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MOLECULAR IDENTIFICATION OF PpHDAC1, THE FIRST HISTONE DEACETYLASE FROM THE SLIME MOLD PHYSARUM POLYCEPHALUM
Cell Biology International 2002, Vol. 26, No. 9, 783–789 doi:10.1006/cbir.2002.0933, available online at http://www.idealibrary.com on
MOLECULAR IDENTIFICATION OF PpHDAC1, THE FIRST HISTONE DEACETYLASE FROM THE SLIME MOLD PHYSARUM POLYCEPHALUM EVA-MARIA BRANDTNER*, THOMAS LECHNER*, PETER LOIDL† and ALEXANDRA LUSSER Institute of Molecular Biology, University of Innsbruck, Fritz-Pregl-Str. 3, 6020 Innsbruck, Austria Received 13 February 2002; accepted 4 June 2002
The dynamic state of post-translational acetylation of eukaryotic histones is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs have been shown to be components of various regulatory protein complexes in the cell. Their enzymatic activities, intracellular localization and substrate specificities are regulated in a complex, cell cycle related manner. In the myxomycete Physarum polycephalum multiple HATs and HDACs can be distinguished in biochemical terms and they exhibit dynamic activity patterns depending on the cell cycle stage. Here we report on the cloning of the first P. polycephalum HDAC (PpHDAC1) related to the S. cerevisiae Rpd3 protein. The expression pattern of PpHDAC1 mRNA was analysed at different time points of the cell cycle and found to be largely constant. Treatment of macroplasmodia with the HDAC inhibitor trichostatin A at several cell cycle stages resulted in a significant delay in entry into mitosis of treated versus untreated plasmodia. No effect of TSA treatment could be observed on PpHDAC1 expression 2002 Elsevier Science Ltd. All rights reserved. itself. K: chromatin modification; gene regulation; cell cycle; histone acetylation.
INTRODUCTION The basic structural unit of eukaryotic chromatin—the nucleosome—consists of approximately 200 bp of DNA wrapped around an octamer of core histone proteins (H2A, H2B, H3, H4). Further compaction is achieved by association of histone H1 with linker DNA leading to a helical superstructure termed the ‘30 nm fiber’. The 30 nm fiber is supposed to be attached by distinct regions within the DNA to proteins of an underlying nuclear matrix, finally resulting in the highly ordered structure of an interphase chromosome. Recent studies have revealed that the modification of chromatin structure is an important mechanism for the regulation of gene transcription (Struhl, 1998) but also for other processes, such as DNA replication, recombination or repair (e.g. Chen et al., 2001). Multisubunit complexes have been identified that mediate the covalent and *Both authors have contributed equally to this work. †To whom correspondence should be addressed: Tel.: +43-512/ 5073600; Fax: +43-512/5072866; E-mail: [email protected] 1065–6995/02/$-see front matter
noncovalent alteration of chromatin. Covalent modifications of chromatin components include histone acetylation and deacetylation, which is carried out by HATs and HDACs, respectively. Increased levels of histone acetylation are often associated with enhanced transcriptional activity (Hebbes et al., 1988), whereas hypoacetylation of histones correlates with transcriptional silencing (Pazin & Kadonaga, 1997). Numerous HATs have been identified that share conserved motifs for acetyl CoA binding and can be grouped into several superfamilies (GNAT, MYST, p300/CBP, nuclear receptor coactivators and components of the basal transcription complex; for review e.g. Sterner & Berger, 2000). All HDACs identified so far belong to either one of four different classes (Gray & Ekstro¨m, 2001; Lusser et al., 2001; Graessle et al., 2001). Class 1 consists of HDACs with similarity to the yeast Rpd3 protein. Class 2 comprises HDACs homologous to yeast Hda1, and the third class contains proteins that are similar to the nucleolar histone deacetylase HD2 from Zea mays (Lusser et al., 1997b). The yeast silencing 2002 Elsevier Science Ltd. All rights reserved.
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protein Sir2 and its homologs in various organisms define a fourth family of HDACs (Imai et al., 2000). Whereas class 1 and 2 are structurally related protein families, class 3 and class 4 HDACs do not share significant sequence homology with either each other or the class 1 and 2 enzymes. Multiple HDACs belonging to class 1 or class 2 have been characterized in animals and were found to interact with a variety of transcriptional regulators (e.g. Mad, NCoR, hunchback, YY1, Rb, etc.; for review see Cress & Seto, 2000), in most cases leading to repression of gene activity. The true slime mold Physarum polycephalum is capable of growing as a uninuclear amoeba as well as a multinuclear plasmodium. The latter is a syncytium and can adopt a size of up to 10 cm in diameter (macroplasmodium) under laboratory conditions, containing roughly 109 nuclei. Nuclear divisions as well as other cell cycle regulated processes within a single macroplasmodium display perfect synchrony. Thus Physarum has been used as a model organism for studies of cell cycle related problems for several decades. A significant body of data on the cell cycle dependence of histone acetylation (Loidl et al., 1983; Loidl & Gro¨bner, 1987a,b; Lang et al., 1993) as well as of HATs and HDACs of Physarum has accumulated in earlier studies (e.g. Loidl, 1988; Lopez-Rodas et al., 1992). It has been previously shown by our laboratory that five distinct HAT activities (Lopez-Rodas et al., 1992; Lusser et al., 1997a) and two HDACs (Brosch et al., 1992) can be distinguished in Physarum macroplasmodia by chromatographic separation and substrate specificity. The activity patterns of both HATs and HDACs revealed to be regulated during the cell cycle. The isolation and molecular characterization of PpHDAC1 shown here to our knowledge represents the first report on the molecular nature of a histone deacetylase in myxomycetes.
METHODS Organism and culture conditions Physarum polycephalum (strain M3bFII) microplasmodia were cultivated as described (Lusser et al., 1997a). For analysis of cell cycle dependent gene expression giant macroplasmodia were prepared by coalescence of 2.5 ml of microplasmodial sediment on filter paper supported by glass beads. Microplasmodia were allowed to fuse for 1 h before adding semi-defined nutrient medium (Daniel & Baldwin, 1964). Mitosis (telophase) was
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determined by phase contrast microscopy in small smear samples taken from the macroplasmodium. Telophase occurred within 3 min in all nuclei over the whole plasmodium. The second post-fusion mitosis (MII) took place 14 to 15 h after inoculation. Cloning of PpHDAC1 cDNA PpHDAC1 encoding cDNA was cloned by a PCR approach. Template cDNA was synthesized from total RNA isolated from exponentially growing microplasmodia using Superscript Reverse Transcriptase (Life Technology) according to the manufacturer’s description. Degenerate oligonucleotide primers were designed from highly conserved parts of human HDAC1 sequence (primer PP1 5 -TTY TGYTAYGTNAAYGA corresponding to aa 147– 152, and primer PP1reverse 5 -GTRTTYTGRTTN GTCATRTT corresponding to aa 346–351 of human HDAC1, respectively, Fig. 1). PCR amplification was carried out under conditions of moderate annealing stringency. The resulting product of 616 bp was cloned into pGEM-T vector (Promega) and sequenced. 3 -RACE (Frohman, 1993) was performed to amplify the 3 -end of the cDNA. For cloning of the 5 -end the GeneRacer kit (Invitrogen) was used by following the manufacturer’s instructions. Northern analysis At several stages of the cell cycle (indicated in Fig. 2) between post-fusion mitoses II (M2) and III (M3), pieces (200 mg wet weight) of a giant macroplasmodium were cut off and harvested into liquid nitrogen for subsequent RNA isolation. Care was taken to leave out the slimy middle part of the plasmodium. Total RNA was prepared using the RNeasy plant Mini Kit (Qiagen), fractionated on 1.2% agarose/2.2 M formaldehyde gels and blotted onto nylon membranes. For hybridization under high stringency conditions DIG-labeled DNA probes were prepared by PCR labeling of PpHDAC1 and histone H4. For PpHDAC1 forward primer 5 -CATCAAACTCGCACAGGG and antisense primer 5 -TTGTTACTATTATTGT CGGC produced a PCR product of 412 bp. The entire coding sequence of histone H4 (257 bp) was amplified with primers 5 -TGGCAAGGGAGGC AAAG (sense) and 5 -CATAGACGACATCCA TTG (antisense), respectively. Trichostatin A (TSA) treatment of P. polycephalum macroplasmodia For TSA treatment slices of a macroplasmodium were transferred onto medium containing 0.6 M
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Fig. 1. Sequence alignment of Physarum polycephalum HDAC1 with Rpd3-type HDACs of human and Aspergillus nidulans. Identical amino acids are boxed and shaded in yellow; gaps are indicated by dashes introduced for alignment purpose. Numbers indicate the amino acid position of each protein. Asterisks mark 21 amino acid residues that are important in known HDACs; circled asterisks indicate amino acids that play an essential role in the catalytic activity.
TSA (Wako Pure Fine Chemicals, Ltd., Osaka, Japan) at the indicated timepoints (Fig. 3) and incubated for one hour. The remaining part of the plasmodium served as a control and was harvested simultaneously with the TSA treated samples for subsequent RNA isolation and Northern analysis.
RESULTS AND DISCUSSION Physarum PpHDAC1 is a putative member of the Rpd3-family of HDACs By a PCR based approach using degenerate oligonucleotide primers corresponding to highly conserved regions of human HDAC1 we isolated a Physarum polycephalum cDNA of 1869 bp in length. Sequence analysis revealed a single open reading frame of 1749 bp encoding a protein of 579 aa with a calculated molecular mass of 64.9 kDa and a pI of 5.11. Alignment of the Physarum sequence with other HDACs in the databases revealed marked identity to class I HDACs from various organisms. Therefore, it was
termed PpHDAC1. From all known sequences in the databases, chicken and human HDAC1 were found to be the most closely related proteins with 57% overal sequence identity (Fig. 1). Similarity is even more pronounced when comparing only the putative catalytic domains (79%) situated in the N-terminal part of the proteins. This suggests that PpHDAC1 indeed functions as an HDAC in vivo. PpHDAC1 is less related to various fungal enzymes (e.g. 44% overall identity to Aspergillus nidulans RpdA; Fig. 1), thus reflecting the intermediate taxonomical position of slime molds between the animal and the fungal kingdom. A unique feature is the presence of multiple consecutive asparagine residues interspersed by serine and threonine residues in the C-terminal domain of the protein (Fig. 1). The significance of this domain is unclear. A recent analysis of sequences in databases for the prevalence of asparagine repeats revealed that very long asparagine repeats (>41) only occurred in Dictyostelium sequences. Shorter repeats appeared more frequently also in other lower eukaryotes and invertebrates but turned out to be significantly underrepresented in mammalian sequences (Kreil
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Fig. 2. Expression of PpHDAC1 during the cell cycle. Total macroplamsodial RNA of Physarum polycephalum was isolated at the indicated time points of the cell cycle between mitosis 2 and 3 (after inoculation of macroplasmodia). RNA was subjected to electrophoresis in 1.2% agarose/2.2 M formaldehyde gels, blotted onto nylon membranes and hybridized with DIG-labeled DNA fragments of PpHDAC1 and Physarum histone H4. Ethidium-bromide stained RNA gels served as an input loading control, showing the amounts of 18S and 26S rRNA. Arrows indicate the time points of harvest of macroplasmodia; approximate duration of cell cycle phases are depicted as colored boxes.
& Kreil, 2000). Since Dictyostelium discoideum also belongs to the myxomycetes, one might speculate that poly-asparagine stretches fulfil certain specialized functions in slime molds. PpHDAC1 expression does not change during the cell cycle as does HDAC activity In earlier studies (Brosch et al., 1992) it was demonstrated, that HDAC activity is fluctuating during the cell cycle of Physarum polycephalum. To test whether this periodic change in enzymatic activity is due to a cell cycle dependent regulation of PpHDAC1 transcription, total RNA of a P. polycephalum macroplasmodium was isolated at different stages of the cell cycle (Fig. 2, upper panel). Hybridization of Northern blots revealed that PpHDAC1 expression did not fluctuate significantly during the cell cycle (Fig. 2). In contrast, the expression of the histone H4 gene, which was used as a control for a cell cycle dependent transcript (Z), was induced in late G2 phase, reached a maximum during mitosis, decreased in S phase and was almost undetectable during late S and early G2 period (Fig. 2, lower panel). Findings on H4
mRNA are in line with earlier obeservations of total histone protein content (Loidl & Gro¨bner, 1987a,b) and H4 mRNA levels (Wilhelm et al., 1988) during the Physarum cell cycle. These results are in line with findings for human and mouse class 1 HDACs (Bartl et al., 1997), but contrast earlier observations that indicated a clear change of enzyme activity profiles in the course of the Physarum cell cycle (Brosch et al., 1992; Lopez-Rodas et al., 1992). This apparent discrepancy can be due to several reasons: (1) as in other organisms there is probably more than one HDAC (Gray & Ekstro¨m, 2001) and one or several of these might be responsible for the cell cycle dependent HDAC activity pattern. (2) PpHDAC1 activity might be regulated on a posttranscriptional and/or posttranslational level by phosphorylation, acetylation etc. in response to cell cycle signals. (3) Mammalian but also yeast class 1 HDACs have been shown to be active mainly in the context of large protein complexes. This might also be the case for Physarum PpHDAC1. Cell cycle dependent changes in HDAC activities might therefore be due to varying complex composition and/or posttranslational modifications of complex components.
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Fig. 3. Effect of TSA on the expression of PpHDAC1 at different phases of the cell cycle. Pieces of Physarum macroplasmodia at different time points of the cell cycle between mitosis 2 and 3 were transfered onto nutrient medium containing 0.6 mM TSA. Plasmodial pieces were incubated for 1 h and harvested simultaneously with untreated control plasmodia at the indicated time points (arrows). Total RNA was isolated, subjected to electrophoresis in 1.2% agarose/2.2 M formaldehyde gels, blotted onto nylon membranes and hybridized with DIG-labeled DNA fragments of PpHDAC1 and Physarum histone H4. Ethidiumbromide stained RNA gels served as an input loading control, showing the amounts of 18S and 26S rRNA.
Analysis of HDAC-dependency of the regulation of PpHDAC1 and histone H4 gene activity In numerous reports HDACs have been implicated in the negative regulation of the transcription of many genes (e.g. Steinbac et al., 2000; Zhang and Jones, 2001). To investigate if the expression of the PpHDAC1 gene itself is a target of HDACmediated gene regulation, Physarum macroplasmodia were treated with the specific HDAC inhibitor Trichostatin A (TSA) at different stages of the cell cycle followed by Northern analysis of PpHDAC1 expression. No effect of TSA treatment on the transcription of the PpHDAC1 gene was observed at any time point tested (Fig. 3). This result strongly suggests that the activity of the PpHDAC1 gene is not a subject to regulation by TSA sensitive HDACs. This finding is consistent with results obtained for maize class 1 HDACs ZmRpd3 and HD1BII (Lechner et al., 2000) but does not correspond with observations from the filamentous fungus Aspergillus nidulans, where a slight induction of the expression of the PpHDAC1 homolog RpdA
was observed after TSA treatment of mycelia (Graessle et al., 2000). Similarly, also the histone H4 genes do not appear to require HDACs for transcriptional regulation at any stage of the cell cycle. However, we cannot exclude that TSAinsensitive HDACs, like Sir2-related proteins (Imai et al., 2000) contribute to the transcriptional control of the PpHDAC1 and H4 genes, respectively, in Physarum polycephalum. Alternatively, autoregulation of the PpHDAC1 gene might not occur within the normal cell cycle of the plasmodial stage but, nevertheless, might be required for differentiation processes (such as sclerotization) or switch to the amoebal stage. Effect of HDAC inhibition on cell cycle progression To investigate whether the inhibition of HDACs affects normal cycling of Physarum macroplasmodia, plasmodial parts were treated with TSA at different time points during the cell cycle. When
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the checkpoint is not yet clear, it has been suggested to involve mechanisms capable of sensing the acetylation state of the chromatin and thus preventing cells from entering mitosis with hyperacetylated chromatin which in turn would interfere with centromere and kinetochore function.
ACKNOWLEDGEMENTS We gratefully acknowledge valuable discussions with Gerald Brosch and Adele Loidl. This work was in part supported by grants from the Dr-Legerlotz-Foundation, the Austrian National Bank (P-7415) and the Austrian Science Foundation (FWF-P-13620) to P.L. REFERENCES Fig. 4. Mitotic delay induced by TSA treatment. A Physarum macroplasmodium was grown until late G2-period (45 min prior to mitosis 2) and divided in 2 parts. One half was transfered onto nutrient medium containing 0.6 mM TSA, the second half served as a control in the absence of TSA. The control plasmodium went through mitosis 45 min after medium change.
TSA was added to macroplasmodia at 45 min before telophase of mitosis II (M2 in the untreated control), macroplasmodia entered mitosis with a pronounced delay of 30–40 min compared to untreated plasmodia (Fig. 4). This is remarkable because the process of mitotic division itself last for approx. 30 min; this means that TSA is able to induce mitotic delay even when it is added 15 min prior to the onset of early prophase. The delay is indicative of the involvement of HDACs in the regulation of the G2/M transition of the cell cycle. HDAC inhibitors have been shown to induce G1 phase arrest and differentiation by upregulating p21CIP/Waf1 in a range of tumor cell types (e.g. Sowa et al., 1997; Archer et al., 1998). Beside this role for G1/S transition several reports show the implication of histone deacetylation also for the progression of the cell through mitosis. In yeast, TSA treatment has been demonstrated to cause missegregation of chromosomes during mitosis (Ekwall et al., 1997). Moreover, mutation of the amino-terminal lysine residues normally acetylated in histone H4 resulted in a G2/M arrest (Megee et al., 1990). In a recent study with different tumor cell lines, the identification of a novel G2 checkpoint sensitive to HDAC inhibitor treatment was described (Qiu et al., 2000). Although the nature of
A SY, M S, S A, H RA, 1998. p21(WAF1) is required for butyrate-mediated growth inhibition of human colon cancer cells. Proc Natl Acad Sci U S A 95: 6791–6796. B S, T J, L G, K H, K K, S C, 1997. Identification of mouse histone deacetylase 1 as a growth factor-inducible gene. Mol Cell Biol 17: 5033–5043. B G, L-R G, G G, L H, G¨ P, L P, 1992. Histone acetyltransferases and histone deacetylases of Physarum polycephalum. Cell Biol. Int. Rep 16: 1103–1109. C H, T M, E RM, 2001. HATs on and beyond chromatin. Curr Opin Cell Biol 13: 218–224. C WD, S E, 2000. Histone deacetylases, transcriptional control, and cancer. J Cell Physiol 184: 1–16. D JW, B HH, 1964. In: Prescott DM, ed. Methods in Cell Physiology, vol. 1. New York, Academic Press. 9–41. E K, O T, T BM, C G, A RC, 1997. Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres. Cell 91: 1021–1032. F MA, 1993. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. Methods Enzymol 218: 340–356. G S, D M, H H, M K, T P, B E-M, W JD, L P, B G, 2000. Characterization of two putative histone deacetylase genes from Aspergillus nidulans. Biochim Biophys Acta 1492: 120–126. G S, L P, B G, 2001. Histone acetylation: plants and fungi as model systems for the investigation of histone deacetylases. Cell Mol Life Sci 58: 704–720. G SG, E¨ TJ, 2001. The human histone deacetylase family. Exp Cell Res 262: 75–83. H TR, T AW, C-R C, 1988. A direct link between core histone acetylation and transcriptionally active chromatin. EMBO J 7: 1395–1402. I S, A CM, K M, G L, 2000. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 17: 795–800.
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K DP, K G, 2000. Asparagine repeats are rare in mammalian proteins. Trends Biochem Sci 25: 270–271. L S, D T, W W, L P, 1993. Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum. J Cell Sci 105: 1121–1130. L T, L A, P A, B G, L A, G-S M, S R, W S, W JD, L P, 2000. RPD3-type histone deacetylases in maize embryos. Biochemistry 39: 1683–1692. L P, 1988. Towards an understanding of the biological function of histone acetylation. FEBS Lett 227: 91–95. L P, G¨ P, 1987a. Postsynthetic acetylation of histones during the cell cycle: a general function for the displacement of histones during chromatin rearrangements. Nucleic Acids Res 15: 8351–8366. L P, G¨ P, 1987b. Histone synthesis during the cell cycle of Physarum polycephalum. Synthesis of different histone species is not under a common regulatory control. J Biol Chem 262: 10195–10199. L-R G, B G, G G, L H, G¨ P, L P, 1992. Enzymes involved in the dynamic equilibrium of core histone acetylation of Physarum polycephalum. FEBS Lett 296: 82–86. L A, B G, L-R G, L P, 1997a. Histone acetyltransferases during the cell cycle and differentiation of Physarum polycephalum. Eur J Cell Biol 74: 102–110. L A, B G, L A, H H, L P, 1997b. Identification of maize histone deacetylase HD2 as an acidic nucleolar phosphoprotein. Science 277: 88–91.
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L A, K¨ D, L P, 2001. Histone acetylation: lessons from the plant kingdom. Trends Plant Sci 6: 59–65. M PC, M BA, M BA, S MM, 1990. Genetic analysis of histone H4: Essential role of lysines subject to reversible acetylation. Science 247: 841–845. P MJ, K JT, 1997. SWI2/SNF2 and related proteins: ATP-driven motors that disrupt protein-DNA interactions? Cell 88: 737–740. Q L, B A, F DP, L H, P PG, G BG, 2000. Histone deacetylase inhibitors trigger a G2 checkpoint in normal cells that is defective in tumor cells. Mol Biol Cell 11: 2069–2083. S Y, O T, M S, N K, M T, N H, S T, 1997. Histone deacetylase inhibitor activates the WAF1/Cip1 gene promoter through the Sp1 sites. Biochem Biophys Res Commun 241: 142–150. S OC, W AP, R RA, 2000. Histone deacetylase activity is required for the induction of the MyoD muscle cell lineage in Xenopus. Biol Chem 381: 1013–1016. S DE, B SL, 2000. Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 64: 435–459. S K, 1998. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev 12: 599–606. W ML, T B, F RA, W FX, 1988. Histone H4 mRNA is stored as a small cytoplasmic RNP during the G2 phase in Physarum polycephalum. Biochem Biophys Res Commun 153: 162–171. Z Y, J C, 2001. The bovine herpesvirus 1 immediateearly protein (bicp0) associates with histone deacetylase 1 to activate transcription. J Virol 75: 9571–9578.
MOLECULAR IDENTIFICATION OF PpHDAC1, THE FIRST HISTONE DEACETYLASE FROM THE SLIME MOLD PHYSARUM POLYCEPHALUM EVA-MARIA BRANDTNER*, THOMAS LECHNER*, PETER LOIDL† and ALEXANDRA LUSSER Institute of Molecular Biology, University of Innsbruck, Fritz-Pregl-Str. 3, 6020 Innsbruck, Austria Received 13 February 2002; accepted 4 June 2002
The dynamic state of post-translational acetylation of eukaryotic histones is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs have been shown to be components of various regulatory protein complexes in the cell. Their enzymatic activities, intracellular localization and substrate specificities are regulated in a complex, cell cycle related manner. In the myxomycete Physarum polycephalum multiple HATs and HDACs can be distinguished in biochemical terms and they exhibit dynamic activity patterns depending on the cell cycle stage. Here we report on the cloning of the first P. polycephalum HDAC (PpHDAC1) related to the S. cerevisiae Rpd3 protein. The expression pattern of PpHDAC1 mRNA was analysed at different time points of the cell cycle and found to be largely constant. Treatment of macroplasmodia with the HDAC inhibitor trichostatin A at several cell cycle stages resulted in a significant delay in entry into mitosis of treated versus untreated plasmodia. No effect of TSA treatment could be observed on PpHDAC1 expression 2002 Elsevier Science Ltd. All rights reserved. itself. K: chromatin modification; gene regulation; cell cycle; histone acetylation.
INTRODUCTION The basic structural unit of eukaryotic chromatin—the nucleosome—consists of approximately 200 bp of DNA wrapped around an octamer of core histone proteins (H2A, H2B, H3, H4). Further compaction is achieved by association of histone H1 with linker DNA leading to a helical superstructure termed the ‘30 nm fiber’. The 30 nm fiber is supposed to be attached by distinct regions within the DNA to proteins of an underlying nuclear matrix, finally resulting in the highly ordered structure of an interphase chromosome. Recent studies have revealed that the modification of chromatin structure is an important mechanism for the regulation of gene transcription (Struhl, 1998) but also for other processes, such as DNA replication, recombination or repair (e.g. Chen et al., 2001). Multisubunit complexes have been identified that mediate the covalent and *Both authors have contributed equally to this work. †To whom correspondence should be addressed: Tel.: +43-512/ 5073600; Fax: +43-512/5072866; E-mail: [email protected] 1065–6995/02/$-see front matter
noncovalent alteration of chromatin. Covalent modifications of chromatin components include histone acetylation and deacetylation, which is carried out by HATs and HDACs, respectively. Increased levels of histone acetylation are often associated with enhanced transcriptional activity (Hebbes et al., 1988), whereas hypoacetylation of histones correlates with transcriptional silencing (Pazin & Kadonaga, 1997). Numerous HATs have been identified that share conserved motifs for acetyl CoA binding and can be grouped into several superfamilies (GNAT, MYST, p300/CBP, nuclear receptor coactivators and components of the basal transcription complex; for review e.g. Sterner & Berger, 2000). All HDACs identified so far belong to either one of four different classes (Gray & Ekstro¨m, 2001; Lusser et al., 2001; Graessle et al., 2001). Class 1 consists of HDACs with similarity to the yeast Rpd3 protein. Class 2 comprises HDACs homologous to yeast Hda1, and the third class contains proteins that are similar to the nucleolar histone deacetylase HD2 from Zea mays (Lusser et al., 1997b). The yeast silencing 2002 Elsevier Science Ltd. All rights reserved.
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protein Sir2 and its homologs in various organisms define a fourth family of HDACs (Imai et al., 2000). Whereas class 1 and 2 are structurally related protein families, class 3 and class 4 HDACs do not share significant sequence homology with either each other or the class 1 and 2 enzymes. Multiple HDACs belonging to class 1 or class 2 have been characterized in animals and were found to interact with a variety of transcriptional regulators (e.g. Mad, NCoR, hunchback, YY1, Rb, etc.; for review see Cress & Seto, 2000), in most cases leading to repression of gene activity. The true slime mold Physarum polycephalum is capable of growing as a uninuclear amoeba as well as a multinuclear plasmodium. The latter is a syncytium and can adopt a size of up to 10 cm in diameter (macroplasmodium) under laboratory conditions, containing roughly 109 nuclei. Nuclear divisions as well as other cell cycle regulated processes within a single macroplasmodium display perfect synchrony. Thus Physarum has been used as a model organism for studies of cell cycle related problems for several decades. A significant body of data on the cell cycle dependence of histone acetylation (Loidl et al., 1983; Loidl & Gro¨bner, 1987a,b; Lang et al., 1993) as well as of HATs and HDACs of Physarum has accumulated in earlier studies (e.g. Loidl, 1988; Lopez-Rodas et al., 1992). It has been previously shown by our laboratory that five distinct HAT activities (Lopez-Rodas et al., 1992; Lusser et al., 1997a) and two HDACs (Brosch et al., 1992) can be distinguished in Physarum macroplasmodia by chromatographic separation and substrate specificity. The activity patterns of both HATs and HDACs revealed to be regulated during the cell cycle. The isolation and molecular characterization of PpHDAC1 shown here to our knowledge represents the first report on the molecular nature of a histone deacetylase in myxomycetes.
METHODS Organism and culture conditions Physarum polycephalum (strain M3bFII) microplasmodia were cultivated as described (Lusser et al., 1997a). For analysis of cell cycle dependent gene expression giant macroplasmodia were prepared by coalescence of 2.5 ml of microplasmodial sediment on filter paper supported by glass beads. Microplasmodia were allowed to fuse for 1 h before adding semi-defined nutrient medium (Daniel & Baldwin, 1964). Mitosis (telophase) was
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determined by phase contrast microscopy in small smear samples taken from the macroplasmodium. Telophase occurred within 3 min in all nuclei over the whole plasmodium. The second post-fusion mitosis (MII) took place 14 to 15 h after inoculation. Cloning of PpHDAC1 cDNA PpHDAC1 encoding cDNA was cloned by a PCR approach. Template cDNA was synthesized from total RNA isolated from exponentially growing microplasmodia using Superscript Reverse Transcriptase (Life Technology) according to the manufacturer’s description. Degenerate oligonucleotide primers were designed from highly conserved parts of human HDAC1 sequence (primer PP1 5 -TTY TGYTAYGTNAAYGA corresponding to aa 147– 152, and primer PP1reverse 5 -GTRTTYTGRTTN GTCATRTT corresponding to aa 346–351 of human HDAC1, respectively, Fig. 1). PCR amplification was carried out under conditions of moderate annealing stringency. The resulting product of 616 bp was cloned into pGEM-T vector (Promega) and sequenced. 3 -RACE (Frohman, 1993) was performed to amplify the 3 -end of the cDNA. For cloning of the 5 -end the GeneRacer kit (Invitrogen) was used by following the manufacturer’s instructions. Northern analysis At several stages of the cell cycle (indicated in Fig. 2) between post-fusion mitoses II (M2) and III (M3), pieces (200 mg wet weight) of a giant macroplasmodium were cut off and harvested into liquid nitrogen for subsequent RNA isolation. Care was taken to leave out the slimy middle part of the plasmodium. Total RNA was prepared using the RNeasy plant Mini Kit (Qiagen), fractionated on 1.2% agarose/2.2 M formaldehyde gels and blotted onto nylon membranes. For hybridization under high stringency conditions DIG-labeled DNA probes were prepared by PCR labeling of PpHDAC1 and histone H4. For PpHDAC1 forward primer 5 -CATCAAACTCGCACAGGG and antisense primer 5 -TTGTTACTATTATTGT CGGC produced a PCR product of 412 bp. The entire coding sequence of histone H4 (257 bp) was amplified with primers 5 -TGGCAAGGGAGGC AAAG (sense) and 5 -CATAGACGACATCCA TTG (antisense), respectively. Trichostatin A (TSA) treatment of P. polycephalum macroplasmodia For TSA treatment slices of a macroplasmodium were transferred onto medium containing 0.6 M
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Fig. 1. Sequence alignment of Physarum polycephalum HDAC1 with Rpd3-type HDACs of human and Aspergillus nidulans. Identical amino acids are boxed and shaded in yellow; gaps are indicated by dashes introduced for alignment purpose. Numbers indicate the amino acid position of each protein. Asterisks mark 21 amino acid residues that are important in known HDACs; circled asterisks indicate amino acids that play an essential role in the catalytic activity.
TSA (Wako Pure Fine Chemicals, Ltd., Osaka, Japan) at the indicated timepoints (Fig. 3) and incubated for one hour. The remaining part of the plasmodium served as a control and was harvested simultaneously with the TSA treated samples for subsequent RNA isolation and Northern analysis.
RESULTS AND DISCUSSION Physarum PpHDAC1 is a putative member of the Rpd3-family of HDACs By a PCR based approach using degenerate oligonucleotide primers corresponding to highly conserved regions of human HDAC1 we isolated a Physarum polycephalum cDNA of 1869 bp in length. Sequence analysis revealed a single open reading frame of 1749 bp encoding a protein of 579 aa with a calculated molecular mass of 64.9 kDa and a pI of 5.11. Alignment of the Physarum sequence with other HDACs in the databases revealed marked identity to class I HDACs from various organisms. Therefore, it was
termed PpHDAC1. From all known sequences in the databases, chicken and human HDAC1 were found to be the most closely related proteins with 57% overal sequence identity (Fig. 1). Similarity is even more pronounced when comparing only the putative catalytic domains (79%) situated in the N-terminal part of the proteins. This suggests that PpHDAC1 indeed functions as an HDAC in vivo. PpHDAC1 is less related to various fungal enzymes (e.g. 44% overall identity to Aspergillus nidulans RpdA; Fig. 1), thus reflecting the intermediate taxonomical position of slime molds between the animal and the fungal kingdom. A unique feature is the presence of multiple consecutive asparagine residues interspersed by serine and threonine residues in the C-terminal domain of the protein (Fig. 1). The significance of this domain is unclear. A recent analysis of sequences in databases for the prevalence of asparagine repeats revealed that very long asparagine repeats (>41) only occurred in Dictyostelium sequences. Shorter repeats appeared more frequently also in other lower eukaryotes and invertebrates but turned out to be significantly underrepresented in mammalian sequences (Kreil
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Fig. 2. Expression of PpHDAC1 during the cell cycle. Total macroplamsodial RNA of Physarum polycephalum was isolated at the indicated time points of the cell cycle between mitosis 2 and 3 (after inoculation of macroplasmodia). RNA was subjected to electrophoresis in 1.2% agarose/2.2 M formaldehyde gels, blotted onto nylon membranes and hybridized with DIG-labeled DNA fragments of PpHDAC1 and Physarum histone H4. Ethidium-bromide stained RNA gels served as an input loading control, showing the amounts of 18S and 26S rRNA. Arrows indicate the time points of harvest of macroplasmodia; approximate duration of cell cycle phases are depicted as colored boxes.
& Kreil, 2000). Since Dictyostelium discoideum also belongs to the myxomycetes, one might speculate that poly-asparagine stretches fulfil certain specialized functions in slime molds. PpHDAC1 expression does not change during the cell cycle as does HDAC activity In earlier studies (Brosch et al., 1992) it was demonstrated, that HDAC activity is fluctuating during the cell cycle of Physarum polycephalum. To test whether this periodic change in enzymatic activity is due to a cell cycle dependent regulation of PpHDAC1 transcription, total RNA of a P. polycephalum macroplasmodium was isolated at different stages of the cell cycle (Fig. 2, upper panel). Hybridization of Northern blots revealed that PpHDAC1 expression did not fluctuate significantly during the cell cycle (Fig. 2). In contrast, the expression of the histone H4 gene, which was used as a control for a cell cycle dependent transcript (Z), was induced in late G2 phase, reached a maximum during mitosis, decreased in S phase and was almost undetectable during late S and early G2 period (Fig. 2, lower panel). Findings on H4
mRNA are in line with earlier obeservations of total histone protein content (Loidl & Gro¨bner, 1987a,b) and H4 mRNA levels (Wilhelm et al., 1988) during the Physarum cell cycle. These results are in line with findings for human and mouse class 1 HDACs (Bartl et al., 1997), but contrast earlier observations that indicated a clear change of enzyme activity profiles in the course of the Physarum cell cycle (Brosch et al., 1992; Lopez-Rodas et al., 1992). This apparent discrepancy can be due to several reasons: (1) as in other organisms there is probably more than one HDAC (Gray & Ekstro¨m, 2001) and one or several of these might be responsible for the cell cycle dependent HDAC activity pattern. (2) PpHDAC1 activity might be regulated on a posttranscriptional and/or posttranslational level by phosphorylation, acetylation etc. in response to cell cycle signals. (3) Mammalian but also yeast class 1 HDACs have been shown to be active mainly in the context of large protein complexes. This might also be the case for Physarum PpHDAC1. Cell cycle dependent changes in HDAC activities might therefore be due to varying complex composition and/or posttranslational modifications of complex components.
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Fig. 3. Effect of TSA on the expression of PpHDAC1 at different phases of the cell cycle. Pieces of Physarum macroplasmodia at different time points of the cell cycle between mitosis 2 and 3 were transfered onto nutrient medium containing 0.6 mM TSA. Plasmodial pieces were incubated for 1 h and harvested simultaneously with untreated control plasmodia at the indicated time points (arrows). Total RNA was isolated, subjected to electrophoresis in 1.2% agarose/2.2 M formaldehyde gels, blotted onto nylon membranes and hybridized with DIG-labeled DNA fragments of PpHDAC1 and Physarum histone H4. Ethidiumbromide stained RNA gels served as an input loading control, showing the amounts of 18S and 26S rRNA.
Analysis of HDAC-dependency of the regulation of PpHDAC1 and histone H4 gene activity In numerous reports HDACs have been implicated in the negative regulation of the transcription of many genes (e.g. Steinbac et al., 2000; Zhang and Jones, 2001). To investigate if the expression of the PpHDAC1 gene itself is a target of HDACmediated gene regulation, Physarum macroplasmodia were treated with the specific HDAC inhibitor Trichostatin A (TSA) at different stages of the cell cycle followed by Northern analysis of PpHDAC1 expression. No effect of TSA treatment on the transcription of the PpHDAC1 gene was observed at any time point tested (Fig. 3). This result strongly suggests that the activity of the PpHDAC1 gene is not a subject to regulation by TSA sensitive HDACs. This finding is consistent with results obtained for maize class 1 HDACs ZmRpd3 and HD1BII (Lechner et al., 2000) but does not correspond with observations from the filamentous fungus Aspergillus nidulans, where a slight induction of the expression of the PpHDAC1 homolog RpdA
was observed after TSA treatment of mycelia (Graessle et al., 2000). Similarly, also the histone H4 genes do not appear to require HDACs for transcriptional regulation at any stage of the cell cycle. However, we cannot exclude that TSAinsensitive HDACs, like Sir2-related proteins (Imai et al., 2000) contribute to the transcriptional control of the PpHDAC1 and H4 genes, respectively, in Physarum polycephalum. Alternatively, autoregulation of the PpHDAC1 gene might not occur within the normal cell cycle of the plasmodial stage but, nevertheless, might be required for differentiation processes (such as sclerotization) or switch to the amoebal stage. Effect of HDAC inhibition on cell cycle progression To investigate whether the inhibition of HDACs affects normal cycling of Physarum macroplasmodia, plasmodial parts were treated with TSA at different time points during the cell cycle. When
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the checkpoint is not yet clear, it has been suggested to involve mechanisms capable of sensing the acetylation state of the chromatin and thus preventing cells from entering mitosis with hyperacetylated chromatin which in turn would interfere with centromere and kinetochore function.
ACKNOWLEDGEMENTS We gratefully acknowledge valuable discussions with Gerald Brosch and Adele Loidl. This work was in part supported by grants from the Dr-Legerlotz-Foundation, the Austrian National Bank (P-7415) and the Austrian Science Foundation (FWF-P-13620) to P.L. REFERENCES Fig. 4. Mitotic delay induced by TSA treatment. A Physarum macroplasmodium was grown until late G2-period (45 min prior to mitosis 2) and divided in 2 parts. One half was transfered onto nutrient medium containing 0.6 mM TSA, the second half served as a control in the absence of TSA. The control plasmodium went through mitosis 45 min after medium change.
TSA was added to macroplasmodia at 45 min before telophase of mitosis II (M2 in the untreated control), macroplasmodia entered mitosis with a pronounced delay of 30–40 min compared to untreated plasmodia (Fig. 4). This is remarkable because the process of mitotic division itself last for approx. 30 min; this means that TSA is able to induce mitotic delay even when it is added 15 min prior to the onset of early prophase. The delay is indicative of the involvement of HDACs in the regulation of the G2/M transition of the cell cycle. HDAC inhibitors have been shown to induce G1 phase arrest and differentiation by upregulating p21CIP/Waf1 in a range of tumor cell types (e.g. Sowa et al., 1997; Archer et al., 1998). Beside this role for G1/S transition several reports show the implication of histone deacetylation also for the progression of the cell through mitosis. In yeast, TSA treatment has been demonstrated to cause missegregation of chromosomes during mitosis (Ekwall et al., 1997). Moreover, mutation of the amino-terminal lysine residues normally acetylated in histone H4 resulted in a G2/M arrest (Megee et al., 1990). In a recent study with different tumor cell lines, the identification of a novel G2 checkpoint sensitive to HDAC inhibitor treatment was described (Qiu et al., 2000). Although the nature of
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