can visually distinguish different cell lineages in organs like the heart. Our zebraRDM reporter therefore serves as a convenient and powerful tool for high-resolution investigation of methylation dynamics in live animals.
“…The PFA xed, and MI -CAN stained sam ples showed char ac ter is tic bind ing to cy to plas mic proteins pri mar ily to RER and DER and a lower bind ing to spots of nu clear pro teins. It de serves no tice that in side the nu cleus, the het e rochro matin dy nam ics gen er ates com pact spots with high u o res cent in ten sity (Ueda et al, 2014;Wreggett et al, 1994;Ishov et al, 2004). MI CAN stain ing fa vored by non po lar con di tions in side the cells could be the rea son why con cen trated spots in the nu cleus be came vis i ble.…”
Section: Mican Staining At DI Erent Densities Of Hacat Cellsmentioning
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