Journal: Nature Communications

Article Title: The maternal to zygotic transition regulates genome-wide heterochromatin establishment in the zebrafish embryo

doi: 10.1038/s41467-019-09582-3

Figure Lengend Snippet: Smarca2 degradation is required for H3K9me3 establishment. a Quantitative reverse transcription-PCR (RT-PCR) showing smarca2 RNA decreases during maternal to zygotic transition (MZT). Error bars indicate SEM. b Quantitative RT-PCR confirming elevated levels of smarca2 transcripts in 5 h post fertilization (hpf) embryos that were injected with 2 ng of miR-430 or dicer morpholino at the one-cell stage. P values were calculated using Student’s t test; error bars indicate SEM. c Western blot for H3K9me3 (top) and α-tubulin (bottom) in embryos that were either injected with 200 pg of in vitro transcribed antisense RNA or Smarca2 mRNA at the one-cell stage. Protein was collected for analysis at 4.5 hpf. d Western blot for H3K9me3 (top) and α-tubulin (bottom) in embryos that were either mock injected or injected with 2 ng of smarca2 morpholino at the one-cell stage. Protein was collected for analysis at 3.5 hpf. e Chromatin immunoprecipitation with sequencing (ChIP) for H3K9me3 enrichment at Satellite-1 (Sat1) sequences in embryos injected with a green fluorescent protein (GFP) control morpholino or with smarca2 morpholino. Analysis was performed at 3.5 hpf. P values were calculated using the Student’s t test; error bars indicate SEM. f – g ) ChIP for H3K9me3-enrichment in embryos injected with a scrambled control morpholino or morpholinos targeting smarca2 . f Heatmap depicting H3K9me3 enrichment in control embryos (left) across all H3K9me3 peak centers (±5000 bp) identified in smarca2 morpholino-injected embryos (right). g Screen shots of two representative genomic regions. Red arrows indicate regions that gain H3K9me3 in smarca2 morpholino-injected samples. The black arrow indicates a region that is enriched for H3K9me3 in both control and morpholino-injected embryos. Source data for panels c and d is provided as a source data files

Article Snippet: A plasmid containing the mouse Smarca2 complementary DNA (cDNA) clone was purchased from Transomic Technologies (clone ID BC075641, IMAGE ID 30544092).

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Injection, Western Blot, In Vitro, Chromatin Immunoprecipitation, Sequencing, Control

Journal: Nature Communications

Article Title: The maternal to zygotic transition regulates genome-wide heterochromatin establishment in the zebrafish embryo

doi: 10.1038/s41467-019-09582-3

Figure Lengend Snippet: Smarca2 inhibition accelerates the timeline of chromatin compaction. a Western blot for H3K9me3 (top), and α-tubulin (bottom) in embryos that were either mock injected or injected with 100 μM of the Smarca2 inhibitor PFI-3 at the one-cell stage. Protein was collected for analysis at 3.5 h post fertilization (hpf). b Western blot using embryos injected with increasing concentrations of PFI-3. Protein was collected for analysis at 3.5 hpf. c Electron micrographs demonstrating increased levels of chromatin compaction in 4.5 hpf embryos that were injected with dimethyl sulfoxide (DMSO) or 100 μM PFI-3 at the one-cell stage. Bottom panels represent higher magnification images (×20,000) of nuclear interior in mock- and PFI-3-injected embryos at specified time points. All scale bars indicate 1 μm. d Quantification of the number of particles per nuclear μm 2 and percent nuclear area. Each data point indicates an individual embryo, for each embryo (four DMSO and nine PFI-3-injected embryos) values for particles per um 2 and percent nuclear area were averaged from five to ten representitive nuclei. P values were calculated using the Student’s t test; error bars indicate SD. Source data for panels a and b is provided as a source data files

Article Snippet: A plasmid containing the mouse Smarca2 complementary DNA (cDNA) clone was purchased from Transomic Technologies (clone ID BC075641, IMAGE ID 30544092).

Techniques: Inhibition, Western Blot, Injection

Journal: Nature Communications

Article Title: The maternal to zygotic transition regulates genome-wide heterochromatin establishment in the zebrafish embryo

doi: 10.1038/s41467-019-09582-3

Figure Lengend Snippet: Model for Smarca2-miR430-dependent heterochromatin formation. The early embryo relies exclusively on maternally deposited RNA transcripts and protein, and only starts transcribing the zygotic genome at the maternal to zygotic transition. This model shows that maternal smarca2 , a catalytic subunit of the BRG1/BRM-associated factor (BAF) complex, inhibits H3K9me3 incorporation and chromatin condensation prior to the maternal zygotic transition (1). At the onset of zygotic transcription, the microRNA miR-430 is transcribed and targets maternal smarca2 for degradation (2). Decreasing levels of smarca2 are sufficient to allow for the incorporation of H3K9me3 and formation of condensed chromatin ultrastructure post maternal to zygotic transition (MZT) (3). Zebrafish embryonic stage schematics are reproduced from Kimmel et al. with permission from John Wiley & Sons Inc

Article Snippet: A plasmid containing the mouse Smarca2 complementary DNA (cDNA) clone was purchased from Transomic Technologies (clone ID BC075641, IMAGE ID 30544092).

Techniques: