rabbit anti smarca5  (Novus Biologicals)

Journal: bioRxiv

Article Title: Maternal SMARCA5 is required for major ZGA in mouse embryos

doi: 10.1101/2023.12.05.570276

Figure Lengend Snippet: A. Immunofluorescence images of wild-type CB57L/6 oocytes and preimplantation mouse embryos stained with anti-SMARCA5 antibody (green): Oocyte (MII stage), zygotes (pronuclear stages PN0, PN1, PN3 and PN5, prometaphase 1-cell embryo), 2-cell stage, 4-cell stage, 8-cell stage and blastocyst stage (32-cell). DNA was counterstained with DAPI (blue). Scale bar 50 μm. B. Left: representative brightfield images of wild-type ( Smarca5 WT ) and maternal knockout embryos ( Smarca5 matKO ) collected at E1.5 from wild-type and conditional knockout females ( Smarca5 fl/fl ; Zp3-Cre ) crossed with wild-type male mice. Red asterisks indicate two-cell stage embryos. Right: box plot showing the numbers of two-cell embryos collected at E1.5 from the specified crosses ( Smarca5 WT n=10 crosses, Smarca5 matKO n=28 crosses). Statistical significance was determined by two tailed Mann-Whitney U test. p-value ****P<0.0001, absence of stars (non-significant, ns): p-value>0.05. Scale bar 100 μm. C. Top: Immunofluorescence images of Smarca5 WT and Smarca5 mKO two-cell embryos stained with α-SMARCA5 (green) and α-MERVL (magenta) antibodies. DNA stained with DAPI (blue), scale bar 50 μm. Bottom: Signal intensity levels for nuclear SMARCA5 and cytoplasmic MERVL in two-cell stage embryos. ****P<0.0001 (Student’s t test). D. Bar plots showing the percentage of embryos that reached each developmental stage, shown by the colours in the legend at the bottom, at the specified time point (E0.5, E1.5 and E2.5). Percentages were calculated based on the total number of zygotes collected at E0.5. E. Scatterplots showing the expression levels (log2 RPM) of maternal transcripts (∼6000, blue), major ZGA genes (3156, red) and MERVL transcripts (732, magenta), in transcriptome data from single embryo NMT-seq libraries, from embryos from one replicate, pooled. Maternal knockout embryos ( Smarca5 matKO ) were compared to wild-type ( Smarca5 WT ) ones.

Article Snippet: rabbit anti-EG5 (Merck-Millipore, HPA010568) rabbit anti-MERVL (Huabio, R1501-2, IF: 1:100) rabbit anti-SMARCA5 (abcam, ab72499, IF: 1:200) rabbit anti-mCHERRY (abcam, ab167453, IF: 1:200) rat anti-HP1β (abcam, ab10811, IF: 1:300) mouse anti-pan-histone (Merck-Millipore, mab3422, IF: 1:400)

Techniques: Immunofluorescence, Staining, Knock-Out, Two Tailed Test, MANN-WHITNEY, Expressing

Journal: bioRxiv

Article Title: Maternal SMARCA5 is required for major ZGA in mouse embryos

doi: 10.1101/2023.12.05.570276

Figure Lengend Snippet: A. Experimental design of Trim-Away. Zygotes collected from wild-type female mice (C57BL/6J) are microinjected with mCherry-Trim21 mRNA and the anti-SMARCA5 antibody. Once the TRIM21 protein is translated it recognises the Fc-region of the antibody which is already bound to its target, and the whole complex (Trim21-antibody-protein) will be degraded. Following injection, the embryos are cultured for assessment of their developmental potential, immunofluorescence stainings and single embryo NMT-seq. B. Representative immunofluorescence images of two-cell stage embryos from the following three conditions are shown: WT (uninjected), IgG and α-SMARCA5 injected embryos. mCherry-TRIM21 (red), SMARCA5 (green), MERVL (magenta). DNA was stained with DAPI (blue). Scale bar represents 25 μm. C. Box plots with individual data points are showing the signal intensity (raw values, not normalised) for nuclear SMARCA5 and cytoplasmic MERVL protein in each embryo (individual points), replicate 3 is shown. WT (uninjected) n=11, IgG n=8, α-SMARCA5 n=11. Bonferroni adjusted p-values are shown for each comparison, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Wilcox test). D. Scatterplots showing the expression levels (log2 RPM) of maternal transcripts (∼6000, blue), major ZGA genes (∼3000, red) and MERVL transcripts (∼700, magenta), in the transcriptome data from single embryo NMT-seq libraries generated from two-cell stage SMARCA5 Trim-Away and IgG injected embryos, 28 h post injection, replicate 3. E. Heatmaps (left) showing all DEGs (IgG vs. α-SMARCA5, list compiled from all three experimental replicates) in two-cell stage embryos of replicate 3. Transcripts that failed to downregulate are shown on top, transcripts failed to upregulate at the bottom. The values shown are normalised across datasets (median subtracted from the log2 RPM value for each gene). Box plots (right) illustrate the expression of the corresponding gene sets in wild-type mouse preimplantation embryos . Boxplots show the median, with the upper and lower extremities of the boxes representing the 25 th and 75 th percentile of the data.

Article Snippet: rabbit anti-EG5 (Merck-Millipore, HPA010568) rabbit anti-MERVL (Huabio, R1501-2, IF: 1:100) rabbit anti-SMARCA5 (abcam, ab72499, IF: 1:200) rabbit anti-mCHERRY (abcam, ab167453, IF: 1:200) rat anti-HP1β (abcam, ab10811, IF: 1:300) mouse anti-pan-histone (Merck-Millipore, mab3422, IF: 1:400)

Techniques: Injection, Cell Culture, Immunofluorescence, Staining, Comparison, Expressing, Generated

Journal: bioRxiv

Article Title: Maternal SMARCA5 is required for major ZGA in mouse embryos

doi: 10.1101/2023.12.05.570276

Figure Lengend Snippet: A. Nucleosome phasing plots showing accessibility around TSS (+/-1 kb) for control promoters (all promoters except for SMARCA5-dependent gene promoters) and the promoters of SMARCA5-dependent ZGA genes. The plot on the right shows the profile of nucleosomes around the centre of ATAC-seq peaks (+/-1 kb), that was used to define distal regulatory regions (5066 regions). The ATAC-seq peaks were defined in the published dataset from Wu et al 2016. The y axis shows the GpC methylation percentage (readout for accessibility). The data was normalised and shows levels relative to background. Profiles were generated by running 50 bp windows across 2 kb regions. This data shows replicate 3 (IgG n=10 and α-SMARCA5 n=7), the other two replicates are shown in Figure S8. B. Nucleosome phasing plots showing the accessibility and resulting nucleosome profiles around the centre of CTCF (61,068 Homer motifs) and YY1 (57,432 Homer motifs) motifs at the two-cell stage in WT, IgG and α-SMARCA5 embryos. The y axis shows the GpC methylation percentage. The data was normalised, showing the levels relative to background. The profiles were generated by 50 bp running windows across the 2 kb regions. This data is from replicate 3 (IgG n=10 and α-SMARCA5 n=7), the other two replicates are shown in Figure S8. C. Violin plots showing the global GpC methylation percentage, indicative of chromatin accessibility. Accessibility (percentage of methylated GpC sites in each window) was calculated for windows of 200 GpC sites across the genome for both WT embryos or IgG and anti-SMARCA5 injected embryos. This data is from replicate 3 (IgG n=10 and α-SMARCA5 n=7), the other two replicates are shown in Figure S8.

Article Snippet: rabbit anti-EG5 (Merck-Millipore, HPA010568) rabbit anti-MERVL (Huabio, R1501-2, IF: 1:100) rabbit anti-SMARCA5 (abcam, ab72499, IF: 1:200) rabbit anti-mCHERRY (abcam, ab167453, IF: 1:200) rat anti-HP1β (abcam, ab10811, IF: 1:300) mouse anti-pan-histone (Merck-Millipore, mab3422, IF: 1:400)

Techniques: Methylation, Generated, Injection

Journal: bioRxiv

Article Title: Maternal SMARCA5 is required for major ZGA in mouse embryos

doi: 10.1101/2023.12.05.570276

Figure Lengend Snippet: A. Immunofluorescence images showing representative Smarca5 wild-type and maternal knockout two-cell embryos stained for HP1-β protein (red). The inserts highlight the nuclei, and the zoomed-in regions are showing the NPBs and the presence (or absence) of their heterochromatic “rings”. Scale bar represents 50 μm. B. Number of nucleolus precursor bodies (NPB count) per Smarca5 wild-type (WT) and maternal knockout (matKO) 2-cell embryo. Statistical significance was determined by two tailed Mann-Whitney U test. p-value *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, absence of stars (non-significant, ns): p-value>0.05 C. Volume of NPBs in Smarca5 wild-type (WT) and maternal knockout (matKO) nuclei as indicated by the longest perimeter. Statistical significance was determined by two tailed Mann-Whitney U test. p-value *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: p-value>0.05 D. HP1-β signal intensity around NPBs (measured around longest perimeter). Statistical significance was determined by two tailed Mann-Whitney U test. p-value *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: p-value>0.05 E. Proposed model for the 2 main functions of SMARCA5 in two-cell stage embryos during ZGA. As a regulator of a proportion of ZGA genes (SMARCA5-dependent ZGA genes), SMARCA5 opens up the cromating at their TSS, and also contributes to the nucleosome phasing around this regions, allowing the transcriptional machinery to transcribe these genes. The second role is global, as SMARCA5 seems to be involved in the organisation and reconfiguration of heterochromatic regions in the early embryos.

Article Snippet: rabbit anti-EG5 (Merck-Millipore, HPA010568) rabbit anti-MERVL (Huabio, R1501-2, IF: 1:100) rabbit anti-SMARCA5 (abcam, ab72499, IF: 1:200) rabbit anti-mCHERRY (abcam, ab167453, IF: 1:200) rat anti-HP1β (abcam, ab10811, IF: 1:300) mouse anti-pan-histone (Merck-Millipore, mab3422, IF: 1:400)

Techniques: Immunofluorescence, Knock-Out, Staining, Two Tailed Test, MANN-WHITNEY