Limits...
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.


Related in: MedlinePlus

DNAseI sensitivity assay.The analysed genomic regions were subjected to a digestion with 50U of DNAseI. Open chromatin is accessible to the DNAseI and gets digested, whereas closed chromatin does not permit access to the enzyme and is refractory to the digestion. The actin region is an euchromatic one and has been used as a control. Every closed region analysed (including the H23 and 1.360 genomic regions) shows a more open chromatin state in the absence of Hth. The ordinate of the histogram represents ΔCt = Ct (50U)- Ct (0U), being Ct (50U) the cycle threshold of the qPCR after treatment with 50U DNAseI and Ct (0U) the cycle threshold of the qPCR after treatment with OU DNAseI.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4368669&req=5

pone.0120662.g007: DNAseI sensitivity assay.The analysed genomic regions were subjected to a digestion with 50U of DNAseI. Open chromatin is accessible to the DNAseI and gets digested, whereas closed chromatin does not permit access to the enzyme and is refractory to the digestion. The actin region is an euchromatic one and has been used as a control. Every closed region analysed (including the H23 and 1.360 genomic regions) shows a more open chromatin state in the absence of Hth. The ordinate of the histogram represents ΔCt = Ct (50U)- Ct (0U), being Ct (50U) the cycle threshold of the qPCR after treatment with 50U DNAseI and Ct (0U) the cycle threshold of the qPCR after treatment with OU DNAseI.

Mentions: The results of the DNAseI sensitivity assays are summarized in Fig. 7. In wild type embryos, all the regions analysed showed a closed chromatin state, reflected by the fact that they are almost insensitive to a treatment with 50U DNAseI, in contrast to what happens with the control euchromatic actin region (see Fig. 7). This is in agreement with their heterochromatic condition. However, in hth mutant embryos, the same regions show a more open chromatin state and become sensitive to a digestion with 50U of DNAseI as shown in Fig. 7. This result is not only observed with the rDNA genomic region and all of the 1.688 satellite subfamilies, but also with other known heterochromatic regions analysed, as the 1.360 transposable element on chromosome four or the heterochromatic region H23 (22,000–24,000 located in the pericentromeric heterochromatin of chr. 2) [29].


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

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

DNAseI sensitivity assay.The analysed genomic regions were subjected to a digestion with 50U of DNAseI. Open chromatin is accessible to the DNAseI and gets digested, whereas closed chromatin does not permit access to the enzyme and is refractory to the digestion. The actin region is an euchromatic one and has been used as a control. Every closed region analysed (including the H23 and 1.360 genomic regions) shows a more open chromatin state in the absence of Hth. The ordinate of the histogram represents ΔCt = Ct (50U)- Ct (0U), being Ct (50U) the cycle threshold of the qPCR after treatment with 50U DNAseI and Ct (0U) the cycle threshold of the qPCR after treatment with OU DNAseI.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120662.g007: DNAseI sensitivity assay.The analysed genomic regions were subjected to a digestion with 50U of DNAseI. Open chromatin is accessible to the DNAseI and gets digested, whereas closed chromatin does not permit access to the enzyme and is refractory to the digestion. The actin region is an euchromatic one and has been used as a control. Every closed region analysed (including the H23 and 1.360 genomic regions) shows a more open chromatin state in the absence of Hth. The ordinate of the histogram represents ΔCt = Ct (50U)- Ct (0U), being Ct (50U) the cycle threshold of the qPCR after treatment with 50U DNAseI and Ct (0U) the cycle threshold of the qPCR after treatment with OU DNAseI.
Mentions: The results of the DNAseI sensitivity assays are summarized in Fig. 7. In wild type embryos, all the regions analysed showed a closed chromatin state, reflected by the fact that they are almost insensitive to a treatment with 50U DNAseI, in contrast to what happens with the control euchromatic actin region (see Fig. 7). This is in agreement with their heterochromatic condition. However, in hth mutant embryos, the same regions show a more open chromatin state and become sensitive to a digestion with 50U of DNAseI as shown in Fig. 7. This result is not only observed with the rDNA genomic region and all of the 1.688 satellite subfamilies, but also with other known heterochromatic regions analysed, as the 1.360 transposable element on chromosome four or the heterochromatic region H23 (22,000–24,000 located in the pericentromeric heterochromatin of chr. 2) [29].

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.


Related in: MedlinePlus