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Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin.

Piacentini L, Fanti L, Berloco M, Perrini B, Pimpinelli S - J. Cell Biol. (2003)

Bottom Line: Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila.By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes.These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, 00185 Roma, Italy.

ABSTRACT
Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.

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HP1 mutations do not affect the formation of puffs and HSF and POLII binding after heat shock treatment. (A) HSF and (B) POLII binding heat shock– induced puffs in polytene chromosomes of wild-type larvae. C and D show that in HP1 mutant larvae, the heat shock treatment induces puffs along with (C) HSF and (D) POLII binding. E and F show an immunostaining with HSP70 antibodies of whole salivary glands from untreated (E) and heat shock–treated (F) HP1 mutant larvae. Note the absence of any immunosignal in (E) untreated nuclei and the accumulation of the HSP70 in (F) heat shock–treated nuclei. (G) Northern blot analysis of Hsp70 transcripts in different times after heat shock induction in wild-type larvae (+), HP1 mutant larvae (−), and wild-type larvae carrying a heat shock–inducible Su(var)2-5 transgene (++). Hsp70 transcripts are not detectable in wild-type and HP1 mutant untreated larvae. 3 h after heat shock induction, HP1 mutants and transgenic larvae show smaller and larger amounts of Hsp70 transcripts, respectively, compared with levels in wild-type larvae. At 7 h, an inverse situation is present. Compared with the controls, mutant and transgenic larvae show larger and smaller amounts of Hsp70 transcripts, respectively. (H) Western blot analysis of HSP70 and HP1 proteins in untreated and heat shock–treated wild-type larvae (+), HP1 mutant larvae (−), and wild-type larvae carrying the heat shock– inducible Su(var)2-5 transgene (++). As expected, in untreated larvae, HSP70 is absent, whereas 3 h after the heat shock treatment, in mutant and transgenic larvae, the protein is, respectively, less and more abundant than in wild-type. The differential abundance of HSP70 is clearly correlated to the absence and the overexpression of HP1. Note that in wild-type larvae, the quantity of HP1 is not affected by the heat shock treatment. The rp49 transcripts and the α-tubulin protein were used as a control.
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fig4: HP1 mutations do not affect the formation of puffs and HSF and POLII binding after heat shock treatment. (A) HSF and (B) POLII binding heat shock– induced puffs in polytene chromosomes of wild-type larvae. C and D show that in HP1 mutant larvae, the heat shock treatment induces puffs along with (C) HSF and (D) POLII binding. E and F show an immunostaining with HSP70 antibodies of whole salivary glands from untreated (E) and heat shock–treated (F) HP1 mutant larvae. Note the absence of any immunosignal in (E) untreated nuclei and the accumulation of the HSP70 in (F) heat shock–treated nuclei. (G) Northern blot analysis of Hsp70 transcripts in different times after heat shock induction in wild-type larvae (+), HP1 mutant larvae (−), and wild-type larvae carrying a heat shock–inducible Su(var)2-5 transgene (++). Hsp70 transcripts are not detectable in wild-type and HP1 mutant untreated larvae. 3 h after heat shock induction, HP1 mutants and transgenic larvae show smaller and larger amounts of Hsp70 transcripts, respectively, compared with levels in wild-type larvae. At 7 h, an inverse situation is present. Compared with the controls, mutant and transgenic larvae show larger and smaller amounts of Hsp70 transcripts, respectively. (H) Western blot analysis of HSP70 and HP1 proteins in untreated and heat shock–treated wild-type larvae (+), HP1 mutant larvae (−), and wild-type larvae carrying the heat shock– inducible Su(var)2-5 transgene (++). As expected, in untreated larvae, HSP70 is absent, whereas 3 h after the heat shock treatment, in mutant and transgenic larvae, the protein is, respectively, less and more abundant than in wild-type. The differential abundance of HSP70 is clearly correlated to the absence and the overexpression of HP1. Note that in wild-type larvae, the quantity of HP1 is not affected by the heat shock treatment. The rp49 transcripts and the α-tubulin protein were used as a control.

Mentions: To examine whether HP1 might accumulate at heat shock–induced puffs, we focused our assays on the best characterized heat shock–inducible puffs located at the 87A, 87C, 93D, and 95D regions on the right arm of the third chromosome. The 87A and 87C regions contain two and three genes, respectively, all coding the HSP70 protein isoforms (Leigh Brown and Ish-Horowicz, 1981), whereas the 95D region contains the gene encoding the HSP68 protein (for review see Pauli et al., 1992). The 93D region contains a noncoding gene whose activity produces untranslated transcripts (for review see Lakhotia and Sharma, 1996). We did not detect a significant presence of HP1 on these loci when larvae were raised under standard laboratory conditions (25°C). However, when larvae were treated for 30 min at 37°C, and their salivary glands immediately processed for immunostaining, we detected a strong association of HP1 with the heat shock loci. As shown in Fig. 2 E, along the right arm of the third chromosome there are two prominent heat shock puffs at 87A and 87C, and one at 93D and 95D. All of these sites show intense antibody staining, with signals for HP1 dispersed throughout the entire puffs. Strictly speaking, the detection of HP1 in the heat shock puffs could be due either to the exposure of masked epitopes as the loci expand into puffs, or to the recruitment of new proteins upon induction. The latter possibility was suggested by the observation that the HP1 accumulation on heat shock–induced puffs was accompanied by a strong reduction of HP1 staining at nearly all euchromatic sites (Fig. 2 F). To further distinguish between the recruitment versus the epitope exposure possibilities, we used a transgene that increases the level of HP1 at the time of heat shock induction. This transgene, P[(neor)HSHP1.83C] places an HP1 cDNA under the control of the Hsp70 promoter (Eissenberg and Hartnett, 1993). With high temperatures, high levels of HP1 are expressed from the transgene, coincident with heat shock puff formation. Under these conditions, we observed an even stronger accumulation of HP1 on the heat shock puffs than observed in nontransgenic larvae and again a reduction of other euchromatic signals (unpublished data). Most significantly, we also performed heat shock experiments in HP1 mutant larvae carrying the heat shock–inducible P[(neor)HSHP1.83C] transgene. As shown in Fig. 2 G, the HP1 immunostaining does not reveal strong staining on polytenes from untreated larvae. However, in mutant polytenes fixed just after heat shock induction, the puffs are already visible and show an abundant HP1 accumulation (Fig. 2 H). Together, the results demonstrate that HP1 is rapidly recruited to the heat shock–induced puffs likely due to the remobilization of this protein from its euchromatic sites. This interpretation is also consistent with observations described below that verify that the heat shock treatment used in our experiments does not affect the amount of HP1 in larvae (see Fig. 4 H).


Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin.

Piacentini L, Fanti L, Berloco M, Perrini B, Pimpinelli S - J. Cell Biol. (2003)

HP1 mutations do not affect the formation of puffs and HSF and POLII binding after heat shock treatment. (A) HSF and (B) POLII binding heat shock– induced puffs in polytene chromosomes of wild-type larvae. C and D show that in HP1 mutant larvae, the heat shock treatment induces puffs along with (C) HSF and (D) POLII binding. E and F show an immunostaining with HSP70 antibodies of whole salivary glands from untreated (E) and heat shock–treated (F) HP1 mutant larvae. Note the absence of any immunosignal in (E) untreated nuclei and the accumulation of the HSP70 in (F) heat shock–treated nuclei. (G) Northern blot analysis of Hsp70 transcripts in different times after heat shock induction in wild-type larvae (+), HP1 mutant larvae (−), and wild-type larvae carrying a heat shock–inducible Su(var)2-5 transgene (++). Hsp70 transcripts are not detectable in wild-type and HP1 mutant untreated larvae. 3 h after heat shock induction, HP1 mutants and transgenic larvae show smaller and larger amounts of Hsp70 transcripts, respectively, compared with levels in wild-type larvae. At 7 h, an inverse situation is present. Compared with the controls, mutant and transgenic larvae show larger and smaller amounts of Hsp70 transcripts, respectively. (H) Western blot analysis of HSP70 and HP1 proteins in untreated and heat shock–treated wild-type larvae (+), HP1 mutant larvae (−), and wild-type larvae carrying the heat shock– inducible Su(var)2-5 transgene (++). As expected, in untreated larvae, HSP70 is absent, whereas 3 h after the heat shock treatment, in mutant and transgenic larvae, the protein is, respectively, less and more abundant than in wild-type. The differential abundance of HSP70 is clearly correlated to the absence and the overexpression of HP1. Note that in wild-type larvae, the quantity of HP1 is not affected by the heat shock treatment. The rp49 transcripts and the α-tubulin protein were used as a control.
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fig4: HP1 mutations do not affect the formation of puffs and HSF and POLII binding after heat shock treatment. (A) HSF and (B) POLII binding heat shock– induced puffs in polytene chromosomes of wild-type larvae. C and D show that in HP1 mutant larvae, the heat shock treatment induces puffs along with (C) HSF and (D) POLII binding. E and F show an immunostaining with HSP70 antibodies of whole salivary glands from untreated (E) and heat shock–treated (F) HP1 mutant larvae. Note the absence of any immunosignal in (E) untreated nuclei and the accumulation of the HSP70 in (F) heat shock–treated nuclei. (G) Northern blot analysis of Hsp70 transcripts in different times after heat shock induction in wild-type larvae (+), HP1 mutant larvae (−), and wild-type larvae carrying a heat shock–inducible Su(var)2-5 transgene (++). Hsp70 transcripts are not detectable in wild-type and HP1 mutant untreated larvae. 3 h after heat shock induction, HP1 mutants and transgenic larvae show smaller and larger amounts of Hsp70 transcripts, respectively, compared with levels in wild-type larvae. At 7 h, an inverse situation is present. Compared with the controls, mutant and transgenic larvae show larger and smaller amounts of Hsp70 transcripts, respectively. (H) Western blot analysis of HSP70 and HP1 proteins in untreated and heat shock–treated wild-type larvae (+), HP1 mutant larvae (−), and wild-type larvae carrying the heat shock– inducible Su(var)2-5 transgene (++). As expected, in untreated larvae, HSP70 is absent, whereas 3 h after the heat shock treatment, in mutant and transgenic larvae, the protein is, respectively, less and more abundant than in wild-type. The differential abundance of HSP70 is clearly correlated to the absence and the overexpression of HP1. Note that in wild-type larvae, the quantity of HP1 is not affected by the heat shock treatment. The rp49 transcripts and the α-tubulin protein were used as a control.
Mentions: To examine whether HP1 might accumulate at heat shock–induced puffs, we focused our assays on the best characterized heat shock–inducible puffs located at the 87A, 87C, 93D, and 95D regions on the right arm of the third chromosome. The 87A and 87C regions contain two and three genes, respectively, all coding the HSP70 protein isoforms (Leigh Brown and Ish-Horowicz, 1981), whereas the 95D region contains the gene encoding the HSP68 protein (for review see Pauli et al., 1992). The 93D region contains a noncoding gene whose activity produces untranslated transcripts (for review see Lakhotia and Sharma, 1996). We did not detect a significant presence of HP1 on these loci when larvae were raised under standard laboratory conditions (25°C). However, when larvae were treated for 30 min at 37°C, and their salivary glands immediately processed for immunostaining, we detected a strong association of HP1 with the heat shock loci. As shown in Fig. 2 E, along the right arm of the third chromosome there are two prominent heat shock puffs at 87A and 87C, and one at 93D and 95D. All of these sites show intense antibody staining, with signals for HP1 dispersed throughout the entire puffs. Strictly speaking, the detection of HP1 in the heat shock puffs could be due either to the exposure of masked epitopes as the loci expand into puffs, or to the recruitment of new proteins upon induction. The latter possibility was suggested by the observation that the HP1 accumulation on heat shock–induced puffs was accompanied by a strong reduction of HP1 staining at nearly all euchromatic sites (Fig. 2 F). To further distinguish between the recruitment versus the epitope exposure possibilities, we used a transgene that increases the level of HP1 at the time of heat shock induction. This transgene, P[(neor)HSHP1.83C] places an HP1 cDNA under the control of the Hsp70 promoter (Eissenberg and Hartnett, 1993). With high temperatures, high levels of HP1 are expressed from the transgene, coincident with heat shock puff formation. Under these conditions, we observed an even stronger accumulation of HP1 on the heat shock puffs than observed in nontransgenic larvae and again a reduction of other euchromatic signals (unpublished data). Most significantly, we also performed heat shock experiments in HP1 mutant larvae carrying the heat shock–inducible P[(neor)HSHP1.83C] transgene. As shown in Fig. 2 G, the HP1 immunostaining does not reveal strong staining on polytenes from untreated larvae. However, in mutant polytenes fixed just after heat shock induction, the puffs are already visible and show an abundant HP1 accumulation (Fig. 2 H). Together, the results demonstrate that HP1 is rapidly recruited to the heat shock–induced puffs likely due to the remobilization of this protein from its euchromatic sites. This interpretation is also consistent with observations described below that verify that the heat shock treatment used in our experiments does not affect the amount of HP1 in larvae (see Fig. 4 H).

Bottom Line: Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila.By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes.These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, 00185 Roma, Italy.

ABSTRACT
Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.

Show MeSH
Related in: MedlinePlus