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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.

Show MeSH
A mutated chromodomain of HP1a does not modify the immunopattern of Histone H3 di-methylated at lysine 9 (H3K9me2).(A) Immunopattern of H3K9me2 on polytene chromosomes of wild type larvae (Ore-R). The immunosignals are mainly present on the chromocenter. (B) H3K9me2 immunopattern on polytene chromosomes of Su(var)2–502/Su(var)2–505 (02/05) larvae. The immunopattern is very similar to that of wild type. (C) H3K9me2 immunopattern on polytene chromosomes of Su(var)2–504/Su(var)2–505 (04/05)  mutant larvae. In this case, the H3K9me2 immunosignals have been redistributed, even on euchromatic regions.
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pgen-1000670-g003: A mutated chromodomain of HP1a does not modify the immunopattern of Histone H3 di-methylated at lysine 9 (H3K9me2).(A) Immunopattern of H3K9me2 on polytene chromosomes of wild type larvae (Ore-R). The immunosignals are mainly present on the chromocenter. (B) H3K9me2 immunopattern on polytene chromosomes of Su(var)2–502/Su(var)2–505 (02/05) larvae. The immunopattern is very similar to that of wild type. (C) H3K9me2 immunopattern on polytene chromosomes of Su(var)2–504/Su(var)2–505 (04/05) mutant larvae. In this case, the H3K9me2 immunosignals have been redistributed, even on euchromatic regions.

Mentions: To see if HP1a upregulates gene expression by binding to RNA, we did a real time RT-PCR analysis of the expression of 17 genes corresponding to the HP1a target transcripts, in S2 cells treated with dsRNA for HP1a and in Su(var)2–502/Su(var)2–505 larvae, which express an HP1a with a functionally inactive chromodomain. We chose 12 genes that comap with HP1a immunosignals on polytene chromosomes and five others located in regions apparently devoid of HP1a immunosignals (Figure 2A). As a negative control, we tested five genes that do not comap with HP1a in salivary glands and whose transcripts were not HP1a targets in S2 cells. As shown in Figure 2B and 2C, we found a significant reduction in all target transcripts of HP1a while we did not observe any effects on the amount of non-target transcripts (Figure 2D). In HP1a mutant larvae, we found a significant reduction in transcripts of the 12 genes that comap with HP1a (Figure 2E). Three of the genes that do not overlap with any HP1a signals did not show any significant variation between mutant and wild type larvae (Figure 2F). Therefore, these genes do not seem to be regulated by HP1a in larval cells. For the other two genes which do not comap with HP1a on polytene chromosomes, we observed a reduction in transcripts as in S2 cells, probably due to a down-regulation of their expression in other larval tissues; it is possible, in fact, that HP1a binds these transcripts in other larval tissues rather than in salivary glands. As observed in S2 cells, the results of RT-PCR analysis in HP1a mutant larvae clearly show no effect on the amount of the non HP1a target transcripts (Figure 2G). This also implies that the lack of HP1a induces a specific effect in gene expression and not a general effect in gene expression due to a larval lethality induced by the mutation. Previous observations have shown a spreading of H3K9 methylation in salivary glands of HP1a mutant larvae [22], suggesting a general effect on gene transcription following the complete loss of HP1a. To test this possibility, we analyzed the H3K9 methylation along the polytene chromosomes in Su(var)2–502/Su(var)2–505 (02/05) mutants compared to the wild type and to the Su(var)2–504/Su(var)2–505 (04/05) mutants. While in the mutants a H3K9me2 euchromatic redistribution is evident (Figure 3C), in the Su(var)2–502 mutants (Figure 3B) the pattern is similar to that of wild type (Figure 3A), with no spreading of H3K9 methylation. This strongly supports the view that the Su(var)2–502 mutation affects the amount of transcripts of specific genes.


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)

A mutated chromodomain of HP1a does not modify the immunopattern of Histone H3 di-methylated at lysine 9 (H3K9me2).(A) Immunopattern of H3K9me2 on polytene chromosomes of wild type larvae (Ore-R). The immunosignals are mainly present on the chromocenter. (B) H3K9me2 immunopattern on polytene chromosomes of Su(var)2–502/Su(var)2–505 (02/05) larvae. The immunopattern is very similar to that of wild type. (C) H3K9me2 immunopattern on polytene chromosomes of Su(var)2–504/Su(var)2–505 (04/05)  mutant larvae. In this case, the H3K9me2 immunosignals have been redistributed, even on euchromatic regions.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000670-g003: A mutated chromodomain of HP1a does not modify the immunopattern of Histone H3 di-methylated at lysine 9 (H3K9me2).(A) Immunopattern of H3K9me2 on polytene chromosomes of wild type larvae (Ore-R). The immunosignals are mainly present on the chromocenter. (B) H3K9me2 immunopattern on polytene chromosomes of Su(var)2–502/Su(var)2–505 (02/05) larvae. The immunopattern is very similar to that of wild type. (C) H3K9me2 immunopattern on polytene chromosomes of Su(var)2–504/Su(var)2–505 (04/05) mutant larvae. In this case, the H3K9me2 immunosignals have been redistributed, even on euchromatic regions.
Mentions: To see if HP1a upregulates gene expression by binding to RNA, we did a real time RT-PCR analysis of the expression of 17 genes corresponding to the HP1a target transcripts, in S2 cells treated with dsRNA for HP1a and in Su(var)2–502/Su(var)2–505 larvae, which express an HP1a with a functionally inactive chromodomain. We chose 12 genes that comap with HP1a immunosignals on polytene chromosomes and five others located in regions apparently devoid of HP1a immunosignals (Figure 2A). As a negative control, we tested five genes that do not comap with HP1a in salivary glands and whose transcripts were not HP1a targets in S2 cells. As shown in Figure 2B and 2C, we found a significant reduction in all target transcripts of HP1a while we did not observe any effects on the amount of non-target transcripts (Figure 2D). In HP1a mutant larvae, we found a significant reduction in transcripts of the 12 genes that comap with HP1a (Figure 2E). Three of the genes that do not overlap with any HP1a signals did not show any significant variation between mutant and wild type larvae (Figure 2F). Therefore, these genes do not seem to be regulated by HP1a in larval cells. For the other two genes which do not comap with HP1a on polytene chromosomes, we observed a reduction in transcripts as in S2 cells, probably due to a down-regulation of their expression in other larval tissues; it is possible, in fact, that HP1a binds these transcripts in other larval tissues rather than in salivary glands. As observed in S2 cells, the results of RT-PCR analysis in HP1a mutant larvae clearly show no effect on the amount of the non HP1a target transcripts (Figure 2G). This also implies that the lack of HP1a induces a specific effect in gene expression and not a general effect in gene expression due to a larval lethality induced by the mutation. Previous observations have shown a spreading of H3K9 methylation in salivary glands of HP1a mutant larvae [22], suggesting a general effect on gene transcription following the complete loss of HP1a. To test this possibility, we analyzed the H3K9 methylation along the polytene chromosomes in Su(var)2–502/Su(var)2–505 (02/05) mutants compared to the wild type and to the Su(var)2–504/Su(var)2–505 (04/05) mutants. While in the mutants a H3K9me2 euchromatic redistribution is evident (Figure 3C), in the Su(var)2–502 mutants (Figure 3B) the pattern is similar to that of wild type (Figure 3A), with no spreading of H3K9 methylation. This strongly supports the view that the Su(var)2–502 mutation affects the amount of transcripts of specific genes.

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