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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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Related in: MedlinePlus

Hrb87F and Pep are dominant suppressors of position effect variegation (PEV).(A) In flies carrying the In(1)wm4 rearrangement and deficiencies for either Hrb87F or Pep, the eyes are significantly more pigmented than in flies carrying only the chromosome inversion. Hrb87FKG02089 and PepKG00294 mutations could not be tested because these mutations are caused by insertions containing a functionally wild type white gene. (B) Heat-shock lac-Z induction in salivary glands of Tp(3;Y)BL2 larvae which were also either wild type or heterozygous for Su(var)2–502, Hrb87FKG02089, Df(Hrb87F), PepKG00294, PepEP(3)3357, PepEP(3)0408, or Df(Pep). The panel shows examples of staining patterns observed: in all heterozygous mutant larvae the glands stained more heavily than those of wild type larvae.
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pgen-1000670-g011: Hrb87F and Pep are dominant suppressors of position effect variegation (PEV).(A) In flies carrying the In(1)wm4 rearrangement and deficiencies for either Hrb87F or Pep, the eyes are significantly more pigmented than in flies carrying only the chromosome inversion. Hrb87FKG02089 and PepKG00294 mutations could not be tested because these mutations are caused by insertions containing a functionally wild type white gene. (B) Heat-shock lac-Z induction in salivary glands of Tp(3;Y)BL2 larvae which were also either wild type or heterozygous for Su(var)2–502, Hrb87FKG02089, Df(Hrb87F), PepKG00294, PepEP(3)3357, PepEP(3)0408, or Df(Pep). The panel shows examples of staining patterns observed: in all heterozygous mutant larvae the glands stained more heavily than those of wild type larvae.

Mentions: The heterochromatic convergence of these hnRNPs suggests that they, like HP1a, have a role in heterochromatin formation. A common test for the involvement of a gene in heterochromatin formation is to analyze its mutations for their effects on the heterochromatin-induced gene silencing called position effect variegation (PEV). The gene for DDP1 has already been shown to be a suppressor of PEV [25]. We tested Hrb87F and Pep mutations for their effects on the variegation of the Stubble (Sb) gene associated with T(2;3)Sbv [41]. In this translocation, the dominant neomorphic Sb mutation is relocated adjacent to the pericentromeric heterochromatin of the second chromosome. Flies carrying the translocation have a mosaic phenotype with Sb and wild type bristles. The normal bristle phenotype is due to the transcriptional repression of the dominant mutation. We crossed T(2;3)Sbv males to either Hrb87FKG02089/TM3, Df(Hrb87F)/TM3, PepKG00294/TM3, Df(Pep)/TM3 females. We compared the number of Sb and Sb+ bristles in flies carrying the T(2;3)Sbv alone with flies who had the translocation and were also heterozygotes for either Hrb87FKG02089, Df(Hrb87F), PepKG00294, Df(Pep) or TM3 balancer chromosome. The results reported in Table 1 clearly show that mutations at Hrb87F and Pep are dominant suppressors of PEV: they significantly increase the frequency of Sb bristles with respect to the control. We also tested Df(Hrb87F) and Df(Pep) for their effects on the variegation of the white (w) gene associated with the In(1)wm4 [42]. In this inversion, the white gene is transferred to a new position in the heterochromatin. In this location white undergoes a cis-heterochromatin inactivation that occurs in a certain proportion of the cells during development giving, for example in the eyes, a mosaic phenotype of mutant and wild-type areas. As shown in Figure 11A, the mutations dominantly suppress this type of PEV as well. We could not test the other Hrb87F and Pep mutations because they were induce by a P transposon insertion, which contains the w+ gene. Instead we used Tp(3;Y)BL2, a Y chromosome rearrangement carrying the Hsp70-lacZ inducible transgene inserted into its centromeric region [43]. The heterochromatic location causes a variegation for the inducible lac-Z in salivary glands of larval males [44]. We constructed heterozygotes carrying either the Su(var)2–502, Hrb87FKG02089, Df(Hrb87F), PepKG00294, PepEP(3)3357, PepEP(3)0408, or Df(Pep) mutations and a balancer carrying an insertion of the GFP gene (to distinguish heterozygous larvae by the lack of GFP fluorescence). These females heterozygotes were crossed to Tp(3;Y)BL2 males. As the examples in Figure 11B show, all the tested mutations dominantly suppress lac-Z variegation.


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)

Hrb87F and Pep are dominant suppressors of position effect variegation (PEV).(A) In flies carrying the In(1)wm4 rearrangement and deficiencies for either Hrb87F or Pep, the eyes are significantly more pigmented than in flies carrying only the chromosome inversion. Hrb87FKG02089 and PepKG00294 mutations could not be tested because these mutations are caused by insertions containing a functionally wild type white gene. (B) Heat-shock lac-Z induction in salivary glands of Tp(3;Y)BL2 larvae which were also either wild type or heterozygous for Su(var)2–502, Hrb87FKG02089, Df(Hrb87F), PepKG00294, PepEP(3)3357, PepEP(3)0408, or Df(Pep). The panel shows examples of staining patterns observed: in all heterozygous mutant larvae the glands stained more heavily than those of wild type larvae.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000670-g011: Hrb87F and Pep are dominant suppressors of position effect variegation (PEV).(A) In flies carrying the In(1)wm4 rearrangement and deficiencies for either Hrb87F or Pep, the eyes are significantly more pigmented than in flies carrying only the chromosome inversion. Hrb87FKG02089 and PepKG00294 mutations could not be tested because these mutations are caused by insertions containing a functionally wild type white gene. (B) Heat-shock lac-Z induction in salivary glands of Tp(3;Y)BL2 larvae which were also either wild type or heterozygous for Su(var)2–502, Hrb87FKG02089, Df(Hrb87F), PepKG00294, PepEP(3)3357, PepEP(3)0408, or Df(Pep). The panel shows examples of staining patterns observed: in all heterozygous mutant larvae the glands stained more heavily than those of wild type larvae.
Mentions: The heterochromatic convergence of these hnRNPs suggests that they, like HP1a, have a role in heterochromatin formation. A common test for the involvement of a gene in heterochromatin formation is to analyze its mutations for their effects on the heterochromatin-induced gene silencing called position effect variegation (PEV). The gene for DDP1 has already been shown to be a suppressor of PEV [25]. We tested Hrb87F and Pep mutations for their effects on the variegation of the Stubble (Sb) gene associated with T(2;3)Sbv [41]. In this translocation, the dominant neomorphic Sb mutation is relocated adjacent to the pericentromeric heterochromatin of the second chromosome. Flies carrying the translocation have a mosaic phenotype with Sb and wild type bristles. The normal bristle phenotype is due to the transcriptional repression of the dominant mutation. We crossed T(2;3)Sbv males to either Hrb87FKG02089/TM3, Df(Hrb87F)/TM3, PepKG00294/TM3, Df(Pep)/TM3 females. We compared the number of Sb and Sb+ bristles in flies carrying the T(2;3)Sbv alone with flies who had the translocation and were also heterozygotes for either Hrb87FKG02089, Df(Hrb87F), PepKG00294, Df(Pep) or TM3 balancer chromosome. The results reported in Table 1 clearly show that mutations at Hrb87F and Pep are dominant suppressors of PEV: they significantly increase the frequency of Sb bristles with respect to the control. We also tested Df(Hrb87F) and Df(Pep) for their effects on the variegation of the white (w) gene associated with the In(1)wm4 [42]. In this inversion, the white gene is transferred to a new position in the heterochromatin. In this location white undergoes a cis-heterochromatin inactivation that occurs in a certain proportion of the cells during development giving, for example in the eyes, a mosaic phenotype of mutant and wild-type areas. As shown in Figure 11A, the mutations dominantly suppress this type of PEV as well. We could not test the other Hrb87F and Pep mutations because they were induce by a P transposon insertion, which contains the w+ gene. Instead we used Tp(3;Y)BL2, a Y chromosome rearrangement carrying the Hsp70-lacZ inducible transgene inserted into its centromeric region [43]. The heterochromatic location causes a variegation for the inducible lac-Z in salivary glands of larval males [44]. We constructed heterozygotes carrying either the Su(var)2–502, Hrb87FKG02089, Df(Hrb87F), PepKG00294, PepEP(3)3357, PepEP(3)0408, or Df(Pep) mutations and a balancer carrying an insertion of the GFP gene (to distinguish heterozygous larvae by the lack of GFP fluorescence). These females heterozygotes were crossed to Tp(3;Y)BL2 males. As the examples in Figure 11B show, all the tested mutations dominantly suppress lac-Z variegation.

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