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Enrichment of H3K9me2 on Unsynapsed Chromatin in Caenorhabditis elegans Does Not Target de Novo Sites.

Guo Y, Yang B, Li Y, Xu X, Maine EM - G3 (Bethesda) (2015)

Bottom Line: Loss of the SET domain protein, MET-2, greatly reduces H3K9me2 abundance and results in germline mortality.These results suggest that MET-2 activity is elevated in him-8 mutants generally as well as targeted preferentially to the unsynapsed X.We hypothesize H3K9me2 may have a structural function critical for germline immortality, and a greater abundance of these marks may be required when a chromosome does not synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Syracuse University, Syracuse, New York 13244.

No MeSH data available.


Related in: MedlinePlus

Histone H3 lysine 9 dimethylation (H3K9me2) abundance is elevated in him-8 and low in the germline relative to the soma. (A, C) Representative protein blots probed with pan-H3 antibody and antibodies against several modified forms of histone H3. The number of whole adult hermaphrodites or dissected adult hermaphrodite gonad arms used to prepare extract for each lane is indicated. As the C. elegans gonad is primarily germ line, most of the histone H3 in gonad extracts is from germ cells (see Impact of H3K9me2 loss on the gonad transcriptome). Aliquots of a single extract were probed for total H3 and an individual H3 modification. Signal was quantified and the ratio of H3 modification to pan-H3 signal was calculated to determine the normalized value for each modification. (B) Histogram represents the ratio of normalized H3K9me2 signal in him-8 relative to wild-type (N2) whole animals and isolated gonads. H3K9me2 signal is greater in him-8 whole animals and gonads. (D) Histogram represents the ratio of normalized signal in isolated gonads relative to whole animals. Note that the Y-axis scale is different in B and D. Data represent three biological replicates for H3K4me1 and two biological replicates for each other mark (see the section Materials and Methods). Error bars, SEM.
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fig6: Histone H3 lysine 9 dimethylation (H3K9me2) abundance is elevated in him-8 and low in the germline relative to the soma. (A, C) Representative protein blots probed with pan-H3 antibody and antibodies against several modified forms of histone H3. The number of whole adult hermaphrodites or dissected adult hermaphrodite gonad arms used to prepare extract for each lane is indicated. As the C. elegans gonad is primarily germ line, most of the histone H3 in gonad extracts is from germ cells (see Impact of H3K9me2 loss on the gonad transcriptome). Aliquots of a single extract were probed for total H3 and an individual H3 modification. Signal was quantified and the ratio of H3 modification to pan-H3 signal was calculated to determine the normalized value for each modification. (B) Histogram represents the ratio of normalized H3K9me2 signal in him-8 relative to wild-type (N2) whole animals and isolated gonads. H3K9me2 signal is greater in him-8 whole animals and gonads. (D) Histogram represents the ratio of normalized signal in isolated gonads relative to whole animals. Note that the Y-axis scale is different in B and D. Data represent three biological replicates for H3K4me1 and two biological replicates for each other mark (see the section Materials and Methods). Error bars, SEM.

Mentions: Western blots were prepared using standard protocols. Gonads were dissected from hermaphrodites at L4 stage + ∼18−24 hr. Animals were washed 2X in M9 + protease inhibitor cocktail (Roche) and dissected in fresh M9 + protease inhibitor. Dissected gonads were transferred to a well with fresh solution; when ∼150 gonads were accumulated, material was centrifuged, excess solution removed, and tissue was frozen on dry ice. For whole worms, the specified number of animals was washed in M9, transferred to a tube, spun down, excess solution removed, and frozen on dry ice. To prepare samples, an appropriate amount of 4× loading buffer was added, and sample was boiled for 10 min, cooled down on ice, and centrifuged. Supernatant was transferred to a new tube and volume was measured. Total volume was adjusted by addition of 1× loading buffer, and appropriate amounts were loaded onto gels for histone detection. Typically, the adjusted volume was 32 μL; 16 μL was used for testing histone H3 modification, and 8 μL was used for testing histone H3. Pierce SuperSignal West Pico (#34080) or Femto (#34094) detection substrate was used to visualize protein. We noted that the Femto substrate consistently detected rarer modifications, H3K9me2 and H3K4me3, at a higher level relative to total H3 than did the Pico substrate. In contrast, we did not observe this difference when assaying protein that is present at a relatively greater level, e.g., gonad H3K4me1 was detected at 80% of whole animal H3K4me1 by both the Pico and Femto substrates. We hypothesize that detection at the lower limit of the Pico substrate sensitivity is unreliable, and included only Femto detection data for H3K9me2 and H3K4me3 in Figure 6. Quantification was performed using Image J software (Gassmann et al. 2009).


Enrichment of H3K9me2 on Unsynapsed Chromatin in Caenorhabditis elegans Does Not Target de Novo Sites.

Guo Y, Yang B, Li Y, Xu X, Maine EM - G3 (Bethesda) (2015)

Histone H3 lysine 9 dimethylation (H3K9me2) abundance is elevated in him-8 and low in the germline relative to the soma. (A, C) Representative protein blots probed with pan-H3 antibody and antibodies against several modified forms of histone H3. The number of whole adult hermaphrodites or dissected adult hermaphrodite gonad arms used to prepare extract for each lane is indicated. As the C. elegans gonad is primarily germ line, most of the histone H3 in gonad extracts is from germ cells (see Impact of H3K9me2 loss on the gonad transcriptome). Aliquots of a single extract were probed for total H3 and an individual H3 modification. Signal was quantified and the ratio of H3 modification to pan-H3 signal was calculated to determine the normalized value for each modification. (B) Histogram represents the ratio of normalized H3K9me2 signal in him-8 relative to wild-type (N2) whole animals and isolated gonads. H3K9me2 signal is greater in him-8 whole animals and gonads. (D) Histogram represents the ratio of normalized signal in isolated gonads relative to whole animals. Note that the Y-axis scale is different in B and D. Data represent three biological replicates for H3K4me1 and two biological replicates for each other mark (see the section Materials and Methods). Error bars, SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555223&req=5

fig6: Histone H3 lysine 9 dimethylation (H3K9me2) abundance is elevated in him-8 and low in the germline relative to the soma. (A, C) Representative protein blots probed with pan-H3 antibody and antibodies against several modified forms of histone H3. The number of whole adult hermaphrodites or dissected adult hermaphrodite gonad arms used to prepare extract for each lane is indicated. As the C. elegans gonad is primarily germ line, most of the histone H3 in gonad extracts is from germ cells (see Impact of H3K9me2 loss on the gonad transcriptome). Aliquots of a single extract were probed for total H3 and an individual H3 modification. Signal was quantified and the ratio of H3 modification to pan-H3 signal was calculated to determine the normalized value for each modification. (B) Histogram represents the ratio of normalized H3K9me2 signal in him-8 relative to wild-type (N2) whole animals and isolated gonads. H3K9me2 signal is greater in him-8 whole animals and gonads. (D) Histogram represents the ratio of normalized signal in isolated gonads relative to whole animals. Note that the Y-axis scale is different in B and D. Data represent three biological replicates for H3K4me1 and two biological replicates for each other mark (see the section Materials and Methods). Error bars, SEM.
Mentions: Western blots were prepared using standard protocols. Gonads were dissected from hermaphrodites at L4 stage + ∼18−24 hr. Animals were washed 2X in M9 + protease inhibitor cocktail (Roche) and dissected in fresh M9 + protease inhibitor. Dissected gonads were transferred to a well with fresh solution; when ∼150 gonads were accumulated, material was centrifuged, excess solution removed, and tissue was frozen on dry ice. For whole worms, the specified number of animals was washed in M9, transferred to a tube, spun down, excess solution removed, and frozen on dry ice. To prepare samples, an appropriate amount of 4× loading buffer was added, and sample was boiled for 10 min, cooled down on ice, and centrifuged. Supernatant was transferred to a new tube and volume was measured. Total volume was adjusted by addition of 1× loading buffer, and appropriate amounts were loaded onto gels for histone detection. Typically, the adjusted volume was 32 μL; 16 μL was used for testing histone H3 modification, and 8 μL was used for testing histone H3. Pierce SuperSignal West Pico (#34080) or Femto (#34094) detection substrate was used to visualize protein. We noted that the Femto substrate consistently detected rarer modifications, H3K9me2 and H3K4me3, at a higher level relative to total H3 than did the Pico substrate. In contrast, we did not observe this difference when assaying protein that is present at a relatively greater level, e.g., gonad H3K4me1 was detected at 80% of whole animal H3K4me1 by both the Pico and Femto substrates. We hypothesize that detection at the lower limit of the Pico substrate sensitivity is unreliable, and included only Femto detection data for H3K9me2 and H3K4me3 in Figure 6. Quantification was performed using Image J software (Gassmann et al. 2009).

Bottom Line: Loss of the SET domain protein, MET-2, greatly reduces H3K9me2 abundance and results in germline mortality.These results suggest that MET-2 activity is elevated in him-8 mutants generally as well as targeted preferentially to the unsynapsed X.We hypothesize H3K9me2 may have a structural function critical for germline immortality, and a greater abundance of these marks may be required when a chromosome does not synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Syracuse University, Syracuse, New York 13244.

No MeSH data available.


Related in: MedlinePlus