EMBO/soda-nel huggingface dataset:
Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast (2020)
Figure 4

Figure 4 - Swap experiments reveal differential functionalities of Ame1 and Mif2 N-termini in vivo.

(A) Schematic introduction of Ame1 and Mif2 swap mutants in which the N-terminus of one protein is exchanged with the N-terminus of the other.

(B) Analysis of Ame1 N-terminal deletion and swap mutant using the anchor-away technique. Ame1-FRB strains containing RPL13-FKBP12 for ribosome anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.

(C) Expression levels of 3xHA tagged Ame1 rescue constructs from (B) analyzed by western blotting.

(D) Tetrad dissection of strains where one endogenous copy of Ame1 was replaced with the indicated construct.

(E) Phenotypic analysis of different Ame1 rescue constructs using fluorescence microscopy. Images of representative large budded cells immediately and 4 h after Rapamycin addition are shown, indicated as t = 0 h and t = 4 h. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. One kinetochore cluster is enlarged in the white box. Scale bar represents 5 µm.

(F) Quantification of (E) in which large budded cells were classified according to their Mtw1-GFP signal into the three indicated groups. Scale bar represents 5 µm. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 3 independent experiments.

(G) Analysis of Mif2 N-terminal and swap mutant using the anchor-away technique as for (B). Phenotypes of the indicated Mif2 constructs were observed in an otherwise wild-type or ctf19∆ background.

(H) Western blotting showing 6xFlag tagged Mif2 expression levels of strains from (G).
Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast (2020)
Figure 5

Figure 5 - The Mif2 N-terminus is prevented from high affinity Mtw1c binding in the full-length molecule.

(A) Co-purification of Strep-Mtw1c through Flag-Mif2 after co-expression in insect cells. Proteins were detected by western blotting using their respective tags.

(B) Western blotting of elution fractions from an SEC experiment of Mif2-wt or Mif2Ame1-N swap in the absence (upper panel) or presence (lower panel) of Mtw1c.

(C) ITC analysis of Mif21-41 or Ame11-20 binding to MN. Experiments were performed 3 or 4 times, respectively. One representative binding curve is shown, whereas the KD value represents the average of all experiments.

(D) Cross-link (XL) MS analysis of the Mif2wt protein in isolation or bound to Cse4n using the cross-linker Bis[sulfosuccinimidyl] glutarate. Mif2wt alone or in combination with Cse4n were subjected to analytical size exclusion chromatography at 10 µM concentration. The corresponding Coomassie-stained SDS-PAGE gels of Mif2-wt alone or in combination with Cse4n are shown. Fractions used for cross-linking are highlighted. Topological map of Mif2 based on the identified intra-molecular cross-links. Common cross links that are found in both samples are shown in red, whereas unique crosslinks that can be only found in one of the samples are highlighted in blue. Conserved regions and their described functions are indicated. A list of cross-links that link N-terminal residues to C-terminal regions of Mif2 is shown for Mif2 alone and bound to Cse4n leading to a proposed conformation of Mif2.
Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast (2020)
Figure 6

Figure 6 - Mif2 auto-inhibition depends on its Cse4 and DNA binding motifs.

(A) Schematic introduction of Mif2wt and Mif2-∆sigAT in which its Cse4 and DNA binding motifs have been removed. Cross linked Lysin residues that connect the N-terminus with residues within the signature motif or AT hook of Mif2 in isolation are depicted in blue and were the basis to create the Mif2-∆sigAT mutant.

(B) Mif2Flag-wt or -∆sigAT directly from insect cell lysate was immobilized on M2 Flag Agarose, washed and incubated with Mtw1-Nnf1 complex (MN). After washing, bound complexes were eluted by adding 3xFlag peptide. Western blotting against the His-tag on Nnf1 confirmed increased binding of MN to Mif2-∆sigAT, already visible in the Coomassie stained SDS-PAGE gel.

(C) Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mif2-wt or Mif2-∆sigAT and MN alone and in combination at 10 µM concentration. All combinations were incubated for 1 h at 4°C prior to the run. MN (blue), Mif2 (red), combination (green). Note that the same MN elution profile and SDS-PAGE (upper panel) is displayed in both sets to improve clarity. Dashed boxes highlighting corresponding fractions were included to improve comparability.
Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast (2020)
Figure 7

Figure 7 - Mif2 auto-inhibition is released upon binding to Cse4 nucleosomes. Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mif2-wt or Mif2Ame-1N swap, Mtw1c and Cse4n alone and in combination at 10 µM concentration. All combinations were incubated for 1 h at 4°C prior to the run.

(A) Cse4n (light blue), Mif2-wt (red), combination (green). Note that the same Cse4n (upper panel) and Mif2 (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in B and C (Mif2) or C (Cse4n) to improve clarity.

(B) Mtw1c (blue), Mif2-wt (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in C to improve clarity.

(C) Mtw1c (blue), Cse4n (light blue), Mif2-wt (red), combination (green).

(D) Cse4n (light blue), Mif2Ame-1N swap (red), combination (green). Note that the same Cse4n (upper panel) and Mif2Ame-1N swap (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in E and F (Mif2Ame-1N swap) or F (Cse4n) to improve clarity.

(E) Mtw1c (blue), Mif2Ame-1N swap (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in F to improve clarity.

(F) Mtw1c (blue), Cse4n (light blue), Mif2Ame-1N swap (red), combination (green).
Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast (2020)
Figure 8

Figure 8 - Auto-inhibition of Mif2 is crucial for chromosome segregation and cell viability.

(A) Phenotypic analysis of Mif2 mutants using a galactose inducible overexpression system. Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.

(B) Serial dilution assay of chromosomally integrated Mif2 variants under the control of the pGAL promoter. Strains were plated on YEP-Glucose and YEP-Raffinose/Galactose plates.

(C) Analysis of chromosome segregation in strains from (B). Distribution of fluorescently labeled Chromosome III was analyzed in large-budded cells 16 hours after switching the medium to Galactose. Scale bar represents 2 μm.

(D) Microscopic analysis of kinetochores in Mif2 overexpressing cells visualizing Mtw1-GFP and Spc42-mCherry. Images of representative large budded cells after 16h in Galactose are shown. One kinetochore cluster is enlarged in the white box for each strain. Scale bar represents 5 µm. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis.

(E) Quantification of (D) in which large budded cells were classified according to their Mtw1-GFP signal into the four indicated groups. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 2 independent experiments.

(F) Co-IP experiment of indicated Mif2-Flag variants and Mtw1-Myc from yeast cell extracts.

(G) Phenotypic analysis of Mif2-∆sigAT mutant using the same galactose inducible overexpression system as in (A). Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.
Profiling MHC II immunopeptidome of blood stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells (2017)
Figure 2

Figure 2. Immunodominance of PbA-derived MHC II peptides during blood stage malaria induced after pRBC infection

(A) Experimental scheme to analyze splenic CD4 T cell responses at day 6 following inoculation of PbA pRBC. (B) Gating strategy to calculate the proportion of CD11a+ CD49d+ activated CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC, OVA peptide (IR17) or ETRAMP10.2 peptide (NL17). (C) Percentage of IFN/TNF-double producing cells among activated CD4 T cells for each peptide. Basal level with MutuDC alone was subtracted. (D) Hierarchy of immunodominance, depicted as the percentage of each peptide-specific response with respect to the total parasite-specific response obtained with PbA pRBC-loaded MutuDC. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.
Profiling MHC II immunopeptidome of blood stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells (2017)
Figure 3

Figure 3. Immunodominance of PbA-derived MHC II peptides during blood stage malaria induced after spz infection

(A) Experimental protocol used to analyze the ex vivo T cell responses in the spleen at day 8 and 14 following spz inoculation, with or without chloroquine (CQ) treatment as indicated. (B) IFN/TNF-double producing cells among activated CD4 T cells, after subtracting the basal level with MutuDC alone (mean +/- SEM). Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.027 ; TIM14, P = 0.84 ; ENO, P = 0.84 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.71; EF1α, P = 0.001 ; M1, P = 0.032 ; MSP1, P = 0.23 ; LDH, P = 0.016 ; ETRAMP, P = 0.064 ; ATPSYN, P = 0.77 ; END70, P = 0.097 ; PDI, P = 0.0052 ; GAPDH.2, P = 0.19 ; pRBC, P = 0.001. N = 15 mice pooled from 3 replicates. (C) Blood parasitemia (mean +/- SEM) monitored by flow cytometry after inoculation of 5x104 PbA.GFP spz in B6 mice, treated (open squares) or not (black squares) with CQ at day 6 and 7 pi, in order to avoid ECM without blunting infection. Day 6, P = 0.29 ; Day 7, P = 0.0013 by multiple unpaired t-tests. (D) IFN/TNF-double producing cells among activated CD4 T cells, at day 14 pi following CQ treatment (mean +/- SEM). Basal level with MutuDC alone was subtracted. GAPDH.1, P = 0.99 ; EF1α, P = 0.062 ; ETRAMP, P = 0.031 ; pRBC, P = 0.031 by paired non-parametric Wilcoxon tests in comparison with OVA peptide.
Profiling MHC II immunopeptidome of blood stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells (2017)
Figure 4

Figure 4. cDC1 are superior to other spleen antigen-presenting cells for MHC II presentation of PbA and non-PbA antigens

(A) Gating strategy for FACS-sorting pDC (Lin- CD11clo B220+ Ly6C+), cDC1 (Lin- CD11c+ CD8α+), cDC2 (Lin- CD11c+ CD8α-) and a mix of macrophages & moDC (Lin- CD11c- CD11b+ Ly6C+) from spleens of mice infected for 6 days with PbA pRBC. (B) I-Ab presentation of ETRAMP10.2 peptide by the indicated APC, measured with BEZ hybridomas. PbA pRBC-loaded MutuDC were used as controls. Representative of 2 experiments with 4 infected mice pooled per replicate. (C) Fold-change surface expression of I-Ab in cDC2 over cDC1 in day 6-infected mice (mean +/- SEM of 5 experiments). Difference is not significant based on unpaired t-test with Welch's correction. (D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (D) I-Ab-AS15 presentation after exogenous addition of AS15 peptide at the indicated concentrations, assessed with the BTg01Z hybridomas. (E) I-Ab-AS15 presentation by DC incubated with heat-killed T. gondii at the indicated ratio, assessed with the BTg01Z hybridomas. (F) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at the indicated ratio, assessed with the OTIIZ hybridomas. PbA pRBC-loaded MutuDC were used as controls. D,E,F: representative of 2 experiments with 4 infected mice pooled per replicate. (G) In vivo pRBC uptake by cDC1 (XCR1+ CD172a-) and cDC2 (XCR1- CD172a+) analyzed at 6 days post-infection with PbA.GFP. Gates were positioned based on the auto-fluorescence of the cDC subset in day 6 PbA-infected mice. As GFP+ events are rare, 107 events were acquired for each mouse. Symbols show the mean percentage +/- SEM (N = 4 mice per group) of GFP+ cells in each DC subset. *, P = 0.0005 by unpaired t-test without assuming consistent SD. (H,I) I-Ab presentation by cDC isolated from naïve mice. (H) I-Ab-NL17 presentation by DC incubated with PbA pRBC at a ratio of 5:1, assessed with the BEZ hybridomas. (I) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at a ratio of 20:1, assessed with the OTIIZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen. (J) Fold-change surface expression of I-Ab in cDC2 over cDC1 in naïve mice (mean +/- SEM of 4 experiments). Difference is not significant based on unpaired t-test with Welch's correction.
Profiling MHC II immunopeptidome of blood stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells (2017)
Figure 5

Figure 5. cDC1 are required for the development of parasite-specific IFN+ TNF+ Th1 responses during PbA blood stage malaria

(A) Experimental protocol to analyze T cell responses in Karma mice at day 6 post-infection with PbA pRBC. Mice were injected with DT prior to infection and repeatedly every 60 h thereafter. (B) Gating strategy to assess efficiency of cDC1 depletion. (C) Percentage (mean +/- SEM) of CD8α+ out of Lin- B220- CD11c+ MHC IIhi cells. *, P = 0.0043 by Mann-Whitney test. (D) FACS plots showing the proportion of CD11a+ CD49d+ CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC or ETRAMP10.2 peptide. (E) Proportion of CD11a+ CD49d+ CD4 T cells that co-produce IFN and TNF (mean +/- SEM) in response to MutuDC loaded with the indicated antigen, isolated from Karma mice treated (open circles) or not (black circles) with DT. Asterisks show significant differences between no DT and + DT group by multiple unpaired t-tests without assuming consistent SD. RBC, P = 0.18 ; GAPDH.1, P = 0.019 ; EF1a, 0.039 ; ETRAMP, P = 0.00054 ; OVA, P = 0.25 ; pRBC, P = 0.015. (F) Absolute numbers of IFN+ TNF+ CD11a+ CD49d+ CD4+ T cells responding to the indicated stimuli (mean +/- SEM). GAPDH.1, P = 0.0047 ; EF1a, 0.00039 ; ETRAMP, P = 0.00022 ; pRBC, P = 0.00019 by multiple unpaired t-tests without assuming consistent SD. (G) Blood parasitemia (mean +/- SEM) at day 4 and 6 pi in Karma mice treated (open circles) or not (black circles) with DT. Day 4, P = 0.022 ; Day 6, P = 0.77 by multiple unpaired t-tests without assuming consistent SD. C,E,F,G: No DT group, N = 5 mice ; + DT group, N = 6 mice. Representative of 3 independent experiments. (H) Absolute numbers of CD4+ T cells producing IFN, TNF or IL-10, either individually or in combination, following restimulation with MutuDC+PbA pRBC (mean +/- SEM). These cells are called cytokine+ in the next panels. *, P = 0.0023 by unpaired t-test without assuming consistent SD. (I) Multifunctionality analysis (IFN, TNF, IL-10) of cytokine+ T cells responding to MutuDC loaded with PbA pRBC. (J) Proportion of IL-10+ IFN- TNF- among cytokine+ T cells, in response to MutuDC loaded with the indicated antigen (mean +/- SEM). GAPDH.1, P = 0.32 ; EF1a, 0.16 ; ETRAMP, P = 8.5x10-5 ; OVA, P = 0.11 ; pRBC, P = 0.0015 by multiple unpaired t-tests without assuming consistent SD. H,I,J: N = 3 mice per group.

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Retromer oligomerization drives SNX-BAR coat assembly and membrane constriction (2022)
Figure 8

Figure 8: In vivo effect of Vps35 dimerization mutants on Vps10 in yeast

A. Vps10yEGFP localization. Yeast cells carrying Vps10yEGFP and expressing the indicated vps35 alleles as the sole source of Vps35 were logarithmically grown in SC medium. Their vacuoles were labelled with FM4-64. Cells were harvested by brief centrifugation and immediately imaged by confocal microscopy. Single confocal planes are shown. A brightfield image was used to outline the cell boundaries (shown in the merged images). Scale bar: 5 µm.

B. Co-localization of Vps10yEGFP and FM4-64 in cells from a was measured using Pearson's coefficient. 15 Confocal planes with 20 to 30 cells each from 3 independent experiments were analyzed. Means and standard error of the mean are shown. P values were calculated by Welch's t-test. ****: P<0.0001

C. CLEM analysis of Vps10yEGFP localization in vps35PISA mutant cells. Logarithmically growing cells were high-pressure frozen and processed by freeze substitution and embedding. Scale bar: 5 µm.
Retromer oligomerization drives SNX-BAR coat assembly and membrane constriction (2022)
Figure 9

Fig. 9: Effects of Vps35 dimerization mutants in human kidney (HK2) cells

A. GLUT1 at the plasma membrane. HK2 cells were treated with siRNA targeting VPS35 or with mock siRNA. Cells were fixed and stained with antibody to GLUT1 (red) and with DAPI (blue). Cells were not detergent permeabilized to preferentially show GLUT1 at the cell surface. Maximum projections of image stacks (step size in z of 300 nm) are shown. Scale bars: 10 μm.

B. Influence of VPS35 variants on GLUT1. HK2 cells silenced for VPS35 were transfected with a plasmid carrying siRNA resistant wildtype or mutant forms of GFP-VPS35. GLUT1 was detected by fixation and immunofluorescence staining as in a. Maximum projections of image stacks (step size in z of 300 nm) are shown. Scale bars: 10 µm.

C. Quantification of GLUT1 immunofluorescence in cells from B. Regions of interest (ROIs) corresponding to cells expressing the indicated VPS35 variants, and some regions outside the cells (background), were manually defined using ImageJ software. Total cell fluorescence was integrated and corrected for background fluorescence. 105 cells per condition stemming from 3 independent experiments were analyzed. Mean and SEM are indicated. P values were calculated by Welch's t-test. The analysis was performed with 99% confidence: *** p < 0.001.

D. Expression of Vps35 variants in cells from B was analyzed by SDS-PAGE and Western blot against Vps35. Tubulin served as loading control.

E. Quantification of the Vps35/tubulin ratio in cells from B. Data stems from three independent experiments and show the mean and SEM. P values were calculated by Welch's t-test. NS: not significant (p>0.01).

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DPPA2 and DPPA4 are necessary to establish a 2C-like state in mouse embryonic stem cells (2019)
Figure 1

Figure 1. DPPA2 and DPPA4 are expressed throughout pre-implantation embryo development

(A) Comparative expression during murine pre-implantation embryo development of Dppa2 (red), Dppa4 (blue), Dux (black), and Zscan4c (ZGA marker, green). Each dot represents the average value of single-cell RNA-seq from 15[] (B) Schematic representation of DPPA2, DPPA4 full-length and DPPA4 truncated proteins. The conserved domains are shown: SAP (light green), C-terminal (dark green). (C) Sashimi plot representing coverage on the Dppa4 gene of an RNA sequencing analysis of murine zygotes, early 2C, mid 2C and late 2C (A). Arcs depict splicing events, and numbers their relative frequency (i.e. reads across junctions).
DPPA2 and DPPA4 are necessary to establish a 2C-like state in mouse embryonic stem cells (2019)
Figure 2

Figure 2. DPPA2 and DPPA4 regulate expression of Dux and other genes in mESCs

(A) Average expression of Dppa2 and Dppa4 in a single-cell RNA-seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population. (B) Fraction of GFP+ cells or (C) Dppa2, Dppa4, Dux and Zscan4 gene expression in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test. RNA-seq analysis of WT and Dppa2 (D) or Dppa4 (E) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3. (F) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and two DUX-independent targets of DPPA2 and DPPA4 (Mael and Tdrd1) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DUX or GFP as a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3 (G) Venn diagram representing the overlap of genes dowregulated in Dppa2 and Dppa4 KO compared to WT cells. (H) Comparative expression of DUX-dependent, DPPA2 and DPPA4-dependent, and the rest of the genes in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population (unpaired t-test).
DPPA2 and DPPA4 are necessary to establish a 2C-like state in mouse embryonic stem cells (2019)
Figure 3

Figure 3. DPPA2 and DPPA4 associate with the promoter of their target genes in mESCs

(A) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and three targets of DPPA2 and DPPA4 independent of DUX (Mael, Tdrd1, Prex2) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DPPA2, DPPA4 or GFP as a control Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test. (B) Average coverage normalized for sequencing depth of the ChIP-seq signal of DPPA2, DPPA4 or DPPA4 truncated of the SAP domain overexpressed in mESCs in a window of 500bp around the Dux gene (n=2). Total input (TI) is shown in gray. Peaks over the Dux gene were called in DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq. (C) Heatmap showing the distribution of DUX, DPPA2, DPPA4 and DPPA4ΔSAP coverage in a ± 5kb window around the TSS of DUX-dependent, DPPA2 and DPPA4-dependent and random genes in mESCs. (D) Boxplot/Jitterplot representing the percentage of GC nucleotide contents in the peaks from DPPA2, DPPA4 and DUX ChIP, and in a random shuffle of peaks.
DPPA2 and DPPA4 are necessary to establish a 2C-like state in mouse embryonic stem cells (2019)
Figure 4

Figure 4. DPPA2 and DPPA4 directly regulate expression of young LINE-1 families in mESCs

Total expression of transposable element subfamilies in (A) Dppa2 KO or (B) Dppa4 KO compared to WT mESC clones. (C) RNA-seq analysis of MERVL-int, L1Md_A and L1Md_T in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population. (D) Heatmap displaying the enrichment of DUX, DPPA2, DPPA4 and DPPA4ΔSAP binding at different transposable element families. Every subfamily enriched (pval < 0.05) in at least one DPPA2, DPPA4 or DPPA4ΔSAP replicate are shown. (E) MSA plot of DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq in mESCs showing enrichment over L1Md_A, L1Md_T and MERVL-int sequences. Gaps are in grey and sequences in white with overlap of color-coded ChIP-seq signals. Upper plot shows average coverage of the signal over the aligned TEs.

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