EMBO/SourceData huggingface dataset:
Figure 6C. Cumulative distribution showing fold changes (FCs) in transcript abundance in control morphants (blue curves) and maternal zRbm14 morphants (red curves) between the indicated stages. Transcript abundances were averaged from two independent experiments.D. Expression patterns of four clusters of representative transcripts, categorized from RNA-seq results of ctrl-MO embryos. The transcripts in the maternal-early and the zygotic-early clusters respectively displayed continual degradation or accumulation from 2.5 hpf, whereas those in the maternal-late and zygotic-late clusters respectively started degradation or accumulation from 4 hpf.E. Cumulative distribution showing FCs of transcript abundance of the indicated clusters between control and maternal zRbm14 morphants.F. Relative transcript levels of three typical maternal genes. FCs were calculated relatively to the levels in 2.5-hpf ctrl-MO embryos. org, oogenesis-related; trip10a, thyroid hormone receptor interactor 10a; dnajc5ga, DnaJ (Hsp40) homolog, subfamily C, member 5 gamma a.G. Whole-mount in situ RNA hybridization of org, trip10a, and dnajc5ga. A representative embryo is shown for each group of at least 26 embryos.EU incorporated into nascent transcripts were visualized through a click reaction. Hoechst 33342 was used to counterstain the nucleus. A representative maximum intensity-projected confocal micrograph, taken from the animal pole, is shown for each group of 10 embryos (I).
Figure 7Depletion of zRbm14 resulted in accumulation of poly(A)-containing maternal mRNAs. Total RNAs purified from the indicated zebrafish embryos were subjected to poly(A) length assays. One set of representative PCR results for org and trip10a (B) As the PCR products of the poly(A)-transcripts mainly emerged as a single band, only intensities of this major band were measured.Depletion of zRbm14 resulted in accumulation of poly(A)-containing maternal mRNAs. Total RNAs purified from the indicated zebrafish embryos were subjected to poly(A) length assays. quantification results from three independent experiments (C) are presented. As the PCR products of the poly(A)-transcripts mainly emerged as a single band, only intensities of this major band were measured.D. zParn associated with zRbm14b. GFP-zRbm14b was co-expressed respectively with the indicated RFP fusion proteins in HEK293T cells. Co-immunoprecipitations (co-IP) were performed using anti-GFP beads. Luciferase (Luc)-RFP served as a negative control.E, F. zParn markedly promoted LLPS of zRbm14b by interplaying with zIDR. Purified His-tagged RFP or zParn-RFP was mixed with His-tagged GFP or GFP-zRbm14b or mutants (E). Parn-RFP was mixed with decreasing concentrations of GFP-zRbm14b (F).G, H. zParn was highly expressed from 4-cell to 32-cell stages and enriched in zRbm14 condensates. Immunoblotting (G) and immunostaining (H) were performed using a rabbit anti-zebrafish Parn antibody. Lysates from 5 embryos were loaded in each lane in (G). α-tubulin served as internal controls. Representative single optical sections are presented in (H). Chromatin DNA was stained with DAPI. Arrows point to typical regions abundant in condensates.J. zRbm14b and zParn efficiently co-phase separated in deadenylation experiments. The addition of SDS disrupted the condensates to terminate the deadenylation reaction.Co-phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn. RNAs extracted from the indicated samples were subjected to immuno-northern blotting using an antibody against m6A (K). were quantified from five sets of independent results.Co-phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn. Relatively proportions of Cap-10m6A-A0 in total RNAs (L) were quantified from five sets of independent results.
Figure 8B. Mouse Rbm14 formed cytoplasmic puncta in blastomeres (1-cell to 8-cell). Maximum intensity-projected confocal immunofluorescent micrographs are presented. Framed regions are magnified to show details of cytoplasmic Rbm14 puncta. Arrows point to Rbm14 condensates accumulated at spindle poles.D. Relative transcript levels of Rbm14 in the indicated E4.5 blastocysts based on RNA deep Sequencing results, normalized to those of Gapdh. Six blastocysts were considered as Rbm14-/- embryos because they contained only trace amount of Rbm14 transcript reminiscent of maternal mRNA.F. Heat map of differentially expressed genes.G. Heat map of representative lineage-specific transcripts. TE, trophectoderm; EPI, epiblast; PrE, primitive endoderm.Severe down-regulation of Gata6 and Nanog in Rbm14-depleted mouse blastocysts. Representative immunofluorescent images (H) were from single option sections. DAPI stained nuclear DNA. As Gata6, Nanog, and Rbm14 localize to the nucleus in blastocystsSevere down-regulation of Gata6 and Nanog in Rbm14-depleted mouse blastocysts. As Gata6, Nanog, and Rbm14 localize to the nucleus in blastocysts, their relative intensity in a cell (I) was quantified as their nuclear immunofluorescent intensity normalized with fluorescent intensity of the nuclear DNA. Five brightest cells were quantified for each embryo.
Figure 1C Example images of TIRFM endpoint colocalization assays. Top panels show CEN DNA (Top-left panel, blue circles) or CDEIIIMUT CEN DNA (top-right panel, blue circles) visualized in lysates containing Ndc10-mCherry. Middle panels show the visualized Ndc10-mCherry on CEN DNA (middle-left panel) or CDEIIIMUT DNA (middle-right panel) with colocalization shown in relation to blue DNA circles. Bottom panels show overlay of DNA channel (magenta) with Ndc10-mCherry (yellow) on CEN DNA (bottom-left panel) or CDEIIIMUT DNA (bottom-right panel). Scale bars 3μm. Graph shows the quantification of Ndc10 endpoint colocalization on CEN DNA and on CDEIIIMUT CEN DNA (70 ± 7.6%, 1.6 ± 0.3% respectively, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).E Example images of TIRFM endpoint colocalization imaging. Top panels show CEN DNA (top-left panel, blue circles) or CDEIIIMUT CEN DNA (top-middle panel, blue circles) visualized in lysates that included Cse4CENP‑A-GFP or CEN DNA in lysates that lacked Scm3HJURP (scm3-AID) (top-right panel, blue circles). Middle panels show Cse4CENP‑A-GFP visualized on CEN DNA (middle-left panel) or CDEIIIMUT CEN DNA (center panel) or on CEN DNA in lysates lacking Scm3HJURP (scm3-AID) (middle-right panel) with colocalization shown in relation to blue DNA circles. Bottom panels show overlay of CEN DNA channel (magenta) with Cse4CENP‑A-GFP (green) on CEN DNA (bottom-left panel) or CDEIIIMUT DNA (bottom-middle panel) or on CEN DNA in lysates lacking Scm3HJURP (scm3-AID) (bottom-right panel). Scale bars 3μm. Graph shows quantification of observed colocalization of Cse4 on CEN DNA and on CDEIIIMUT CEN DNA or on CEN DNA in lysates that lacked Scm3HJURP (47 ± 2.9%, 3.5 ± 3.0% and 0.6 ± 0.4% respectively, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).
Figure 2B Estimated survival function plots of Kaplan-Meier analysis of CEN DNA residence lifetimes of Ndc10 (magenta - median lifetime of 102 s, n=3231 over 3 experiments of ~1000 DNA molecules using different extracts) and Cse4CENP‑A (red - median lifetime of 79 s, n=1049 over 3 experiments of ~1000 DNA molecules using different extracts). Significant difference between ternary Ndc10 residence lifetime survival plots (***) compared to Cse4CENP‑A (two-tailed p-value of 0 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.E Kaplan-Meier analysis of TernaryNdc10 residence lifetimes of Cse4CENP‑A on CEN DNA (purple - median lifetime of 88 s, n=539 over 3 experiments of ~1000 DNA molecules using different extracts) and of Non-TernaryNdc10 Cse4CENP‑A residence lifetimes (teal - median lifetime of 69 s, n=612 over 3 experiments of ~1000 DNA molecules using different extracts). There is a significant difference (**) between TernaryNdc10 and Non-TernaryNdc10 lifetime survival plots (two-tailed p-value of 7.4e-4 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.G Timing of all non-simultaneous Ndc10 and Cse4CENP‑A ternary residence events. The proportion when Ndc10 precedes Cse4CENP‑A is 0.83 ± .06 and when Cse4CENP‑A precedes Ndc10 is 0.17 ± .06 (avg ± s.d., n=504 over 3 experiments of ~1000 DNA molecules using different extracts). Simultaneous arrival defined as residence initiation within 5 s of each other and comprise .07 ± .04 of all ternary residence events (avg ± s.d., n=35 over 3 experiments of ~1000 DNA molecules using different extracts).
Figure 3F Longer residence lifetimes are measured for Cse4CENP‑A after Scm3HJURP colocalizes. Estimated survival function plots of Kaplan-Meier analysis of the lifetimes of TernaryScm3 Cse4CENP‑A residences on CEN DNA (purple - median lifetime of 108 s, n=305 over 3 experiments of ~1000 DNA molecules using different extracts) and Non-TernaryScm3 Cse4CENP‑A residences on CEN DNA (green - of 79 s, n=1230 over 3 experiments of ~1000 DNA molecules using different extracts). Significant difference (**) between TernaryScm3 and Non-TernaryScm3 lifetime survival plots (two-tailed p-value of 2.1e-3 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.
Figure 4B Kinetochores were assembled on beads containing either a 250 bp single-tether, a 250 bp double-tether or a 250 bp CDEIIImut CEN DNA. They were then treated with MNase, and the remaining DNA was visualized on an agarose gel. Black arrow indicates Cse4CENP‑A nucleosome protected DNA (~150 bp); white arrow indicates theoretical location of undigested template DNA (250 bp).C Example images of TIRFM endpoint colocalization assays. Top panels show Cse4CENP‑A/Ndc10 ternary colocalizations visualized on single-tethered CEN DNA (top-left panel) or on double-tethered CEN DNA (top-right panel) with colocalization shown in relation to Ndc10 in yellow circles. Bottom panels show overlay of Ndc10 channel (magenta) with Cse4CENP‑A (green). Scale bars 3 μm. D Quantification of observed ternary colocalization of Cse4CENP‑A with Ndc10 (right) on single-tethered CEN DNA containing Ndc10 (19.9± 2.4%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts) and on double-tethered CEN DNA (7.3 ± 1.1%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).F Residence times for Cse4CENP‑A on double-tethered CEN DNAs are shorter than on single-tethered CEN DNAs and equivalent to those on non-functional mutant CEN DNAs. Estimated survival function plots of Kaplan-Meier analysis of ternary residences of Cse4CENP‑A with Ndc10 on single-tethered CEN DNA (blue - median lifetime of 79 s, n=675 over 3 experiments of ~1000 DNA molecules using different extracts), on double-tethered CEN DNA (red - median lifetime of 67 sec, n=3175 over 3 experiments of ~1000 DNA molecules using different extracts) or Cse4CENP‑A lifetimes on CDEIII-80 bp CEN DNA (purple - median lifetime of 71 sec, n=901 over 3 experiments of ~1000 DNA molecules using different extracts). No significant difference (n.s.) between double-tethered 250 bp CEN DNA and 80 bp CDEIII CEN DNA lifetime survival plots (two-tailed p-value of 0.06 as determined by log-rank test). Significant difference (***) between double-tethered 250 bp CEN DNA and 250 bp single-tether DNA lifetime survival plots (two-tailed p-value of 1.4e-10 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.
Figure 5B Immunoblot analysis of whole cell extracts from WT, chl4-K13S and okp1-AID cells using indicated antibodies.C Example images of TIRFM endpoint colocalization assays. Top panels show visualized Cse4CENP‑A-GFP on CEN DNA in extracts from chl4-K13S (top-left panel) or auxin-treated okp1-AID strains (okp1-AID, top-right panel) with colocalization shown in relation to identified CEN DNA in blue circles. Bottom panels show overlay of CEN DNA channel (magenta) with Cse4CENP‑A-GFP (green), Scale bars 3 μm. D Quantification of Cse4CENP‑A endpoint colocalization with CEN DNA in extracts from WT, chl4-K13S, or okp1-AID genetic backgrounds (19 ± 1.1%, 8 ± 0.7%, 5 ± 0.9%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).F Kaplan-Meier analysis of Cse4CENP‑A residence lifetimes on CEN DNA in extracts from WT (blue - median lifetime of 82 s, n=1419 over 3 experiments of ~1000 DNA molecules using different extracts), chl4-K13S (red - median lifetime of 70 s, n=546 over 3 experiments of ~1000 DNA molecules using different extracts) and okp1-AID (purple - median lifetime of 61 s, n=348 over 3 experiments of ~1000 DNA molecules using different extracts) genetic backgrounds. Significant difference (***) between WT extract and chl4-K13S extract residence lifetime plots (two-tailed p-value of 3.4e-5 as determined by log-rank test). No significant difference (n.s.) between chl4-K13S and okp1-AID residence lifetimes in (two-tailed p-value of .40 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.
Figure 6B Example images of TIRFM endpoint colocalization assays. Visualized Cse4CENP‑A-GFP on unstable1 CDEII-mutant DNA (top panel) or on v-unstable1 CDEII-mutant DNA (bottom panel) with colocalization shown in relation to identified CEN DNA in blue circles. Scale bars 3 μm. C Quantification of endpoint colocalization of Cse4CENP‑A on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (19.3 ± 1.1%, 13.8 ± 0.6%, 7.8 ± 0.5%, 5.5 ± 1.0%, 5.5 ± 1.1%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).E Very unstable CDEII mutants have reduced average Cse4CENP‑A binding when compared to unstable counterparts. Average residences of Cse4CENP‑A per CEN DNA (left) on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (1.03 ± 0.12, 0.88 ± 0.22, 0.58 ± 0.41, 0.61 ± 0.18, 0.41 ± 0.09, avg ± s.e.m. n=3 experiments of ~1000 DNA molecules using different extracts) and average residences of Scm3HJURP per CEN DNA (right) on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (0.57 ± 0.07, 0.35 ± 0.10, 0.52 ± 0.16, 0.53 ± 0.12, 0.46 ± 0.02, avg ± s.e.m. n=3 experiments of ~1000 DNA molecules using different extracts).
Figure 1(A) Excision of endogenous Hi in plants with and without HiTG obtained from the same parental plant.(B) Patterns of DNA methylation for each cytosine context in an endogenous Hi locus calculated from WGBS data. Vertical dashed lines indicate the termini of Hiun.
Figure 2(A-C) Excision of endogenous Hi in cmt23 (A), drm12 (B), and ddcc (C) mutant backgrounds when HiTG is segregated away. Plants with * and ** were used for WGBS, and the results of those with ** were used for the analysis shown in Figures.(D) Excision of endogenous Hi in mutant backgrounds of epigenetic regulators. drm12: drm1 drm2 double mutant, ddc: drm1 drm2 cmt3 triple mutant, cmt23: cmt2 cmt3 double mutant, and ddcc: drm1 drm2 cmt2 cmt3 quadruple mutant. Histone acetyltransferase HDA6/RTS1 is reported as important for silencing of TEs, and TEs activated in hda6 mutant are shared with those activated in met1
Figure 3(A-C) Patterns of DNA methylation in endogenous Hi (A), AT2TE05755 (B), and AT2TE06955 (C) in the drm12 mutant background. '(-HiTG)' indicates that HiTG was segregated. Vertical dashed lines indicate termini of TEs.
Figure 4(A) Patterns of DNA methylation in flanking regions of VANC21 targets when HiTG was segregated in drm12 (left) and cmt23 (right) mutant background. Grey dashed lines indicate the points of VANC targets.(C) Patterns of DNA methylation around VANC21 (left), VANC6 (middle), and VANC1 (right) targets.
Figure 5(C) Hi excision in F2 plants obtained from met1 x GK345. Genotyping results of MET1 and Hi loci are also shown. a-h indicate individuals who are analyzed for their DNA methylation status in (D).(D) DNA methylation status in endogenous Hi calculated from the bisulfite sequencing results.
Figure 6(A) Expression of VANB21 (At2g23490) and VANC21 (At2g23480) measured by RT‒qPCR. The values were normalized to the expression levels in the ddm1 mutant. For each genotype, the average values and standard deviations of the three technical replicates are shown. Note that we could not detect the expression of VANC21 in drm12 (-HiTG), although endogenous Hi retained mobility (Fig. 6C). This finding can likely be explained by the expression of VANA21, a transposase of VANDAL21, but we failed to compare the expression levels of this gene because of its very low expression level.(B) DNA methylation status of coding and noncoding regions of five VANDAL21 TEs The average values and standard deviations for each region are shown. One-way ANOVA with Tukey's multiple comparisons test was used to determine the significant differences (p<0.01).(C) Excision of endogenous Hi detected by PCR. For RT-qPCR shown in (A) and excision analysis shown in (C), RNA and DNA, respectively, were extracted from the same plants.
Figure 1C-D) Representative western blots and respective densitometries (right panel) for the selected SWI/SNF/BAF complex subunits (n=3-5 biological replicates). Arrow heads indicate non-specific bands, while # symbol highlights BAF53B.F) Representative immunoblots of co-immunoprecipitation (co-IP) experiments in eNPCs (top panels) and smNPCs (bottom panels). Co-IPs were performed with antibodies against BAF170 (left) and BRG1(right). Inputs are homogenates from eNPCs (left) and smNPCs (right). IgG indicates IgG-bound Sepharose beads (as controls), while IP indicates immunoprecipitated samples after elution from the beads. Arrow head indicates BCL7A band at the correct MW.G) Immunofluorescence staining of BCL7A (purple) along with actin, nestin and β-III tubulin (green) as well as GFAP (white) in iPSCs, smNPCs, and smNPC-derived neurons and astrocytes, respectively. Arrow heads indicate GFAP-positive glial cells with low BCL7A expression.H) Western blot analysis of BCL7A and BAF170 in cortex and hippocampus of embryonic (E17.5), early postnatal (PD0 and PD7) and adult (PD120) mouse brain tissue showing a progressive temporal reduction of protein levels in the analyzed tissues.I) Immunofluorescence staining of BCL7A (purple), EdU (green) and NeuN (white) in embryonic (E17.5) and adult (PD120) hippocampal brain sections. EdU was used to label proliferating NPCs, whereas NeuN was used as a neuronal marker.
Figure 2A) Immunoblot and immunofluorescence staining of BCL7A (red) in eNPCs isolated from Bcl7awt/wt (wt) and Bcl7ako/ko (ko) embryos (E13.5). Nestin (green) was used as NPC marker, whereas DAPI (blue) was used to stain nuclei.B) Immunoblot and immunofluorescence staining of BCL7A in smNPCs. Two different BCL7A knockout clones (KO1 and KO2) were used along with the parental (P) cell line.C-D) Representative western blots and respective densitometries (right panel) for selected SWI/SNF/BAF complex subunits in (C) eNPCs (n=4 biological replicates) and (D) smNPCs (n=3-6 biological replicates).E-G) BCL7A KO and wt smNPCs were used for (F) nuclear isolation followed by density sedimentation Top panels show representative immunoblots for selected SWI/SNF/BAF complex subunits and respective densitometries are shown below (n=3 biological replicates).BCL7A KO and wt smNPCs were used for differential salt extraction. Top panels show representative immunoblots for selected SWI/SNF/BAF complex subunits and respective densitometries are shown below (n=3 biological replicates).H-I) Heatmaps of SMARCA4/BRG1 and H3K27me3 ChIP-seq in wt and BCL7A KO eNPCs. J) Average density plots for SMARCA4/BRG1 occupancy around the TSS in wt and BCL7A KO eNPCs.K-L) Heatmaps of SMARCA4/BRG1 and H3K27me3 ChIP-seq in parental and BCL7A KO smNPCs. M) Average density plots for SMARCA4/BRG1 occupancy around the TSS in parental and BCL7A KO smNPCs.
Figure 3A) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs. Samples were collected at the indicated differentiation time points. Respective densitometries are shown on the right (n=6-7 biological replicates).B) Sholl analysis of β-III tubulin-positive immature wt and BCL7A KO neurons following 7 days of spontaneous eNPC differentiation (n=4 biological replicates, each experiment with 10-15 cells per condition).C) Immunofluorescence staining (central panel) and quantification (right panel) of β-III tubulin- and GFAP-positive cells as well as RT-PCR analysis (right panels) of spontaneously differentiated iPSC-derived parental and BCL7A KO smNPCs (n=3-7 biological replicates).D) Immunofluorescence staining of BCL7A expression in hippocampal sections from adult control and Bcl7afl/fl; Nestin-Cretg/wt mice.E) EdU experiments in adult mice. Animals were injected with EdU twice daily for three days and sacrificed 21 days thereafter. Images show immunofluorescence stainings for EdU (green), doublecortin (DCX, white), and S100β (red) in hippocampal brain sections from Bcl7awt/wt; Nestin-Cretgt/wt (as control) and Bcl7afl/fl; Nestin-Cretg/wt mice. Representative EdU+ DCX+ (yellow triangles) or EdU+ S100β+ (yellow asterisk) double-labelled cells are indicated in the insets. Scale bar= 50 μm and 20 μm (for insets).F) Quantification of EdU+ (right panel), EdU+ DCX+ (middle panel) and EdU+ S100β+ (left panel) cells within the hippocampal dentate gyrus (DG) of adult control (n=4) and Bcl7afl/fl; Nestin-Cretg/wt (n=5) animals.G) Immunofluorescence staining for DCX+ cells within the DG region of adult Bcl7afl/fl; Nestin-Crewt/wt (as control) and Bcl7afl/fl; Nestin-Cretg/wt animals. Confocal imaging analysis shows a lower number, misalignment and reduced neuritic complexity of DCX+ labelled cells in BCL7A KO mice compared to controls. Yellow arrows indicate misaligned DCX+ cells.H) Immunofluorescence staining of BCL7A (red) of hippocampal sections from Bcl7afl/fl; Baf53b-Crewt/wt (as control) and Bcl7afl/fl; Baf53b-Cretg/wt mice.I) Immunofluorescence staining of BCL7A (red) and DCX (green) in hippocampal sections of control and Bcl7afl/fl; Baf53b-Cretg/wt mice. Yellow arrow indicates dividing cells, whereas yellow asterisks mark differentiating immature neurons.J) Immunofluorescence staining for DCX (white) within the DG region of adult control and Bcl7afl/fl; Baf53b-Cretg/wt mice. Representative images show no obvious difference in number, morphology or alignment of DCX+ labelled cells. Quantification of DCX+ labelled cells in control and Bcl7afl/fl; Baf53b-Cretg/wt mice is shown on the right (n=3 per genotype).