ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

The is7 transgene recapitulates key features of constitutive heterochromatin. (A–B) Brightfield (top) and fluorescence (bottom) images of representative wild-type (A) and is7/+(B) larva at 3 days post-fertilization (dpf). Scale bar indicates 1 mm. (C–D) Levels of the histone modifications H3K9me3, H4K20me3 and H3K27me3 at the is7 transgene array (C) or Sat1 pericentromeric repeats (D) as assessed by chromatin immunoprecipitation quantitative PCR (ChIP-qPCR). (E) 5-methycytosine (5 mC) enrichment (filled circles) at the is7 transgene array in two representative larval pools as detected by sodium bisulfite sequencing. Error bars indicate standard deviation.

FIGURE 2. Morpholino depletion of the histone H3K9me3 methyltransferase Suv39h1b reactivates expression from the silenced is7 transgene array. (A–C) Representative brightfield (left) and fluorescence (right) images of is7/+ transgenic larvae at 3 dpf. (A) Uninjected control, (B) larva that had been injected with 4 ng Suv39h1b morpholino at the one-cell stage, (C) larva that had been coinjected with Suv39h1b morpholino and 400 pg Suv39h1 mRNA (MO = morpholino). (D) Percent Suv39h1b morpholino injected larvae, Suv39h1b morpholino plus Suv39h1b mRNA injected larvae, and sibling uninjected control larvae that show high, medium or low dsRed fluorescence at 3 dpf (U = uninjected). Roughly 50% of larva from crosses between is7/+ males and wild-type females will be transgenic, therefore a maximum of ∼50% of larvae have the capacity for increased is7 expression. n = 40–50 larvae per experimental condition (E) Mean fluorescent intensity of the three larvae showing the highest dsRed fluorescence in Suv39h1b morpholino injected larval pools, morpholino plus RNA injected larval pools and uninjected sibling control pools at 3 dpf. (F–H) Brightfield (left) and fluorescence (right) images of representative is7/+ transgenic larvae that were uninjected (F), injected with 4 ng of morpholino targeting Ehmt2 (G) or 4 ng of morpholino targeting Setdb2 (H) at the one-cell stage and imaged at 3 dpf. (I) Percent larvae showing high, medium or low dsRed fluorescence after injection with 4 ng Ehmt2 or Setdb2 morpholino compared to sibling uninjected controls. n = 50–60 larvae per experimental condition. All scale bars indicate 1 mm. Error bars indicate standard deviation.

Morpholinos designed to deplete additional proteins involved in heterochromatic repression reactivate expression from the is7 transgene array. (A–D) Representative brightfield (left) and fluorescence (right) images of is7/+ transgenic larvae at 3 dpf. (A) Uninjected control, (B) larva injected with 4 ng of Cbx5 morpholino at the one-cell stage, (C) larva injected with 4 ng of Zbtb24 morpholino at the one-cell stage, (D) larva injected with 4 ng of Suv420h2 morpholino at the one-cell stage. (MO = morpholino). (E) Percent larvae injected with each morpholino that show high, medium or low dsRed fluorescence at 3 dpf compared to sibling uninjected controls (U = uninjected). n = ∼30 embryos per condition (F) Mean fluorescent intensity of larvae showing the highest dsRed fluorescence in morpholino injected larval pools, compared to uninjected sibling control pools at 3 dpf. All scale bars indicate 1 mm. Error bars indicate standard deviation.

Morpholinos designed to deplete the H3K36 methyltransferases Nsd1a and Nsd1b increase expression from the is7 transgene array. (A–B) Levels of the histone modifications H3K36me2 and H3K36me3 at the is7 transgene array (A) and Sat1 pericentromeric repeats (B) as assessed by ChIP-qPCR (C–F) Brightfield (left) and fluorescence (right) images of representative is7/+ transgenic larvae at 3 dpf. (C) Uninjected control, (D) larva injected with 4 ng of Nsd1a morpholino at the one-cell stage, (E) larva injected with 4 ng of Nsd1b morpholino at the one-cell stage, (F) larva injected with 3 ng each of Nsd1a and Nsd1b morpholino at the once cell stage (MO = morpholino). (G) Percent larvae that show high, medium or low dsRed fluorescence at 3 dpf compared to sibling uninjected controls (U = uninjected) n = 50-70 embryos per condition. (H) Mean fluorescent intensity of larvae showing the highest dsRed fluorescence in morpholino injected larval pools, compared to uninjected sibling control pools at 3 dpf. All scale bars indicate 1 mm. Error bars indicate standard deviation.

Combined homozygous deletion of nsd1a/b causes derepression of endogenous pericentromeric Sat1 repeats without impacting key molecular markers of heterochromatic silencing. (A) Survival plot comparing wild-type and MZnsd1a/b homozygous deletion mutants over the first 12 months of development (n = ∼60 animals per genotype). (B) Bright field image of representative wild-type and MZnsd1a/b homozygous mutant adult zebrafish at 6 months post fertilization. (C) Expression from pericentromeric Sat1 repeats in wild-type and MZnsd1a/b homozygous mutant larvae at 3 dpf as assessed by qPCR. (D) ChIP-qPCR for IgG, H3K3me2, H3K36me3 and H3K9me3 at Sat1 pericentromeric sequences in wild-type and MZnsd1a/b homozygous mutant larvae at 3 dpf. (E) Southern blot of genomic DNA isolated from wild-type and MZnsd1a/b homozygous mutant larvae at 3 dpf, digested with the methylation sensitive restriction enzyme HpyCH4IV and probed with Sat1 sequence. Equivalent digestion suggests 5 mC levels at Sat1 repeats are unaffected in MZnsd1a/b homozygous mutant larvae. (Wt = Wildtype). Error bars indicate standard deviation.