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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 associates with and colocalizes on polytene chromosomes with DDP1, HRB87F, and PEP hnRNP proteins.(A–C) A part of the right arm of a wild type polytene second chromosome simulteneously immunostained with the anti-HP1a antibody and an antibody against: (A) DDP1, (B) PEP, and (C) HRB87F. There is an extensive colocalization of HP1a with each of the other proteins (arrows). We could not perform simultaneous immunostaining among the DDP1, PEP and HRB87F because the available specific antibodies were made in mouse. However, the colocalization of each protein with HP1a in same regions indicated that all the proteins colocalize in such regions. (For simultaneous immunostainings on whole polytene chromosomes, see Figure S3). (D) Coimmunoprecipitation of HP1a with DDP1 by an anti-DDP1 antibody. (E) Coimmunoprecipitation of HP1a with DDP1, PEP, and HRB87F proteins by the C1A9 anti-HP1a antibody.
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pgen-1000670-g004: HP1a associates with and colocalizes on polytene chromosomes with DDP1, HRB87F, and PEP hnRNP proteins.(A–C) A part of the right arm of a wild type polytene second chromosome simulteneously immunostained with the anti-HP1a antibody and an antibody against: (A) DDP1, (B) PEP, and (C) HRB87F. There is an extensive colocalization of HP1a with each of the other proteins (arrows). We could not perform simultaneous immunostaining among the DDP1, PEP and HRB87F because the available specific antibodies were made in mouse. However, the colocalization of each protein with HP1a in same regions indicated that all the proteins colocalize in such regions. (For simultaneous immunostainings on whole polytene chromosomes, see Figure S3). (D) Coimmunoprecipitation of HP1a with DDP1 by an anti-DDP1 antibody. (E) Coimmunoprecipitation of HP1a with DDP1, PEP, and HRB87F proteins by the C1A9 anti-HP1a antibody.

Mentions: The direct association of HP1a with RNA transcripts suggests that HP1a could be part of one or more ribonucleic complexes. Previous data have shown an interaction between HP1a and DDP1, a multi-KH-domain vigilin that binds single-stranded nucleic acids with high affinity in vitro [24],[25]. The KH-domain is a motif identified for the first time in the human heterogeneous nuclear ribonucleoprotein K (hnRNP K) [26]. In Drosophila, HP1a coimmunoprecipitates (Figure 4D) and extensively colocalizes with DDP1 protein on sub-regions of the heterochromatin and in many sites along the euchromatic arms of polytene chromosomes [24] (see also Figure 4A and Figure S2B). In fact, the binding of HP1a to heterochromatin seems to depend on DDP1 [25]. Treatment with RNase abolishes the DDP1 euchromatic immunopatterns but does not significantly affect the heterochromatin localization (data not shown). It may be that DDP1 binds single-stranded DNA in the chromocenter and RNA transcripts along the euchromatic arms. We asked whether HP1a might also interact with the hnRNP proteins HRB87F and PEP. Both these proteins associate with Hrb57A, another hnRNP protein that, like DDP1, is closely related to the human hnRNP K [27]. Both these proteins, like HP1a, colocalize with the active form of Pol II along polytene chromosomes [28]. HRB87F is the closest Drosophila homolog to mammalian A/B type hnRNP [29], which can bind both RNA and single-stranded DNA [30]. Like HP1a [17], HRB87F undergoes a dramatic chromosomal redistribution after heat-shock [31]. PEP (Peptide on Ecdysone Puffs) is a unique zinc finger protein that, like HP1a, is found preferentially associated on ecdysone-induced puffs as well as other regions of polytene chromosomes [32]. It has been shown that PEP can bind DNA and, with higher affinity, RNA [33].


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 associates with and colocalizes on polytene chromosomes with DDP1, HRB87F, and PEP hnRNP proteins.(A–C) A part of the right arm of a wild type polytene second chromosome simulteneously immunostained with the anti-HP1a antibody and an antibody against: (A) DDP1, (B) PEP, and (C) HRB87F. There is an extensive colocalization of HP1a with each of the other proteins (arrows). We could not perform simultaneous immunostaining among the DDP1, PEP and HRB87F because the available specific antibodies were made in mouse. However, the colocalization of each protein with HP1a in same regions indicated that all the proteins colocalize in such regions. (For simultaneous immunostainings on whole polytene chromosomes, see Figure S3). (D) Coimmunoprecipitation of HP1a with DDP1 by an anti-DDP1 antibody. (E) Coimmunoprecipitation of HP1a with DDP1, PEP, and HRB87F proteins by the C1A9 anti-HP1a antibody.
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Related In: Results  -  Collection

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

pgen-1000670-g004: HP1a associates with and colocalizes on polytene chromosomes with DDP1, HRB87F, and PEP hnRNP proteins.(A–C) A part of the right arm of a wild type polytene second chromosome simulteneously immunostained with the anti-HP1a antibody and an antibody against: (A) DDP1, (B) PEP, and (C) HRB87F. There is an extensive colocalization of HP1a with each of the other proteins (arrows). We could not perform simultaneous immunostaining among the DDP1, PEP and HRB87F because the available specific antibodies were made in mouse. However, the colocalization of each protein with HP1a in same regions indicated that all the proteins colocalize in such regions. (For simultaneous immunostainings on whole polytene chromosomes, see Figure S3). (D) Coimmunoprecipitation of HP1a with DDP1 by an anti-DDP1 antibody. (E) Coimmunoprecipitation of HP1a with DDP1, PEP, and HRB87F proteins by the C1A9 anti-HP1a antibody.
Mentions: The direct association of HP1a with RNA transcripts suggests that HP1a could be part of one or more ribonucleic complexes. Previous data have shown an interaction between HP1a and DDP1, a multi-KH-domain vigilin that binds single-stranded nucleic acids with high affinity in vitro [24],[25]. The KH-domain is a motif identified for the first time in the human heterogeneous nuclear ribonucleoprotein K (hnRNP K) [26]. In Drosophila, HP1a coimmunoprecipitates (Figure 4D) and extensively colocalizes with DDP1 protein on sub-regions of the heterochromatin and in many sites along the euchromatic arms of polytene chromosomes [24] (see also Figure 4A and Figure S2B). In fact, the binding of HP1a to heterochromatin seems to depend on DDP1 [25]. Treatment with RNase abolishes the DDP1 euchromatic immunopatterns but does not significantly affect the heterochromatin localization (data not shown). It may be that DDP1 binds single-stranded DNA in the chromocenter and RNA transcripts along the euchromatic arms. We asked whether HP1a might also interact with the hnRNP proteins HRB87F and PEP. Both these proteins associate with Hrb57A, another hnRNP protein that, like DDP1, is closely related to the human hnRNP K [27]. Both these proteins, like HP1a, colocalize with the active form of Pol II along polytene chromosomes [28]. HRB87F is the closest Drosophila homolog to mammalian A/B type hnRNP [29], which can bind both RNA and single-stranded DNA [30]. Like HP1a [17], HRB87F undergoes a dramatic chromosomal redistribution after heat-shock [31]. PEP (Peptide on Ecdysone Puffs) is a unique zinc finger protein that, like HP1a, is found preferentially associated on ecdysone-induced puffs as well as other regions of polytene chromosomes [32]. It has been shown that PEP can bind DNA and, with higher affinity, RNA [33].

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