Telomeric puffing induced by heat shock in Chironomus ... - CiteSeerX

J. Biosci., Vol. 21, Number 2, April 1996, pp 247-257.© Printed in India.

Telomeric puffing induced by heat shock in Chironomus thummi G MORCILLO* and J L DIEZ† Centro de Investigaciones Biológicas. CSIC, Velazquez 144, 28006 Madrid, Spain *Universidad Nacional de Education a Distancia, Senda del Rcy s/n, 28040 Madrid, Spain MS received 6 December 1995; revised 22 January 1996 Abstract. We summarize the most remarkable features of the heat shock inducible large telomeric puffs (T-BRs) in polytene chromosomes of Chironomus thummi. Kinetic aspects of formation of T-BRs as well as their transcriptional behaviour clearly support the view that T-BRs are components of the heat shock response in Chironomus. Available molecular data indicate T-BRs to include long arrays of 176 bp tandem repeats. A large transcript (> 10 kb) encompassing the telomere associated repeat has been detected. Several other similarities between T-BRs of Chironomus and the hsrω genes of Drosophila suggest the T-BRs to be hsrω counterpart in Chironomus. Keywords.

1.

Heat Shock; telomere; polytene chromosomes, Τ-BR; hsrω.

Introduction

The rapid changes in the pattern of gene expression in any cell in response to environmental damage (e.g., supraoptimal temperatures) reveal the existence of a universal and conserved cellular mechanism which works from prokaryotic to eukaryotic cells. It is known as the heat shock response. A group of genes, named heat shock (HS) genes, is activated upon heat shock. In all eukaryotic HS-genes so far examined, a specific heat shock transcription factor (HSF) interacting with 5’ upstream heat shock regulatory elements (HSE) has been found. This leads to a rapid, transient and coordinated transcriptional regulation of HS-gene expression. Subsequently, the cells carry out an efficient and massive synthesis of the socalled heat shock proteins (HSPs), which account for the physiological and protective role of the response which is not fully understood yet. Drosophila polytene chromosomes provided the first insight into the heat shock response through the observation of the HS-activated puffs (Ritossa 1962). Later, the molecular organization of HS-genes was analysed and their encoding HSPs were characterized (reviewed in Ashburner and Bonner 1979). The response to heat has been thereafter studied in many different organisms as a model system to investigate the regulation of gene activity. On the other hand, this ubiquitous response from bacteria to man, pointed to a basic process of fundamental importance to all living cells. Thus, surprisingly one HSP, the HSP70, is the most conserved protein in phylogeny so far known (Gupta and Singh 1994). The evidence that a broad range of physiological stress conditions lead to the synthesis of HSPs opened even more the general interest of this research field. Nowadays, medical and therapeutic implications of the heat shock proteins are also being actively investigated (Morimoto et al 1990). *Corresponding author (Fax, 3391 -5627518; Email, CIBMOJP(a cc.csic.es).

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248 2.

G Morcillo and J L Díez The heat shock response in Chironomus

Chironomus, a genus of Diptera with larval polytene chromosomes, has been much less studied with respect to the heat shock response. Nevertheless, early studies in Chironomus tentans revealed a rapid and transient repression of normally active genes, a phenomenon that has received less attention but that is concomitant with the activation of HS-genes in most species. This was clearly observed in the regression of the giant RNA transcribing puffs named Balbiani rings (BR1, BR2 and BR3) encoding giant secretory proteins of the larvae (Yamamoto 1970). The first insight into the cellular localization of HSPs was also obtained in microdissected nuclei of C. tentans (Vincent and Tanguay 1979), showing a rapid entry of some newly synthesized HSPs into the nuclei following heat shock. This was later confirmed in other systems. The heat shock response has been described in some Chironomus species. A set of loci became puffed and transcribe actively (C. tentans: Lezzi et al 1981; Lezzi 1984; C. thummi: Morcillo et al 1981, 1982; C. striatipennis: Nath and Lakhotia 1989), while a new group of polypeptides, which fairly well correspond to the HSPs described in Drosophila species, are actively synthesized under heat shock (C. tentans: Tanguay and Vincent , 1981; C. thummi: Carretero et al 1986; C. striatipennis: Nath and Lakhotia 1989). With the species-specific characteristics, these results agreed with those expected for a typical heat shock response. The only exception and the most striking feature is the puffing of some telomeres (figure 1) we have found systematically in C. thummi polytene chromosomes (Morcillo et al 1981; Santa-Cruz et al 1981). In this article we will summarize some characteristics of these giant telomeric puffs, which resemble a Balbiani ring, named T-BRs. The significance of T-BRs in the context of the heat

Figure 1. Salivary gland chromosome complement from a heat shocked larva (1 h 35°C). Telomeric puffing at IIIR is induced (T-BRIII), while BR1 and BR2, on chromosome IV, are collapsed.

Telomeric puffing induced by heat shock in Chironomus thummi

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shock response will be discussed in relation to possible equivalents in Drosophila and other Chironomids. 3.

Induction of telomeric puffs: T-BRs

The most remarkable effect of elevated temperatures on C. thummi polytene chromosomes is the induction of a giant Balbiani ring-like structure at the right telomere of chromosome III (figure 2). Electron microscopy showed that T-BRIII is formed at the very end of the chromosome (Santa-Cruz et al 1984). No dense material appeared bordering the puff as it occurs in subterminal puffs, e.g., the 2-48BC HS-locus in Drosophila hydei delimited by the telomere (Berendes and Meyer 1968; Derksen and Willart 1976). This justified the term telomeric puffing to describe these structures.

Figure 2. Heat shock induced changes at III R telomere, (a) Control conditions, (b) T-BRIII induced after 3 h 35°C heat shock and (c) electron micrograph of T-BRIII after 3 h 35°C treatment.

Besides the T-BRIII, other telomeres may appear additionally puffed but in a rather variable way. Nevertheless, a frequency gradient can be established, ranging from the IIIR that is always induced, to the IL that does rather infrequently, and finally chromosome IV where neither the telomere nor the telocentromere is seen to puff (figure 3). There is also a difference among telomeres in the puffing size. This is clearly manifested at the two ends of chromosome III, the right being the major T-BR while the left, when puffed, reached always a much smaller size. The temperature necessary to induce T-BRs ranges from 33°C to 37°C, while the larvae are cultivated at 18°C in the laboratory. At lower temperatures we could not observe either induction of T-BRs or other changes in HS-loci, whereas slight modifications in the pattern of protein synthesis with appearance of HSPs were obtained even at 28°C (Carretero et al 1986). Higher temperatures were lethal for the larvae (Carretero et al 1991). Nevertheless, a short shock of 5’ at 39°C can induce, during the recovery at 18°C, the T-BRs as well as the typical pattern of HS-puffs and HSPs.

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Figure 3. Differential HS inducibility of C. thummi telomeres. All the telomeres share the 176 bp TA repeat ( ) except IV left which corresponds to the kinetochoric end. The frequency of T-BR induction is ranged in four categories: high, medium, low and null. The T-BR size is ranged in two categories: large and small.

The kinetics of T-BRs induction-regression also parallels the heat shock response, that is the induction-regression of the rest of HS-puffs. Moreover, regression of T-BRs is always concomitant with reactivation of constitutive genes, i.e., BR1 and BR2. After a 3 h treatment at 35°C the T-BRs reach their maximum size, regressing after 7h of recovery at 18°C, when reactivation of BR1 and BR2 takes place. From these conditions larvae can recover in a matter of hours. In a continuous 35°C treatment, T-BRs reached their maximum size in 3 h, regressing after 10 h at 35°C when there was a partial reactivation of BR1 and BR2. But finally the larvae die in 16–20h at this temperature. As a consequence of a short shift at sublethal temperatures (5’ at 39°C) the T-BRs reach a maximum in about 3 h at 18°C, regressing after 8 h of recovery when the normal pattern of puffing was reached. With this treatment most larvae survived. Therefore, the kinetics of T-BR expansion-regression suggest a physiological process, positively correlated with the heat shock response (other HS-puffs and HSPs) and negatively correlated with the nromal pattern of puffing (BR1, BR2). This result argues against the T-BRs as the manifestation of a process of irreversible cell damage that precedes lysis as was interpreted by some authors (Reznik et al 1984). A broad variety of stress conditions are able to mimic the effect of heat on gene expression, although with some species-specificity of inducers. Nevertheless, different conditions reported to induce the heat shock response in Drosophila failed to induce it


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in C. thummi, including the activation of T-BRs. Among the treatments assayed, only during recovery from CO2 have we observed a response similar to that previously described after a heat treatment: induction of HS-puffs, synthesis of HSPs and T-BR puffing (Barettino et al 1988b). This fact reinforces the idea of T-BRs as a functional component of the stress response, and not a consequence of the effect of temperature on the local decondensation of telomeric chromatin. Finally, activation of telomeres to form T-BRs is not limited to the polytene chromosomes of salivary glands as it has been also observed in other larval tissues as Malpighian tubules (Morcillo et al 1994). Heat shock-induced telomeric puffs have also been observed in the sibling species C. piger (Morcillo et al 1988). In this case, unlike the situation in C. thummi only the telomere IVR formed a Τ-BR, while the telomere IIIR did not puff. An intriguing property of the T-BRs regarding their inducibility is worth noting. They could be induced to puff upon heal shock even in the presence of different RNA synthesis inhibitors (aclinomycin D, α-amanitin, DRB) although, under these conditions, there was neither any transcriptional activity nor detection of other HS-puffs. (Barettino et al 1982). 4. Transcription at T-BRs T-BR formation involves transcriptional activity, the most typical feature of puffing. Transcription at T-BRs could be detected by different methods. The local labelling all over the induced T-BR after a short pulse with [3H]-uridine undoubtedly means transcription at this structure under heat shock. Labelling appeared also heavily concentrated over specific loci that correspond to other heat shock-induced puffs (Morcillo et al 1981, 1982). The presence of RNA polymerase II as well as DNA-RNA hybrids was also detected at T-BRs by immunolocalization with specific antibodies with the localization being limited to the T-BRs and other HS-puffs induced at elevated temperatures (Diez and Barettino 1984; Barettino et al 1988a, b). Also the HSF has been found at T-BR and other heal shock loci in C. thummi by immunocytochemistry using the Drosophila HSF-antibody (Morcillo et al 1994). HSF binds selectively to specific sequences, the HSE, in the promoters of all known heat shock genes (Westwood et al 1991). Therefore, this result suggests the existence of HSE in the promoter region of T-BR that triggered its activation by heat shock, characterizing the T-BR as a real heat shock puff despite its peculiar telomeric localization. T-BRIII was observed to contain 250Å RNP, EDTA-positive, particles forming either linear arrays or very frequently large aggregates in a clustered form (Santa-Cruz et al 1984). These granules were absent in controls or when the T-BR was formed but not transcriptionally active after a heat shock in the presence of actinomycin D. Therefore these RNP granules appeared related to the product of transcription of these loci under heat shock. 5. Molecular characterization of T-BRs: the T-BR transcript T-BRIIIR specific sequences were obtained by microcloning this chromosome region (Carmona et al 1985). A 176 bp sequence, representing a basic repeat unit clustered in

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tandem arrays, was isolated. Sequence analysis showed tracks of AT rich segments but no open reading frame. Although originally obtained from the right telomere of chromosome III, this or related sequences appear shared by all the telomeres, except for the telocentromere at chromosome IV. They are also present at two interstitial bands in chromosome II, as revealed by in situ hybridization to chromosomal DNA (Carmona et al 1985). This sequence represents about 0·9% of the genome of C. thummi, so that around 6,900 copies of the repeat should be present per haploid genome, which should correspond to about 1000 copies in each telomere. As will be discussed later (§7), these DNA blocks represent “telomere associated sequences” (TA), similar to those described in other chironomids as C. pallidivitatus and C. tentans (Saiga and Edström 1985; Nielsen et al 1990). Northern blot analysis, using the repeat as probe, showed a transcript (larger than 10 kb) in control salivary glands suggesting that TA sequences are constitutively transcribed (Carmona et al 1985). This transcript has been also detected in other non polytenic tissues (Morcillo et al 1994). The level of transcripts is modified by heat shock in a rather variable fashion. Only in some Northern blot experiments was their level clearly increased. However, in situ hybridization of the 176 bp repeat to chromosomal RNA always showed a clear increase in labelling at the induced T-BRIII, and other telomeres when puffed, either after heat shock or CO2 induction (Carmona et al 1985; Barettino et al 1988b). Transcription at telomeres was only observed in controls after long exposures after in situ hybridization with radioactive probes (Botella et al 1991). Transcriptional activation of TA sequences, either by heat shock or CO2, seems to be associated with T-BR formation suggesting that this RNA is the transcription product of the T-BRs. This does not exclude the possibility of other transcripts at T-BRs not detected up to now. In situ localization of the transcript in frozen sections after a heat shock shows a clear nuclear location in a rather clustered pattern (G Morcillo, unpublished results). Nevertheless, in Northern blot analysis of cellular fractions, transcripts were found both in nuclei and cytoplasm (Botella et al 1991). The function of these transcripts, either in controls or under heat shock, is largely unknown at present. It does not seem to encode for any protein as there is no open reading frame at least in the sequence representing the repeat (Carmona et al 1985). 6.

Significance of T-BR in the context of the heat shock response

The experimental data suggest that T-BRs behave as component of the heat shock response in C. thummi. The HSF-mediated transcription, its activation following CO2 treatments, a condition that mimics the heat shock response in C. thummi, and the kinetics of induction-regression that parallels other HS-puffs support this view. Moreover, cis regulatory elements appear to be implicated in the activation of telomeres as heterozygotic expression of telomeric puffing was obtained in chromosomes of C. thummi × C. piger hybrids (Morcillo et al 1988). The question is whether this represents a special telomeric location of a HS-gene with counterparts in other systems. In this hypothesis, a gene functionally and/or structurally equivalent must be present at least in other dipterans. Searching for a counterpart, the 93D locus in Drosophila melanogaster has been long considered as the first candidate. This locus reveals a very unusual gene with properties


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Table 1. Comparison of some characteristics of hsr-ω genes in Drosophila and T-BRs in Chironomus.

rather reminiscent of T-BRs. Functionally conserved in all species of Drosophila investigated so far (Lakhotia and Singh 1982; Ryseck et al 1987), 93D and 93D-like HS-loci have been termed “hsrω” (Bendena et al 1989). While diverging in their base sequence, these genes consist of a 5’ unique region followed by approximately 10kb stretch of tandem repeats of 115–280 bp repeat unit in different species (Garbe et al 1986). ω-n is the full length untranslated transcript, which is constitutively transcribed and heat shock activated, appearingg restricted to the nucleus (Fini et al 1989; Bendena et al 1989, 1991). Most of these characteristics are unique to hsrω genes in Drosophila and differ largely from other well known and evolutionary conserved HS-genes. Surprisingly, most of these features are also shared by T-BRs (table 1). Nevertheless, none of the tested agents, known to cause specific induction of 93D and its homologous loci in Drosophila, have been effective in Chironomus (Barettino et al 1988b; Nath and Lakhotia 1991). There are other properties of hsrω genes that hold them as unusual HS-genes, but they have not been analysed in detail in Chironomus (for review see Lakhotia 1987, 1989). We have recently found another common feature. The HSP83, a member of the 90 kDa HSP family, specifically associates with the hsrω puff in D. melanogaster and D. hydei and to T-BRs in C. thummi following heat shock (Morcillo et al 1993). This greatly reinforces the hypothesis of some functional homology between hsrω and T-BRs. Moreover, this protein also binds preferentially to the heat shock locus I-1C in C. tentans suggesting the existence of T-BR equivalents with a non-telomeric location in Camptochironomus. If this is true, we would be faced with a new gene function to be discerned within the heat shock response, perhaps hidden in other systems where an untranslated gene with divergent sequence cannot be easily identified.

254 7.

G Morcillo and J L Diez T-BRs and telomere organization

The organization of telomeres is highly conserved among eukaryotes. Very short and tandemly repetitive sequences (5–10 bp in length), characterized by strand asymmetry and replicated via telomerase, are found at the termini. Internally, more complex and repetitive sequences have also been localized (Greider 1994; Pardue 1994). In Dipteran species, telomeric short repeats have not been detected. Instead, more complex repetitive telomere-associated DNA (TA), like the subtelomeric DNA in other species, has been found. Different families of TA sequences have been described in Drosophila(Biessmann el al 1993; Levis et al 1993) and Chironomus (Nielsen et al 1990), at or close to the chromosome ends. This may suggest a different mechanism for telomere maintenance than the widely conserved telomerase based mechanism. Different alternatives have been proposed to balance the terminal loss (see Mason and Biessmann 1995). The blocks of repeated sequences obtained directly by microcloning the T-BRIII and implicated in puffing and transcription at this site, can be considered as telomericassociated sequences (TA) and therefore a component of the telomere. Their size and highly repeated organization, the fact of being shared by most telomeres (all except the kinetochoric one), and their terminal location frequently seen as intertelomeric bridges, allow to classify them as TA sequences. Nevertheless, little is known about the arrangement of terminal regions in C. thummi chromosomes to assess the significance or even the precise location of these sequences in the telomere. Interestingly, TA sequences differing in base sequence but similar in size and number have been described in other Chironomid species. A complex 340 bp sequence is tandemly repeated also in 7 out of 8 telomeres in C. tentans and C. pallidivitatus (Cohn and Edström 1991). This repeat has an internally subrepeated and conserved structure. The linker regions, differing even within a species, can be grouped into four different subfamilies differentially distributed between and within telomeres (Cohn and Edström 1992). These sequences are organized in 50 to 200 kb blocks which are highly variable in amount even within stocks (Zhang et al 1994). Constitutive transcription of at least some TA sequences is a common feature in Chironomids, since large RNA molecules (10–15 kb) hybridizing to ΤA repeats have been detected in all species so far studied (Saiga and Edström 1985; Carmona et al 1985). Additionally in C. thummi and C. piger, a class of TA sequences with high homology and some of these sequences being located in specific telomeres, are heat shock transcribed and develop a T-BR structure. As suggested by HSF binding to T-BRs, this inducibility seems to be due to the presence of a HS promoter upstream of the transcribed block of TA repeats. The multiple appearance as well as the variability observed in the number of HS-activated telomeres (see § 3) suggest a more complex regulation of the T-BR formation. Possible explanations could lie in differences in the efficiency of HS-promoters (either by differences in composition or conformation).Or. assuming a single HS-inducible locus, the multiple appearance of T-BRs could be associated to a high incidence of telomere rearrangements probably related to the dynamics of telomere formation and maintenance. The direct participation of TA sequences, a component of telomeres whatever is its function, in T-BRs makes an accurate interpretation of the nature and meaning of these HS-puffs somewhat elusive. The question is whether the thermal inducibility of some TA sequences is actually due to the existence of a HS-gene functionally integrated in the heat shock response or it


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represents the result of an evolutionary event restricted to some Chironomus species that accidentaly rendered heat shock inducibility to some TA sequences. The similarities between T-BRs and hrsω genes support the first view. This could be reinforced by the possible existence of a T-BR-related non telomeric locus in C. tentans (locus I-1C) (Morcillo et al 1993). As a working hypothesis, T-BRs as members of the hsrω family is the most acceptable. However, the present state of knowledge does not allow to completely discard the view of T-BRs as “accidental” members of the HS-gene family. 8.

Conclusion

The formation of T-BRs is a peculiar manifestation of the heal shock response restricted, at present, to two species of the C. thummi group. T-BRs show similarities with the 93D locus in Drosophila which makes their study interesting since they may represent the hsrω counterpart of Chironomus, a dipteran distantly related to Drosophila. The possible existence of hsrω genes in Chironomus raises the possibility of their presence in other organisms as a universal component of the heat shock response. The particular features of hsrω genes would have been difficult to be identified in those organisms lacking polytene chromosomes and, consequently, the phenomenon of puffing as a cytological reference of specific gene activity. Further comparative study of 93D and T-BR may strengthen the similarities between these two genes and could potentially provide some clues to look for hsrω equivalents in other systems. However, the involvement of telomeric components in the T-BR adds some difficulties to the mere conception of T-BRs as a singular localization of a heat shock gene. Further studies on the molecular structure of T-BRs are needed to integrate the available data into a single conceptual framework. Acknowledgements We thank Eduardo Gorab for suggestions and helpful discussions on this work, and to L Μ Botella and J L Martinez for critically reading the manuscript. We also thank A Partearroyo and Ρ Cabrera for technical assistance. Financial support was obtained from DGICYT of Spain (DP94/0024 A). References Ashburner Μ and Bonner J J 1979 The induction of gene activity in Drosophila by heal shock; Cell 17 241–254 Barettino D, Morcillo G and Diez J L 1982 Induction of heat-shock Balbiani rings after RNA synthesis inhibition in polytene chromosomes of Chironomus thummi; Chromosoma 87 507–517 Barettino D, Morcillo G, Diez J L, Carretero Τ and Carmona Μ J 1988a Correlation between the activity of a 5,6-dichloro-l-ßD-ribofuronosylbenzimidazole-insensitive puff and the synthesis of major heat-shock polypeptide, hsp70, in Chironomus thummi; Biochem. Cell Biol. 66 1177–1185 Barettino D, Morcillo G and Diez J L 1988b Induction of the heat-shock response by carbon dioxide in Chironomus thummi; Cell Differ. 23 27–36 Bendena W G, Ayme-Southgate A, Garbe J C and Pardue Μ L 1991 Expression of hear-shock locus hsr-omega in nonstressed cells during development in Drosophila melanogaster; Dev. Biol. 144 65–67 Bendena W G, Garbe J C, Traverse Κ L, Lakhotia S C and Pardue Μ L 1989 Multiple inducers of the Drosophila heat shock locus 93D (hsrω): inducer specific pattern of the three transcripts; J. Cell Biol. 108 2017–2028

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