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:

Journal: Science Advances

Article Title: Human SAMD9 is a poxvirus-activatable anticodon nuclease inhibiting codon-specific protein synthesis

doi: 10.1126/sciadv.adh8502

Figure Lengend Snippet: ( A ) HeLa cells with a Dox-inducible FTSJ1 –wild type (WT) allele were induced to express different amount of FTSJ1 with varied Dox concentrations (0.1 to 1 μg/ml) for various time (3 to 24 hours). The FTSJ1 protein (arrowhead) and a nonspecific protein were detected by the anti-FTSJ1 antibody. The position of the 37-kDa molecular weight (MW) marker is shown. The abilities of the cells to restrict a SAMD9-sensitive mutant VACV (vK1 − C7 − ) were determined by measuring the viral titers [plaque-forming units (PFU) per milliliter] at 0 and 24 hours postinfection (hpi) with plaque assays on Vero cells. ( B ) HeLa cells were engineered to express no FSTJ1 (ΔFTSJ1) or different FTSJ1 mutants under a Dox-inducible promoter. FTSJ1-K28A mutant is catalytically inactive, while FTSJ1-A26P mutant is defective of methylating at position 34 of certain tRNAs. 2′- O -methylations are indicated by the red lines in the tRNA schematics. ( C ) WT and ΔFTSJ1 HeLa cells were infected with vK1 − C7 − for the indicated time (2 to 8 hours). 8 + A, infection for 8 hours in the presence of cytosine arabinoside (AraC). Separately, total RNAs from uninfected HeLa cells were incubated with recombinant SAMD9 134–385 protein. RNAs resolved on a denaturing gel were stained with SYBR Gold. Lane M contains an RNA ladder, the sizes of which (in bases) are indicated on the left. Northern blots were performed with two probes complementary to 5′ or 3′ end of tRNA Phe or one probe complementary to 3′ end of tRNA Leu(CAA) . Numbers below the gel are relative band intensities with respect to that in uninfected cells. Black or white arrowheads point to the tRNA Phe cleavage products. ( D ) Total RNAs from HeLa cells were incubated with SAMD9 134–385 protein for the indicated time (0.5 to 4 hours). FL, full-length tRNA. ( E ) Total RNAs from HeLa (WT) or ΔFTSJ1 (ΔFt1) cells were incubated with SAMD9 134–385 protein for 1 hour.

Article Snippet: Lentiviral plasmids for expressing FTSJ1 were constructed by PCR amplification of a FTSJ1 complementary DNA (cDNA) clone (Transomic Technologies) and subcloning the PCR fragment into Nhe I and BamH I sites of pCW57.1 (Addgene, no. 41393), a doxycycline-inducible lentiviral vector with rtTA-VP16-2A-puro.

Techniques: Molecular Weight, Marker, Mutagenesis, Infection, Incubation, Recombinant, Staining, Northern Blot

Journal: Science Advances

Article Title: Human SAMD9 is a poxvirus-activatable anticodon nuclease inhibiting codon-specific protein synthesis

doi: 10.1126/sciadv.adh8502

Figure Lengend Snippet: ( A ) RNA-seq was performed on HeLa cells that were uninfected or infected with either WT or vK1 − C7 − (mut) VACV for 8 hours. Numbers of genes in mut VACV and WT VACV–infected cells that showed RPKM (reads per kilobase of transcript, per million mapped reads) fold change (FC) greater than 4 with respect to uninfected cells are shown. A common set of nine genes were induced by SAMD9 R1293W in BT20 cells and by infection with the mut VACV (but not the WT VACV) in HeLa cells. The fold changes are shown in the heatmap. ( B ) Depletion of cellular tRNA Phe and induction of ATF3 in VACV-infected cells in a SAMD9- and FTSJ1-dependent manner. HeLa cells with SAMD9 and SAMD9L KO (ΔhSAMD9&L) or FTSJ1 KO (ΔFTSJ1) were infected with vK1 − C7 − or WT VACV for 8 hours. tRNA Phe and ATF3 levels in the cells were determined by Northern blot and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. ATF3 level is normalized with a mitochondrial mRNA level in the cells. ( C ) SAMD9 R1293W induces ATF3 expression and eIF2a phosphorylation. BT20 cell lines with a Dox-inducible SAMD9 allele (WT or SAMD9 R1293W ) were either uninduced or induced with Dox for 24 hours. Proteins were detected with the indicated antibodies in immunoblots. ( D ) Knockdown of cellular tRNA Phe induces ATF3 expression. Short hairpin RNA (shRNA) against tRNA Phe was transduced into BT20 cells. tRNA Phe level was determined by RT-qPCR, and ATF3 and phosphorylated eIF2a levels were determined by immunoblots.

Article Snippet: Lentiviral plasmids for expressing FTSJ1 were constructed by PCR amplification of a FTSJ1 complementary DNA (cDNA) clone (Transomic Technologies) and subcloning the PCR fragment into Nhe I and BamH I sites of pCW57.1 (Addgene, no. 41393), a doxycycline-inducible lentiviral vector with rtTA-VP16-2A-puro.

Techniques: RNA Sequencing Assay, Infection, Northern Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, shRNA