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ciegli
ska1
1 Departments of Tumor Biology, and
2 Experimental and Clinical Radiobiology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, Poland
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The mouse Hsp70.2 gene and its rat counterpart Hst70 are constitutively expressed specifically in spermatocytes and spermatids (Krawczyk et al. 1988; Zakeri et al. 1988,). The genes encode proteins with 99.7% identity. Also organization of the promoters is very similar (87% identity in the most essential for regulation fragment: up to 380 bp upstream of the translation start codons). HSP70.2 protein expression appears in leptotene and is largely accumulated in pachytene primary spermatocytes (Dix et al. 1997). This is a molecular chaperone that is required for the assembly of a functional Cdc2/cyclin B1 complex in pachytene spermatocytes, which is necessary for completion of meiotic division (Zhu et al. 1997). The protein is also essential for desynapsis of synaptonemal complexes (Dix et al. 1997). In Hsp70.2(-/-) male mice synaptonemal complexes fail to disassemble, and primary spermatocytes arrest in meiosis I and undergo apoptosis, which leads to infertility (Dix et al. 1996). The other testis-specific Hsp70 gene, termed Hsc70t, is activated in round spermatids. However, this gene is less abundant compared to Hsp70.2, and its regulation and functions remain largely elusive (Matsumoto & Fujimoto 1990).
The major regulators of heat shock genes are heat shock transcription factors (HSFs), which in vertebrates comprise a multimember protein family (Pirkkala et al. 2001). Most probably different HSFs have specialized functions in response to distinct stimuli under normal or stress conditions. HSF1 is the primary transcription factor responsible for the transcriptional response to different forms of cellular stress (e.g., heat shock) (Baler et al. 1993). Under non-stress conditions, HSF1 exists in an inactive form as a monomer, while in response to cellular stress HSF1 forms homotrimers. Trimers of HSF1 activate transcription of Hsp genes by binding to cis-acting DNA elements termed heat shock elements (HSEs), which are present in the promoters of heat shock genes (Fernandes et al. 1994). The HSE consensus is composed of several contiguous inverted repeats of the 5-base pair sequence nGAAn (Perisic et al. 1989). Although three repeats are thought to be required for heat regulation in vivo, the binding of HSF to HSEs is cooperative and some deviations from the canonical sequence are functionally tolerated (Santoro et al. 1998). The HSE-like element present in the promoter of Hsp70.2/Hst70 gene consists of two such pentameric repeats. Because HSF1 can recognize variations in the HSE consensus sequence (Kroeger & Morimoto 1994), one cannot exclude potential functional interactions between HSF1 and the HSE-like element in the promoter of the Hsp70.2/Hst70 gene, which we aimed to investigate in this study.
In male gonads HSF1 acts as a cell-survival factor only in somatic and premeiotic germ cells, but not in meiotic and postmeiotic ones (Izu et al. 2004). Primary spermatocytes are the most sensitive germ cells to heat stress (Yin et al. 1997). In the majority of mammals male gonads are located outside the main body cavity so as to provide a lower testicular temperature, which is required for correct spermatogenesis and fertility. Transcription of inducible Hsp70 genes is permanently blocked in spermatocytes and expression of constitutively active HSF1 in such cells leads to caspase-3 dependent apoptosis and male infertility (Nakai et al. 2000; Widlak et al. 2003a; Vydra et al. 2006). Because this apoptotic phenotype is very similar to that exhibited by Hsp70.2(-/-) mice, we investigated here the effects of expression of constitutively active HSF1 on Hsp70.2/Hst70 gene expression in spermatocytes. We found that prior to the onset of HSF1-induced apoptosis in spermatocytes a marked reduction in HSP70.2 protein and mRNA levels occurs. Germ cells in the final stages of apoptosis are essentially deficient in HSP70.2, which leads us to propose a cause-vs.-effect relationship between down-regulation of Hsp70.2/Hst70 and the degeneration of seminiferous epithelium caused by active HSF1.
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Results |
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To directly assess the role of HSF1 in regulating the Hsp70.2/Hst70 gene we have analyzed the levels of Hsp70.2 gene transcripts (by RT-PCR) and HSP70.2 proteins (by Western blot) in testes of either wild-type mice or transgenic mice expressing a constitutively active form of HSF1 (the pHST-HSF1
RD transgene). Because the Hsf1 transgene is under the control of the rat Hst70 promoter (Widlak et al. 2003a; Vydra et al. 2006), both endogenous Hsp70.2 and the active form of HSF1 are expressed simultaneously during postnatal development, starting from prophase of the first meiotic division (leptotene spermatocytes). It has been previously observed that expression of this constitutively active form of HSF1 in spermatocytes of transgenic mice induces massive apoptosis (Nakai et al. 2000; Widlak et al. 2003a; Vydra et al. 2006). Considering that, we have analyzed Hsp70.2 expression in testes of 15-, 18- and 22-day-old animals before the seminiferous epithelium becomes destroyed. We have unexpectedly found that the levels of Hsp70.2 transcripts (Fig. 1A) and HSP70.2 protein (Fig. 1B) were markedly reduced in the presence of co-expressed active HSF1. Densitometry quantification of Western-blots revealed at least 10-fold reduction of HSP70.2 protein level in 18- and 22-day-old HSF1 transgenic animals as compared to control ones. Pachytene spermatocytes and round spermatids, where HSP70.2 protein normally accumulates, are predominant spermatogenic cells in testes of wild-type 18- and 22-day-old mice, respectively. Importantly however, the Hsp70.2 gene was not down-regulated in testes of 15-day-old mice (Fig. 1A) where leptotene/zygotene spermatocytes are the predominant spermatogenic cells (Bellve et al. 1977).
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We have analyzed by immunohistochemistry the localization of HSP70.2 protein in mouse testes during postnatal development and observed that in the presence of constitutively active HSF1, endogenous, HSP70.2 had a different subcellular distribution (Fig. 2A). In seminiferous tubules of 18-day-old wild-type males, HSP70.2 protein was localized mostly in the cytoplasm (up to 95% of HSP70.2–positive cells). In such animals the majority of HSP70.2–positive cells are pachytene spermatocytes. In spermatocytes of HSF1 transgenic mice the HSP70.2 protein was detected in both the cytoplasm and the nucleus (up to 75% of HSP70.2–positive cells). Additionally, HSP70.2–positive cells of 18-day-old transgenic males were markedly smaller, and HSP70.2–negative cells could also be detected centrally within the seminiferous tubules of transgenic males (Fig. 2A). Consequently, the presence of co-expressed active HSF1 induced a subcellular redistribution of HSP70.2 protein and apparently affected the development of spermatocytes.
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HSP70.2 protein is not present in the final stages of apoptosis induced by HSF1
Active HSF1 expressed in spermatocytes induces caspase-dependent apoptosis (Vydra et al. 2006). Here we aimed to determine the temporal and spatial relationships between HSF1-dependent down-regulation of Hsp70.2 and the HSF1-dependent apoptosis in spermatocytes. Seminiferous tubules of adult animals expressing active HSF1 contain a large fraction of spermatocytes undergoing the terminal stages of apoptosis, which is evidenced by the TUNEL assay. The presence of HSP70.2 protein was analyzed in the same specimens. We have found that HSP70.2 essentially was not present in such TUNEL-positive cells (Fig. 3). Importantly, the same observation was made in histological preparations from the testes of animals at different ages (18- to 42-day-old, data not shown). These results may suggest a cause-vs.-effect relationship between down-regulation of Hsp70.2/Hst70 and the degeneration of seminiferous epithelium caused by active HSF1.
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Knowing that expression of constitutively active HSF1 results in down-regulation of the Hsp70.2 gene expression we aimed to identify a putative cis-acting element in the Hsp70.2/Hst70 promoter that might be responsible for interactions with HSF1. The HSE-like element in this gene's promoter was the most obvious candidate. We have previously shown that efficient tissue-specific expression of the CAT reporter gene driven by the rat Hst70 gene promoter requires sequences localized between the T2 transcription start site and box B (see Fig. 4A). A comparison of results obtained with different fragments of the Hst70 gene promoter, either intact or partially replaced with the minimal thymidine kinase (tk) promoter, suggested a functional importance for the HSE-like element localized in the Hst70 promoter upstream of the TATA box (position –181/–172) (Scieglinska et al. 2004). To directly study the role of this HSE-like element in spermatocyte-specific expression of a CAT reporter gene, we have generated new lines of transgenic mice carrying a CAT reporter gene under the control of the Hst70 promoter. The HSE-like element (sequence tTTCcaGAAg) in parental pHST(368/62)-CAT6, which has the highest testis-specific activity (Widlak et al. 1995), was replaced by tGGCcaTGGg, a sequence that could be recognized by BalI or NcoI restriction enzymes (Fig. 4A). We have obtained four lines of such pHST (368/62)HSE-CAT6 transgenic mice that express the transgene. Surprisingly, the CAT activity in extracts from testes of mature pHST(368/62)HSE-CAT6 and pHST(368/62)-CAT6 transgenic mice was similar (Fig. 4B).
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HSE-CAT6 transgenic males with transgenic females expressing constitutively active HSF1 in spermatocytes (both types of transgenes are expressed simultaneously during postnatal development). Double transgenic males 15 and 22 days old were selected for analysis; previous studies have shown that in testes of 15-day-old animals transgenes are expressed but only few "physiological" apoptotic cells are visible, while in testes of 22-day-old animals the expression of HSF1 reaches higher levels and clusters of apoptotic spermatocytes are detected, yet seminiferous epithelium is not completely destroyed (Vydra et al. 2006). Although in testes of double transgenic males the CAT activity was markedly lower than in corresponding CAT single transgenic animals, which was in accordance with the finding that HSF1 down-regulates Hsp70.2/Hst70, there was not a significant difference between these levels in the [pHST(368/62)-CAT6]x[pHST-HSF1RD] and [pHST(368/62)HSE-CAT6]x[pHST-HSF1RD] animals (Fig. 4C). In other words, expression of constitutively active HSF1 prevents the stage-dependent activation of the Hsp70.2/Hst70 gene irrespective of the presence of the HSE-like element. These results demonstrate directly that Hst70 promoter sequences other than the HSE-like segment are those which respond to HSF1-mediated repression. The HSF1 is not associated with the Hsp70.2 gene promoter in vivo
The results obtained with the CAT reporter gene (Fig. 4) show that the HSE-like sequence does not affect Hsp70.2/Hst70 gene expression. However, to determine further whether HSF1 interacts with this element and/or any other sequences within the promoter, we have assayed for HSF1 association in vivo. Testes of 15-day-old males, either wild-type or pHST-HSF1RD transgenic, were selected for chromatin immunoprecipitation experiments (Fig. 5). The promoter of the Hsp105 gene, which has been shown to be up-regulated in the testes of HSF1 transgenic mice (Vydra et al. 2006), was used as a positive control. Sequences specific for the Hsp70.2 gene promoter were not immunoprecipitated with anti-HSF1 antibody (the PCR primers used would amplify sequences from –299 to +34, covering almost the entire promoter tested in transgenic studies). In marked contrast, the fragment of the Hsp105 gene promoter containing a HSE was highly enriched in the HSF1-immunoprecipitated fraction, in either wild-type or transgenic HSF1-expressing animals. These results conclusively demonstrate that HSF1 does not interact with the HSE-like element or any other sequences within the Hst70 promoter.
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We have analyzed global gene expression profiles in testes of 15-day-old males constitutively expressing active HSF1 and identified 713 genes whose expression was repetitively modulated in transgenic animals as compared to non-transgenic controls (Welch t-test non-corrected P < 0.05) (Vydra et al. 2006). Among those the most differentially regulated there were 85 genes known to be involved in different aspects of transcription regulation (61 up-regulated and 24 down-regulated). This included 67 genes encoding known or putative transcription factors; however, none of them contained HSE sequences. Three down-regulated genes (Fig. 6) encoded for transcription factors potentially binding to cis-elements present in the Hsp70.2/Hst70 promoter (Fig. 4A): C/EBP
(Cebpd) that could possibly bind to CAAT-boxes, GATA5 that could possibly bind to a GATA-box and an Oct -2 family member Pou2f2 that could possibly bind to the Oct sequence. Additionally, HSF4 that could hypothetically bind to the HSE-like sequence was also down-regulated in transgenic males; however, this element is not essential for gene regulation in the CAT assay (Fig. 4). Among the up-regulated transcription factors there was the Sp1 family member Copeb (core promoter element binding protein, also termed Klf6 or Zf9) that could possibly bind to several Sp1-binding sites (GC-boxes) present in the Hsp70.2/Hst70 promoter, and p300/CBP-associated factor (Pcaf) (a putative p300-binding sequence is present in the analyzed promoter). However, the functional importance of these factors for the Hsp70.2/Hst70 gene regulation, if any, remains to be verified experimentally.
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A hypothetical HSF1-dependent block of spermatocyte differentiation at early prophase of meiosis I could possibly contribute to down-regulation of HSP70.2 observed in testes of transgenic mice. Assuming this possibility one should expect that other developmental stage-specific genes were down-regulated in testes of HSF1 transgenic animals. To address this question we have analyzed expression patterns of several spermatocyte- and spermatid-specific transcripts in such testes. We have selected genes that become active at the same developmental stages as Hsp70.2 (histone H1t, Drabent et al. 1993; pyruvate dehydrogenase E1 alpha 2, Iannello & Dahl 1992; lactate dehydrogenase C, Thomas et al. 1990), in late pachytene spermatocytes (proacrosin, Kashiwabara et al. 1990; phosphoglycerate kinase 2, Robinson & Simon 1991; cyclin A1, Sweeney et al. 1996; sperm 1 POU-domain transcription factor, Andersen et al. 1993) or in spermatids (Hsc70t, Matsumoto & Fujimoto 1990). These transcripts were analyzed by semiquantitative RT-PCR in testes of 15-, 18-, 22- and 35-day-old mice, either from control or pHST-HSF1RD transgenic (Fig. 7A). Spermatocytes at early prophase of meiosis I (leptotene, zygotene, early pachytene) and mid- to late pachytene spermatocytes were predominant spermatogenic cells in testes of normal 15- and 18-day-old mice, respectively. In testes of 22-day-old mice round spermatids began to accumulate (Bellve et al. 1977). The expression patterns of genes activated in leptotene/zygotene spermatocytes were similar to those of Hsp70.2– in 15-day-old mice comparable amounts of transcripts could be detected in either control or transgenic testes, while in testes of older animals markedly lower amounts of transcripts were detected in transgenic tissues (levels of such transcripts in older transgenic animals barely increased compared to 15-day-old mice). More importantly, transcripts of genes activated in late pachytene spermatocytes or spermatids could not be easily detected in testes of HSF1 transgenic mice at any stages. These data indicate that expression of constitutively active HSF1 results in down-regulation of different spermatocyte- and spermatid-specific genes and/or down-regulation of Hsp70.2 and other testis-specific genes results from a HSF1-dependent blockade of development of spermatogenic cells in testes of transgenic animals.
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Discussion |
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The Hsp70.2/Hst70 gene promoter that drives expression of both endogenous HSP70.2 and transgenic, mutated HSF1 becomes active in leptotene spermatocytes yet Hsp70.2 transcripts and protein accumulate largely in pachytene spermatocytes and later developmental stages (Dix et al. 1997). A putative HSF1-dependent block of spermatocyte development at early prophase of meiosis I could possibly contribute to down-regulation of Hsp70.2 and other testis-specific developmentally regulated genes. Morphological differences visible in seminiferous tubules of 15- and 18-day-old wild-type and HSF1 transgenic mice (Fig. 2A) apparently support this possibility. Alternatively, active HSF1 could misdirect a network of transcription factors required for proper regulation of Hsp70.2 and other spermatocyte- or spermatid-specific genes, which in turn could disturb spermatogenesis. To further elucidate this question the influence of transiently activated HSF1 upon expression of Hsp70.2 gene was analyzed in testes of wild-type mice exposed to the heat shock (Fig. 7B). Obtained data suggest that transcriptional activity of HSF1 but not HSF1-dependent blockade of spermatocyte differentiation could be the major cause for down-regulation of Hsp70.2 gene in testes of HSF1 transgenic mice. Interestingly, in testes of transgenic mice that express constitutively active HSF1 under the control of the actin promoter, whose testicular activity is detected mostly in spermatids but not spermatocytes (Ventela et al. 2000), Nakai and coworkers observed down-regulation of spermatid-specific genes, including Hsc70t, but not those specific for earlier developmental stages, including Hsp70.2 (Nakai et al. 2000). However, hypothetical HSF1-dependent factor(s) involved in negative regulation of Hsp70.2/Hst70 and other testis-specific genes remain to be identified. Apparently, elimination of spermatocytes by apoptosis cannot contribute significantly to down-regulation of HSP70.2 detected in 18-day-old pHST-HSF1RD transgenic males because massive HSF1-dependent apoptosis and degeneration of seminiferous epithelium are observed in older animals (Widlak et al. 2003a; Vydra et al. 2006).
The HSP70.2 protein is functionally important in the nucleus where it takes part in desynapsis of synaptonemal complexes. However, the vast majority of cellular HSP70.2 has a cytoplasmic localization in normal spermatocytes (Dix et al. 1997). Here we observed that in spermatocytes expressing transcriptionally active HSF1, a large fraction of HSP70.2 became relocalized to the nucleus. Other HSP70 proteins are also known to exhibit a nuclear redistribution in heat-shocked cells (Milarski & Morimoto 1989). The HSC70 protein, which has 95% similarity to the HSP70.2 protein, possesses three regions functionally responsible for cytoplasm/nucleus trafficking: a nuclear localization signal involved in the binding of cargo proteins (NLS; residues 246–262) (Lamian et al. 1996), a nuclear localization-related signal (NLRS; residues 473–472) and a nuclear export signal (NES; residues 394–401) (Tsukahara & Maru 2004). Almost identical sequences are present in other HSP70 proteins, including the HSP70.2, that apparently regulate their cytoplasmic or nuclear localization. Interesting future questions are whether the HSP70.2 protein indeed shuttles between the nucleus and the cytoplasm, and whether HSF1-inducible gene products are involved in down-regulating nuclear export of the HSP70.2 protein. Also the importance of nuclear retention for functions of the HSP70.2 protein remains to be established.
Activation of HSF1 in spermatocytes does not activate inducible HSPs but leads instead to a massive caspase-3-dependent apoptosis and degeneration of seminiferous tubules in testes of 3-week-old and older mice (Widlak et al. 2003a; Vydra et al. 2006). Here we demonstrate that HSF1-induced apoptosis is preceded by a marked down-regulation in Hsp70.2 gene expression, and that spermatocytes undergoing the terminal stages of apoptosis (DNA fragmentation) do not contain HSP70.2 protein. Given that this phenotype is very similar to that exhibited by Hsp70.2(-/-) knockout mice (Dix et al. 1996), the induction of apoptosis by HSF1 might be directly linked to the HSF1-mediated down-regulation of the Hsp70.2 gene. The permanent heat shock induced by cryptorchidism results in both degeneration of germ cells and down-regulation of Hsp70.2/Hst70 gene expression. However, it was not clear what phenomenon starts earlier and what the functional relationship is between them (Krawczyk et al. 1987: Guo et al. 2001; Kon & Endoh 2001; Barqawi et al. 2004; Chaki et al. 2005). Our data suggest that HSP70.2/HST70 protein may provide anti-apoptotic functions in spermatocytes and a cause-vs.-effect relationship between down-regulation of Hsp70.2/Hst70 and the degeneration of seminiferous epithelium.
In addition, down-regulation of the Hsp70.2/Hst70 promoter by HSF1 possibly contributed to the lack of Hsp70i-mediated protection against HSF1-induced degeneration of spermatogenic cells observed in transgenic mice expressing the Hsp70i gene under control of this promoter (Vydra et al. 2006).
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Experimental procedures |
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Total RNA was prepared using the guanidine isothiocyanate method (Chomczynski & Sacchi 1987). DNA was removed from RNA samples by digestion with RNase-free DNase I (Scieglinska et al. 1997). After inactivation of the enzyme, control PCR assays were performed to assess for DNA contamination using primers for a glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH; GENBANK accession no. M32599; amplified a 299 bp fragment from position 340 to 639). RT-PCR assays were performed essentially according to Singer-Sam et al. (1990). At the reverse transcription step the reaction mixture (final volume 50 µL) contained RNA (0.5–1 µg), dNTPs (0.2 mM each), primers (0.4 µM each), RNase Inhibitor (40 U, Fermentas), MMLV reverse transcriptase (50 U, Gibco BRL) and Taq polymerase (2 U, Fermentas). The reverse transcription step was performed for 10 min at 50 °C and after inactivation of reverse transcriptase at 94 °C for 4 min, 20–35 cycles of the PCR were performed (94 °C for 30 s; 52 °C or 55 °C for 30 s; 72 °C for 45 s). Products were analyzed on 2% agarose gels stained with ethidium bromide. Primers used in the analysis specifically amplified transcripts of the Hsp70.2 gene (GENBANK accession no. M20567; 343 bp fragment from position 2505 to 2848), histone H1t (GENBANK accession no. X72805; 231 bp fragment from position 2429 to 2659), Pdha2 (GENBANK accession no. NM 008811; 201 bp fragment from position 1555 to 1755), Ldhc (GENBANK accession no. NM 013580; 218 bp fragment from position 1056 to 1273), Pgk2 (GENBANK accession no. NM 031190; 228 bp fragment from position 1256 to 1483), Ccna1 (GENBANK accession no. NM 007628; 242 bp fragment from position 1010 to 1251), Sprm-1 (GENBANK accession no. AK005948; 228 bp fragment from position 759 to 986), Acr (GENBANK accession no. NM 013455; 235 bp fragment from position 1025 to 1259), Hsc70t (GENBANK accession no. D85732; 511 bp fragment from position 38 to 549), Hsp105 (GENBANK accession no. D67016; 326 bp fragment from position 201 to 526) and inducible Hsp70 (Hspa1a—GENBANK accession no. NM 010479; 199 bp fragment from position 2098 to 2296; primers complementary also to Hspa1b—GENBANK accession no. M35021; 208 bp fragment from position 2707 to 2914). Primer's sequences are available upon request.
Protein extraction and Western blotting
Excised tissues were frozen on dry ice and then homogenized in five volumes of buffer containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM dithiothreitol (DTT), 1 mM PMSF, a protease inhibitor mixture (CompleteTM, Roche). After centrifugation at 15 300 g for 10 min at 4 °C, total protein content in supernatants was determined using a Protein Assay Kit (Bio-Rad). Protein species (80 µg) were separated by SDS-PAGE on 8% polyacrylamide gels and blotted to nitrocellulose (BA85 Schleicher and Schuell, Dassel, Germany). The filter was blocked for 60 min with 5% non-fat milk in TTBS (0.25 M Tris–HCl pH 7.5, 0.1% Tween-20, 0.15 M NaCl). Anti-HSP70.2 rabbit polyclonal antibody was generated (by Sigma) using the NH2-SKLYQGGPGGGG SSGGPT peptide corresponding to amino acids 611–628 in the HSP70.2 sequence (Rosario et al. 1992). The primary antibody was detected by horse-radish peroxidase-conjugated secondary antibody (Roche) and visualized by enhanced chemiluminescence (Pierce).
Immunohistochemistry and TUNEL assay
Testes from 15-, 18-, 22-, 35- and 42-day-old wild-type and HSF1 transgenic mice were dissected and fixed overnight in 10% buffered formaldehyde at 4 °C, washed in PBS at 4 °C, dehydrated, paraffin-embedded and sectioned. Tissue sections (6 µm) incubated with anti-HSP70.2 antibody (described above) were stained with an ABC Vectastain kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's guidelines. All steps were performed in a humid chamber at room temperature. DAB was used as a chromogen for visualization of immunohistochemical reactions and hematoxylin was used for counterstaining. For simultaneous analysis of HSP70.2 and DNA breaks on the same specimen, an antigen retrieval step in 0.01 M citrate buffer pH 6.0 was performed before the immunohistochemistry procedure. Immediately after staining for HSP70.2 the specimen sections were processed for TUNEL assays. Prepared sections were incubated with a mixture composed of terminal transferase (TdT) and labeled nucleotides (fluorescein dUTP) at 37 °C for 60 min according to the supplier's protocol (Roche). The samples were washed twice with PBS and incubated with DAKOTM fluorescent medium before analysis (counterstaining by hematoxylin was omitted in this case).
Recombinant plasmids
For the construction of the pHST(1195/62)-EGFP:HST70 transgene, a Bsp1407I site was created by PCR at the 5' end and a NotI site was created at the 3' end of the Hst70 coding sequence. This modified DNA was ligated through the Bsp1407I and NotI sites to the pHST(1195/62)-EGFP plasmid containing a KpnI(-1195)-DraII(-62) fragment of the Hst70 promoter inserted in front of the promoterless EGFP gene cloned in the pEGFP-1 plasmid (GENBANK accession no. U55761; Clontech Laboratories Inc., PaloAlto, CA). A spacer encoding 10 amino acids was introduced between the EGFP and the HST70 genes’ sequences to eliminate possible interference between fused proteins.
The pHST(368/62)-CAT6 plasmid was constructed by inserting a BstXI(-368)-DraII(-62) fragment of the rat Hst70 gene (GENBANK accession no. X15705) in front of the promoterless CAT gene cloned in the plasmid pBLCAT6 (GENBANK accession no. M80484), as described earlier (Widlak et al. 1995). The pHST(368/62)HSE-CAT6 plasmid was obtained from the pHST(368/62)-CAT6 plasmid by PCR-mediated site-directed mutagenesis (Fig. 4A). The primers used to introduce BalI and NcoI restriction sites in place HSE-like sequence (–180, –171) were sense, 5'-TGA GAG TGG CCA TGG GGC AGG-3'; anti-sense, 5'-CCT GCC CCA TGG CCA CTC TCA-3'. Coordinates of the restriction sites refer to the A[
+1] in the ATG codon downstream of the Hst70 gene promoter.
Generation of transgenic mice
Constructs used for microinjection were digested in order to remove vector sequences. To generate transgenic mice appropriate gel-purified restriction fragments were microinjected into the pronuclei of zygotes from FVB/N females by standard procedures. Transgenic founders were screened by PCR using genomic DNA isolated from tail biopsies and primers complementary to the Hst70 promoter and CAT sequences or EGFP and Hst70 coding sequence. PCR products amplified from DNA of pHST(368/62)HSE-CAT6 founders were digested by NcoI or BalI to confirm the existence of the mutated HSE-like sequence. pHST-HSFRD transgenic mice that carry the constitutively active mutant form of human HSF1 under control of the rat Hst70 gene promoter were engineered as described elsewhere (Widlak et al. 2003a). All animal experiments were approved by the Committee of Ethics and Animal Experimentation.
Heat shock of testes and monitoring of EGFP expression
Testes of EGFP-HST70 transgenic males were monitored for EGFP fluorescence under a microscope (ECLIPSE E800 Nicon) without any fixation. For heat shock, whole excised testes were immersed in KSOM medium and exposed to 42 °C for 1 h in a CO2 incubator. Segments of the control or heat shocked seminiferous epithelium were dissected in M2 medium and carefully squashed between a microscope slide and coverslip (Kotaja et al. 2004). Pictures were recorded with an image analyzer equipped with a Hamamatsu Color Chilled 3 CCD camera.
Analysis of CAT activity
Tissue homogenates were prepared according to Pothier et al. (1992). Briefly, excised tissues were frozen in liquid nitrogen and then homogenized in five volumes of a solution consisting of 0.15 M Tris–HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.15 mM spermine, 1 mM DTT and 0.4 mM PMSF. Crude lysates were clarified by centrifugation (15 300 g, 5 min at 4 °C), supernatants were heated at 60 °C for 10 min and centrifuged again (15 300 g, 10 min at 4 °C). To perform CAT assays an aliquot of 100 µL of extract was added to 100 µL of a reaction mixture containing 0.25 M Tris–HCl, pH 7.8, with 1 mM EDTA, 4 mM acetyl-CoA (Sigma) and 6.25 µCi of [14C] chloramphenicol [2.5 mCi/mL, ICN]. CAT assay mixtures contained 10–100 µg of protein and samples were incubated for 10–60 min at 37 °C. The acetylated forms of chloramphenicol were separated by thin-layer chromatography with a chloroform : methanol mixture in proportion 19 : 1. CAT activity was expressed as a percentage of acetylated products formed per 1 h per milligram of extract protein as previously described (Pothier et al. 1992). Total protein content in supernatants after heating of extracts was determined using a Protein Assay Kit (Bio-Rad).
Chromosomal immunoprecipitation (ChIP) assay
ChIP assays were performed using a kit from Upstate Biotechnology Inc. (Lake Placid, NY) according to the supplied protocol. In brief, testes from 15-day-old wild-type and pHST-HSF1RD transgenic mice were chopped into small pieces and fixed with 1% formaldehyde in PBS. After 15 min of incubation, the pelleted samples were disaggregated in PBS using a dounce homogenizer. The cell pellets were collected by centrifugation and then lyzed on ice in SDS-containing buffer. The samples were sonicated on ice in the presence of glass beads (212- to 330-µm diameter) to generate chromatin fragments with an average DNA length of 1000 bp and then cell lysates were clarified by centrifugation. The lysates were diluted and precleared by incubation with salmon sperm DNA saturated protein A-agarose beads, and then incubated with rabbit anti-HSF1 antibody (Stressgen) or mock-treated. Immunoprecipitation was carried out at 4 °C overnight, and immune complexes were collected with salmon sperm DNA saturated protein A-agarose beads. After extensive washing the immunoprecipitated complexes were eluted with 0.1 M NaHCO3 and 1% SDS, and then protein-DNA cross-links were reversed by incubating at 65 °C for 4 h. DNA was purified using proteinase K digestion, phenol : chloroform extraction and ethanol precipitation. PCR was performed (40 cycles: 94 °C for 45 s, 55 °C for 30 s, 72 °C for 30 s) using primers specific for the murine Hsp70.2 sequence between –299 and +34 (where +1 is A in the ATG codon): 5'-AAGCGCCTCACCCAACTA-3' (forward) and 5'-GGTCGATGCCGATAGCCGGG-3' (reverse). As a positive control, immunoprecipitated DNA was also amplified using primers specific for an HSE-containing, 175-nt fragment of the murine Hsp105 promoter, nucleotides 907–1081 according to numbering in GENBANK (accession no. AB005282): 5'-CTGTCACCATGGCAACTCAG-3' (forward) and 5'-CCAATCGCTCAGCCTTATGT-3' (reverse) using the same PCR conditions as for Hsp70.2, except for a 50 °C annealing temperature.
Analysis of expression of transcription factors
A global gene expression profiling was performed on RNA isolated from testes of 15-day-old males, either pHST-HSF1RD transgenic or non-transgenic, using the Affymetrix high-density mouse genome 430 A array as described in details elsewhere (Vydra et al. 2006). Putative transcription factor binding sites within the Hsp70.2/Hst70 gene promoter were predicted thanks to the TESS system available at (<http://www.cbil.upenn.edu>).
Whole body hyperthermia and PCR-based nuclear run-on assay
Adult FVB/N males (10- to 12-week-old, five for each experimental point) were anesthetized with i.p. injection of avertin, and then the lower half of the torso of each animal was submerged in a water bath at 42 °C for 30 min. Testes were collected from control animals and after 2 and 4 h of recovery after the heat shock. Testicular tubules were released by an incision in the tunica albuginea, then individual cells were isolated after mechanical dispersion in ice-cold low salt buffer supplemented with 0.5% Nonidet P-40 by a passage through an 18-gauge needle and nuclei were isolated as described earlier (Scieglinska et al. 2004). Nuclei were isolated from equal amounts of tissues (0.75 g in each case; total RNA was isolated from remaining material); completeness of cell lysis and quality of nuclei preparation were controlled under microscope. For additional standardization total protein contents were determined using a Protein Assay Kit (Bio-Rad). Nuclei were processed for PCR-based run-on assay as described in Rolfe et al. (1997). Briefly, nuclei suspended in 1 mL of the run-on buffer were incubated for 30 min at 30 °C with 0.5 mM each of rNTP. Transcription was terminated, RNA isolated, then subjected to RT-PCR as described above.
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Acknowledgements |
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RD DNA and Dr Michal Jarzab for help with analysis of microarray data. This work was supported by the Polish Ministry of Science, Grants 3P04A03323 and 2P04A04030 (to W.W.). V. Dudaladava was sponsored by the NCI Fellowship, N. Vydra was sponsored by The Foundation for Polish Science (Grant for Young Scientists).
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Footnotes |
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* Correspondence: E-mail: wwidlak{at}io.gliwice.pl
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Received: 1 August 2006
Accepted: 3 January 2007
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