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The TALE transcription factor homothorax functions to assemble heterochromatin during Drosophila embryogenesis.

Zaballos MA, Cantero W, Azpiazu N - PLoS ONE (2015)

Bottom Line: We were able to show that hth mutants exhibit a drastic overall reduction in the tri-methylation of H3 in Lys9, with no reduction of the previous di-methylation.One phenotypic outcome of such a reduction is a genome instability visualized by the many DNA breaks observed in the mutant nuclei.This work indicates that there is an important role of transcription of non-coding RNAs for constitutive heterochromatin assembly in the Drosophila embryo, and suggests that Hth plays an important role in this process.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, C/ Nicolás Cabrera, 1 Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
We have previously identified Homothorax (Hth) as an important factor for the correct assembly of the pericentromeric heterochromatin during the first fast syncytial divisions of the Drosophila embryo. Here we have extended our studies to later stages of embryonic development. We were able to show that hth mutants exhibit a drastic overall reduction in the tri-methylation of H3 in Lys9, with no reduction of the previous di-methylation. One phenotypic outcome of such a reduction is a genome instability visualized by the many DNA breaks observed in the mutant nuclei. Moreover, loss of Hth leads to the opening of closed heterochromatic regions, including the rDNA genomic region. Our data show that the satellite repeats get transcribed in wild type embryos and that this transcription depends on the presence of Hth, which binds to them as well as to the rDNA region. This work indicates that there is an important role of transcription of non-coding RNAs for constitutive heterochromatin assembly in the Drosophila embryo, and suggests that Hth plays an important role in this process.

No MeSH data available.


Tri-methylation of histone H3 in Lys9 is severely impaired in hth mutant embryos.A, A´) Cycle 14 wild type embryo stained with a specific antibody that recognizes the H3K9me3 mark (green/grey). The signal is detected in the apical domain of the nuclei, which corresponds to the topro3 dense domain. B,B´) Cycle 14 hth mutant embryo stained as in A. Note the lack of signal in the nuclei. C, D) Stage 8 embryo stained as in A. High levels of H3K9me3 are observed in all the mitotic cells and lower levels are detected in the heterochromatin domains of the interphase nuclei (see inset C´, D´). E, F) In a stage 8 mutant embryo the levels of H3K9me3 are very much reduced when compared to wild type embryos (see inset F´). (Embryos were staged as in [61]). G) Western blot analysis of chromatin extracts confirms that the chromatin extracted from hth mutants show reduced levels of the H3K9me3 methyl mark. The graph represents the quantification of the signal observed. The western was performed three times with three different extractions (p-value: 0,00025212, calculated using T-Test). Error bars in the graph show the standard deviation and have being calculated as in Fig. 1 E. Anti-H3 was used as a loading control. Band quantification was done using ImageJ.
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pone.0120662.g002: Tri-methylation of histone H3 in Lys9 is severely impaired in hth mutant embryos.A, A´) Cycle 14 wild type embryo stained with a specific antibody that recognizes the H3K9me3 mark (green/grey). The signal is detected in the apical domain of the nuclei, which corresponds to the topro3 dense domain. B,B´) Cycle 14 hth mutant embryo stained as in A. Note the lack of signal in the nuclei. C, D) Stage 8 embryo stained as in A. High levels of H3K9me3 are observed in all the mitotic cells and lower levels are detected in the heterochromatin domains of the interphase nuclei (see inset C´, D´). E, F) In a stage 8 mutant embryo the levels of H3K9me3 are very much reduced when compared to wild type embryos (see inset F´). (Embryos were staged as in [61]). G) Western blot analysis of chromatin extracts confirms that the chromatin extracted from hth mutants show reduced levels of the H3K9me3 methyl mark. The graph represents the quantification of the signal observed. The western was performed three times with three different extractions (p-value: 0,00025212, calculated using T-Test). Error bars in the graph show the standard deviation and have being calculated as in Fig. 1 E. Anti-H3 was used as a loading control. Band quantification was done using ImageJ.

Mentions: We then looked at the tri-methylation of the same histone residue. In wild type embryos, the first accumulation of H3K9me3 is visible in nuclei of early cycle 14 [33] (see Fig. 2 A, A´). This accumulation fails to occur in Dfhth embryos at that same stage of embryonic development (Fig. 2 B, B¨) and hth mutants show a drastic reduction of H3K9me3 throughout embryonic development (Fig. 2 E, F compare with C, D). The same reduction was observed in the hthP2 mutant embryos (S3 Fig.). Western blot analysis of chromatin extracts and band quantifications confirm this result (Fig. 2 G).


The TALE transcription factor homothorax functions to assemble heterochromatin during Drosophila embryogenesis.

Zaballos MA, Cantero W, Azpiazu N - PLoS ONE (2015)

Tri-methylation of histone H3 in Lys9 is severely impaired in hth mutant embryos.A, A´) Cycle 14 wild type embryo stained with a specific antibody that recognizes the H3K9me3 mark (green/grey). The signal is detected in the apical domain of the nuclei, which corresponds to the topro3 dense domain. B,B´) Cycle 14 hth mutant embryo stained as in A. Note the lack of signal in the nuclei. C, D) Stage 8 embryo stained as in A. High levels of H3K9me3 are observed in all the mitotic cells and lower levels are detected in the heterochromatin domains of the interphase nuclei (see inset C´, D´). E, F) In a stage 8 mutant embryo the levels of H3K9me3 are very much reduced when compared to wild type embryos (see inset F´). (Embryos were staged as in [61]). G) Western blot analysis of chromatin extracts confirms that the chromatin extracted from hth mutants show reduced levels of the H3K9me3 methyl mark. The graph represents the quantification of the signal observed. The western was performed three times with three different extractions (p-value: 0,00025212, calculated using T-Test). Error bars in the graph show the standard deviation and have being calculated as in Fig. 1 E. Anti-H3 was used as a loading control. Band quantification was done using ImageJ.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4368669&req=5

pone.0120662.g002: Tri-methylation of histone H3 in Lys9 is severely impaired in hth mutant embryos.A, A´) Cycle 14 wild type embryo stained with a specific antibody that recognizes the H3K9me3 mark (green/grey). The signal is detected in the apical domain of the nuclei, which corresponds to the topro3 dense domain. B,B´) Cycle 14 hth mutant embryo stained as in A. Note the lack of signal in the nuclei. C, D) Stage 8 embryo stained as in A. High levels of H3K9me3 are observed in all the mitotic cells and lower levels are detected in the heterochromatin domains of the interphase nuclei (see inset C´, D´). E, F) In a stage 8 mutant embryo the levels of H3K9me3 are very much reduced when compared to wild type embryos (see inset F´). (Embryos were staged as in [61]). G) Western blot analysis of chromatin extracts confirms that the chromatin extracted from hth mutants show reduced levels of the H3K9me3 methyl mark. The graph represents the quantification of the signal observed. The western was performed three times with three different extractions (p-value: 0,00025212, calculated using T-Test). Error bars in the graph show the standard deviation and have being calculated as in Fig. 1 E. Anti-H3 was used as a loading control. Band quantification was done using ImageJ.
Mentions: We then looked at the tri-methylation of the same histone residue. In wild type embryos, the first accumulation of H3K9me3 is visible in nuclei of early cycle 14 [33] (see Fig. 2 A, A´). This accumulation fails to occur in Dfhth embryos at that same stage of embryonic development (Fig. 2 B, B¨) and hth mutants show a drastic reduction of H3K9me3 throughout embryonic development (Fig. 2 E, F compare with C, D). The same reduction was observed in the hthP2 mutant embryos (S3 Fig.). Western blot analysis of chromatin extracts and band quantifications confirm this result (Fig. 2 G).

Bottom Line: We were able to show that hth mutants exhibit a drastic overall reduction in the tri-methylation of H3 in Lys9, with no reduction of the previous di-methylation.One phenotypic outcome of such a reduction is a genome instability visualized by the many DNA breaks observed in the mutant nuclei.This work indicates that there is an important role of transcription of non-coding RNAs for constitutive heterochromatin assembly in the Drosophila embryo, and suggests that Hth plays an important role in this process.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, C/ Nicolás Cabrera, 1 Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
We have previously identified Homothorax (Hth) as an important factor for the correct assembly of the pericentromeric heterochromatin during the first fast syncytial divisions of the Drosophila embryo. Here we have extended our studies to later stages of embryonic development. We were able to show that hth mutants exhibit a drastic overall reduction in the tri-methylation of H3 in Lys9, with no reduction of the previous di-methylation. One phenotypic outcome of such a reduction is a genome instability visualized by the many DNA breaks observed in the mutant nuclei. Moreover, loss of Hth leads to the opening of closed heterochromatic regions, including the rDNA genomic region. Our data show that the satellite repeats get transcribed in wild type embryos and that this transcription depends on the presence of Hth, which binds to them as well as to the rDNA region. This work indicates that there is an important role of transcription of non-coding RNAs for constitutive heterochromatin assembly in the Drosophila embryo, and suggests that Hth plays an important role in this process.

No MeSH data available.