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Heterochromatin protein 1 (HP1a) positively regulates euchromatic gene expression through RNA transcript association and interaction with hnRNPs in Drosophila.

Piacentini L, Fanti L, Negri R, Del Vescovo V, Fatica A, Altieri F, Pimpinelli S - PLoS Genet. (2009)

Bottom Line: To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes.Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation.This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Genetica e Biologia Molecolare, Università La Sapienza, Istituto Pasteur, Fondazione Cenci Bolognetti, Roma, Italy.

ABSTRACT
Heterochromatin Protein 1 (HP1a) is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3K9me), creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms.

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HP1a does not seem to be involved in transcript elongation, RNA export, or RNA surveillance.(A) Wild type heat-shocked polytene chromosomes treated with DRB. The treatment does not affect HP1a binding to the induced puffs (see arrows for examples). The absence of immunosignals in the insert shows that DRB does affects the Pol II Phospho Ser2 binding to heat-shocked puffs (arrowheads). (B) FISH detection of poly(A)+ RNA by a Cy3-labeled oligo(dT) probe in salivary glands from wild type and Su(var)2–504/Su(var)2–505 HP1a mutant larvae. The tissues of both types of larvae show similar patterns (arrowheads). (C) Top, in polytene chromosomes of wild type larvae, the immunolocalization of the nuclear exosome component Rrp6. Note that the protein is present on many euchromatic sites including telomeres (arrows) and region 31, but absent on the heterochromatic chromocenter (big arrowhead). In the insert, a confocal microscopy image shows similar Rrp6 immunopattern also in not squashed polytenes. Bottom, the simultaneous immunolocalization of HP1a and Rrp6. Note that the two proteins colocalize mainly on telomeric regions and region 31 (small arrowheads); along the euchromatin there is little overlap (arrows indicate examples of regions that show positive HP1a and negative Rrp6 immunosignals).
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pgen-1000670-g008: HP1a does not seem to be involved in transcript elongation, RNA export, or RNA surveillance.(A) Wild type heat-shocked polytene chromosomes treated with DRB. The treatment does not affect HP1a binding to the induced puffs (see arrows for examples). The absence of immunosignals in the insert shows that DRB does affects the Pol II Phospho Ser2 binding to heat-shocked puffs (arrowheads). (B) FISH detection of poly(A)+ RNA by a Cy3-labeled oligo(dT) probe in salivary glands from wild type and Su(var)2–504/Su(var)2–505 HP1a mutant larvae. The tissues of both types of larvae show similar patterns (arrowheads). (C) Top, in polytene chromosomes of wild type larvae, the immunolocalization of the nuclear exosome component Rrp6. Note that the protein is present on many euchromatic sites including telomeres (arrows) and region 31, but absent on the heterochromatic chromocenter (big arrowhead). In the insert, a confocal microscopy image shows similar Rrp6 immunopattern also in not squashed polytenes. Bottom, the simultaneous immunolocalization of HP1a and Rrp6. Note that the two proteins colocalize mainly on telomeric regions and region 31 (small arrowheads); along the euchromatin there is little overlap (arrows indicate examples of regions that show positive HP1a and negative Rrp6 immunosignals).

Mentions: Different classes of hnRNP proteins are involved in different aspects of RNA metabolism, such as transcript elongation, packaging and stability of mRNAs, RNA splicing, RNA surveillance and RNA export. In which of these functions is HP1a implicated? The cytological colocalization and the direct interaction of HP1a with active Pol II seem to be compatible with an involvement of HP1a in transcript elongation. We tested this hypothesis using the transcription inhibitor DRB on heat-shock induced puffs on polytene chromosomes. This treatment is known to precociously remove the elongation factors [34]. However, as shown in Figure 8A, HP1a remains at all the puffs even after the block of transcription. We also observed that the immunopattern of active Pol II is not affected on HP1a mutant polytene chromosomes (data not shown) indicating that RNA Pol II elongation is not disrupted in the absence of HP1a. Another suggested role for some hnRNPs is a shuttle function for the export of RNA from the nucleus to the cytoplasm [35],[36]. We analyzed the nuclear and cytoplasmic localization of poly(A)+ RNAs in wild type and HP1a mutants using a FISH detection of poly(A)+ RNAs in salivary glands from wild type and HP1a mutant larvae. The localization patterns of poly(A)+ RNAs observed in wild type are not changed in the absence of HP1a (Figure 8B). However, we cannot exclude specific changes in localization of HP1a-bound transcripts due to precocious release prior proper processing by hnRNPs or loading of stabilizing proteins. It is also unlikely that HP1a has a relevant role in RNA surveillance mechanisms. It is well known that mutations in nuclear exosome components increase the amount of transcripts [37]; HP1a mutations have the opposite effect. However, we further tested this suggestion by a simultaneous immunofluorescence staining of polytene chromosomes from wild type larvae, with C1A9 antibody and a specific antibody against the Rrp6 protein which is a nuclear component of the exosome [38]. We found little overlap of the two proteins. HP1a and Rrp6 colocalize at only a few euchromatic sites and at the telomeres (Figure 8C). Although in mammals the hnRNPA/B proteins seem to be important in pre-mRNA splicing, at least two lines of evidence exclude a relevant role for HP1a in this process. HP1a mutations have the same effects on genes with or without introns. Piacentini et al. (2003) have shown that HP1a mutations have a strong quantitative effect on Hsp70 transcripts, though the Hsp70 gene lacks introns and its transcripts do not require pre-mRNA splicing for their maturation. In addition, alterations in the levels of HRB87F have limited effects in alternative splicing in Drosophila [39].


Heterochromatin protein 1 (HP1a) positively regulates euchromatic gene expression through RNA transcript association and interaction with hnRNPs in Drosophila.

Piacentini L, Fanti L, Negri R, Del Vescovo V, Fatica A, Altieri F, Pimpinelli S - PLoS Genet. (2009)

HP1a does not seem to be involved in transcript elongation, RNA export, or RNA surveillance.(A) Wild type heat-shocked polytene chromosomes treated with DRB. The treatment does not affect HP1a binding to the induced puffs (see arrows for examples). The absence of immunosignals in the insert shows that DRB does affects the Pol II Phospho Ser2 binding to heat-shocked puffs (arrowheads). (B) FISH detection of poly(A)+ RNA by a Cy3-labeled oligo(dT) probe in salivary glands from wild type and Su(var)2–504/Su(var)2–505 HP1a mutant larvae. The tissues of both types of larvae show similar patterns (arrowheads). (C) Top, in polytene chromosomes of wild type larvae, the immunolocalization of the nuclear exosome component Rrp6. Note that the protein is present on many euchromatic sites including telomeres (arrows) and region 31, but absent on the heterochromatic chromocenter (big arrowhead). In the insert, a confocal microscopy image shows similar Rrp6 immunopattern also in not squashed polytenes. Bottom, the simultaneous immunolocalization of HP1a and Rrp6. Note that the two proteins colocalize mainly on telomeric regions and region 31 (small arrowheads); along the euchromatin there is little overlap (arrows indicate examples of regions that show positive HP1a and negative Rrp6 immunosignals).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743825&req=5

pgen-1000670-g008: HP1a does not seem to be involved in transcript elongation, RNA export, or RNA surveillance.(A) Wild type heat-shocked polytene chromosomes treated with DRB. The treatment does not affect HP1a binding to the induced puffs (see arrows for examples). The absence of immunosignals in the insert shows that DRB does affects the Pol II Phospho Ser2 binding to heat-shocked puffs (arrowheads). (B) FISH detection of poly(A)+ RNA by a Cy3-labeled oligo(dT) probe in salivary glands from wild type and Su(var)2–504/Su(var)2–505 HP1a mutant larvae. The tissues of both types of larvae show similar patterns (arrowheads). (C) Top, in polytene chromosomes of wild type larvae, the immunolocalization of the nuclear exosome component Rrp6. Note that the protein is present on many euchromatic sites including telomeres (arrows) and region 31, but absent on the heterochromatic chromocenter (big arrowhead). In the insert, a confocal microscopy image shows similar Rrp6 immunopattern also in not squashed polytenes. Bottom, the simultaneous immunolocalization of HP1a and Rrp6. Note that the two proteins colocalize mainly on telomeric regions and region 31 (small arrowheads); along the euchromatin there is little overlap (arrows indicate examples of regions that show positive HP1a and negative Rrp6 immunosignals).
Mentions: Different classes of hnRNP proteins are involved in different aspects of RNA metabolism, such as transcript elongation, packaging and stability of mRNAs, RNA splicing, RNA surveillance and RNA export. In which of these functions is HP1a implicated? The cytological colocalization and the direct interaction of HP1a with active Pol II seem to be compatible with an involvement of HP1a in transcript elongation. We tested this hypothesis using the transcription inhibitor DRB on heat-shock induced puffs on polytene chromosomes. This treatment is known to precociously remove the elongation factors [34]. However, as shown in Figure 8A, HP1a remains at all the puffs even after the block of transcription. We also observed that the immunopattern of active Pol II is not affected on HP1a mutant polytene chromosomes (data not shown) indicating that RNA Pol II elongation is not disrupted in the absence of HP1a. Another suggested role for some hnRNPs is a shuttle function for the export of RNA from the nucleus to the cytoplasm [35],[36]. We analyzed the nuclear and cytoplasmic localization of poly(A)+ RNAs in wild type and HP1a mutants using a FISH detection of poly(A)+ RNAs in salivary glands from wild type and HP1a mutant larvae. The localization patterns of poly(A)+ RNAs observed in wild type are not changed in the absence of HP1a (Figure 8B). However, we cannot exclude specific changes in localization of HP1a-bound transcripts due to precocious release prior proper processing by hnRNPs or loading of stabilizing proteins. It is also unlikely that HP1a has a relevant role in RNA surveillance mechanisms. It is well known that mutations in nuclear exosome components increase the amount of transcripts [37]; HP1a mutations have the opposite effect. However, we further tested this suggestion by a simultaneous immunofluorescence staining of polytene chromosomes from wild type larvae, with C1A9 antibody and a specific antibody against the Rrp6 protein which is a nuclear component of the exosome [38]. We found little overlap of the two proteins. HP1a and Rrp6 colocalize at only a few euchromatic sites and at the telomeres (Figure 8C). Although in mammals the hnRNPA/B proteins seem to be important in pre-mRNA splicing, at least two lines of evidence exclude a relevant role for HP1a in this process. HP1a mutations have the same effects on genes with or without introns. Piacentini et al. (2003) have shown that HP1a mutations have a strong quantitative effect on Hsp70 transcripts, though the Hsp70 gene lacks introns and its transcripts do not require pre-mRNA splicing for their maturation. In addition, alterations in the levels of HRB87F have limited effects in alternative splicing in Drosophila [39].

Bottom Line: To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes.Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation.This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Genetica e Biologia Molecolare, Università La Sapienza, Istituto Pasteur, Fondazione Cenci Bolognetti, Roma, Italy.

ABSTRACT
Heterochromatin Protein 1 (HP1a) is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3K9me), creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms.

Show MeSH
Related in: MedlinePlus