Heterochromatin Protein 1 (HP1a) Positively Regulates Euchromatic Gene Expression through RNA Transcript Association and Interaction with hnRNPs in Drosophila

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.

[1]  J. C. Eissenberg,et al.  Overlapping domains of the heterochromatin-associated protein HP1 mediate nuclear localization and heterochromatin binding , 1993, The Journal of cell biology.

[2]  H. Saumweber,et al.  The protein Hrb57A of Drosophila melanogaster closely related to hnRNP K from vertebrates is present at sites active in transcription and coprecipitates with four RNA-binding proteins. , 2000, Gene.

[3]  L. Fanti,et al.  DDP1, a single‐stranded nucleic acid‐binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy , 1999, The EMBO journal.

[4]  B. van Steensel,et al.  High-Resolution Mapping Reveals Links of HP1 with Active and Inactive Chromatin Components , 2007, PLoS genetics.

[5]  T. Jenuwein.  Re-SET-ting heterochromatin by histone methyltransferases. , 2001, Trends in cell biology.

[6]  L. Fanti,et al.  Chromosomal Distribution of Heterochromatin Protein 1 (HP1) in Drosophila: a Cytological Map of Euchromatic HP1 Binding Sites , 2003, Genetica.

[7]  E. Izaurralde,et al.  NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila. , 2001, RNA.

[8]  R. Paro,et al.  The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[9]  P. Chambon,et al.  Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins. , 2001, Molecular cell.

[10]  T. Grigliatti,et al.  Genes which suppress position-effect variegation in Drosophila melanogaster are clustered , 1983, Molecular and General Genetics MGG.

[11]  G. Reuter,et al.  The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation. , 1992, Genetics.

[12]  C. Bishop,et al.  Developmental timing and tissue specificity of heterochromatin‐mediated silencing. , 1996, The EMBO journal.

[13]  M. Frasch,et al.  Nonpackaging and packaging proteins of hnRNA in Drosophila melanogaster , 1983, Cell.

[14]  L. Wallrath,et al.  Role of Drosophila HP1 in euchromatic gene expression , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[15]  D. Johnson,et al.  The Drosophila Hrb87F gene encodes a new member of the A and B hnRNP protein group , 1991, Nucleic Acids Res..

[16]  S. Elgin,et al.  Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. , 1989, European journal of cell biology.

[17]  T. James,et al.  Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene , 1986, Molecular and cellular biology.

[18]  E. Mack,et al.  Drosophila snRNP associated protein P11 which specifically binds to heat shock puff 93D reveals strong homology with hnRNP core protein A1. , 1991, Nucleic acids research.

[19]  A. Waggoner,et al.  Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes. , 1997, Nucleic acids research.

[20]  G. Dreyfuss,et al.  The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein , 1997, The EMBO journal.

[21]  M. Wolf,et al.  The Nuclear Exosome Contributes to Autogenous Control of NAB2 mRNA Levels , 2005, Molecular and Cellular Biology.

[22]  R. Paro,et al.  A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants. , 1991, Nucleic acids research.

[23]  A. Beyer,et al.  A unique zinc finger protein is associated preferentially with active ecdysone-responsive loci in Drosophila. , 1991, Genes & development.

[24]  L. Fanti,et al.  HP1 controls telomere capping, telomere elongation, and telomere silencing by two different mechanisms in Drosophila. , 2004, Molecular cell.

[25]  C. Ling,et al.  Ku70: a candidate tumor suppressor gene for murine T cell lymphoma. , 1998, Molecular cell.

[26]  P. Spierer,et al.  The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1. , 2000, Journal of cell science.

[27]  R. Losson,et al.  Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family , 1999, The EMBO journal.

[28]  A. L. Beyer,et al.  Independent deposition of heterogeneous nuclear ribonucleoproteins and small nuclear ribonucleoprotein particles at sites of transcription. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Beyer,et al.  Heterogeneous nuclear ribonucleoprotein complexes and proteins in Drosophila melanogaster , 1992, Molecular and cellular biology.

[30]  G. Giovinazzo,et al.  The heterochromatin protein 1 prevents telomere fusions in Drosophila. , 1998, Molecular cell.

[31]  Prim B. Singh,et al.  Conservation of Heterochromatin Protein 1 Function , 2000, Molecular and Cellular Biology.

[32]  G. Dreyfuss,et al.  A nuclear export signal in hnRNP A1: A signal-mediated, temperature-dependent nuclear protein export pathway , 1995, Cell.

[33]  M. Botchan,et al.  Association of the Origin Recognition Complex with Heterochromatin and HP1 in Higher Eukaryotes , 1997, Cell.

[34]  N. Andrews,et al.  A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. , 1991, Nucleic acids research.

[35]  A. Stewart,et al.  The chromo shadow domain, a second chromo domain in heterochromatin-binding protein 1, HP1. , 1995, Nucleic acids research.

[36]  C. Caggese,et al.  Transposable elements are stable structural components of Drosophila melanogaster heterochromatin. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. Beyer,et al.  Altered levels of the Drosophila HRB87F/hrp36 hnRNP protein have limited effects on alternative splicing in vivo. , 1996, Molecular biology of the cell.

[38]  L. Fanti,et al.  The trithorax group and Pc group proteins are differentially involved in heterochromatin formation in Drosophila , 2008, Chromosoma.

[39]  J. C. Eissenberg,et al.  Functional analysis of the chromo domain of HP1. , 1995, The EMBO journal.

[40]  Andrea Barta,et al.  Strategies for RNA folding and assembly , 2004, Nature Reviews Molecular Cell Biology.

[41]  Laura Fanti,et al.  Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin , 2003, The Journal of cell biology.

[42]  G. Blobel,et al.  Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin. , 2005, Molecular cell.

[43]  Jordan M. Komisarow,et al.  RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts , 2006, Nature Protocols.

[44]  H. Cooke,et al.  hnRNP A2/B1 binds specifically to single stranded vertebrate telomeric repeat TTAGGGn. , 1992, Nucleic acids research.

[45]  G. Dreyfuss,et al.  The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. , 1993, Nucleic acids research.

[46]  P. Brown,et al.  Global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[47]  F. Sun,et al.  HP1 modulates the transcription of cell-cycle regulators in Drosophila melanogaster , 2005, Nucleic acids research.

[48]  P. Georgiev,et al.  Heterochromatin Protein 1 Is Involved in Control of Telomere Elongation in Drosophila melanogaster , 2002, Molecular and Cellular Biology.

[49]  H. Muller.  Types of visible variations induced by X-rays inDrosophila , 1930, Journal of Genetics.

[50]  J. Casanova,et al.  Drosophila DDP1, a Multi-KH-Domain Protein, Contributes to Centromeric Silencing and Chromosome Segregation , 2004, Current Biology.

[51]  D. Herschlag.  RNA Chaperones and the RNA Folding Problem (*) , 1995, The Journal of Biological Chemistry.

[52]  Prim B. Singh,et al.  Mammalian chromodomain proteins: their role in genome organisation and expression. , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[53]  Stephen Rea,et al.  Central role of Drosophila SU(VAR)3–9 in histone H3‐K9 methylation and heterochromatic gene silencing , 2002, The EMBO journal.

[54]  W. Strätling,et al.  Specific binding of Drosophila nuclear protein PEP (protein on ecdysone puffs) to hsp70 DNA and RNA. , 1998, Nucleic acids research.

[55]  J. Lis,et al.  The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila , 2002, Nature.