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 Previous Article

Eukaryotic Cell, May 2005, p. 981-990, Vol. 4, No. 5
1535-9778/05/$08.00+0     doi:10.1128/EC.4.5.981-990.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Class I Histone Deacetylase Thd1p Affects Nuclear Integrity in Tetrahymena thermophila

Emily A. Wiley,1,2,3* Tamara Myers,2 Kathryn Parker,2 Theodore Braun,1 and Meng-Chao Yao3*

W. M. Keck Joint Science Department, Claremont Colleges, Claremont, California 91711,1 Department of Biology, Mount Holyoke College, South Hadley, Massachussetts 01075,2 Fred Hutchinson Cancer Research Center, Seattle, Washington 981093

Received 3 December 2004/ Accepted 24 February 2005


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ABSTRACT
 
Class I histone deacetylases (HDACs) participate in the regulation of DNA-templated processes such as transcription and replication. Members of this class can act locally at specific sites, or they can act more globally, contributing to a baseline acetylation state, both of which actions may be important for genome maintenance and organization. We previously identified a macronuclear-specific class I HDAC in Tetrahymena thermophila called Thd1p, which is expressed early in the development of the macronucleus when it initially becomes transcriptionally active. To test the idea that Thd1p is important for global chromatin integrity in an active macronucleus, Tetrahymena cells reduced in expression of Thd1p were generated. We observed phenotypes that indicated loss of chromatin integrity in the mutant cells, including DNA fragmentation and extrusion of chromatin from the macronucleus, variable macronuclear size and shape, enlarged nucleoli, and reduced phosphorylation of histone H1 from bulk chromatin. Macronuclei in mutant cells also contained more DNA. This observation suggests a role for Thd1p in the control of nuclear DNA content, a previously undescribed role for class I HDACs. Together, these phenotypes implicate Thd1p in the maintenance of macronuclear integrity in multiple ways, probably through site-specific changes in histone acetylation since no change in the acetylation levels of bulk histones was detected in mutant cells.


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INTRODUCTION
 
The eukaryotic genome is organized into dynamic chromatin structures, the basic repeated unit of which is the nucleosome, composed of 146 bp of DNA wrapped around an octamer of the four core histones H2A, H2B, H3, and H4. DNA folding into compact structures is mediated by nucleosome interactions. These are dependent on other factors, including the binding of linker histone H1 to the DNA between nucleosomes, and the activities of chromatin-associated proteins (29). The amino terminal "tails" of histones are subject to an array of posttranslational modifications including acetylation, methylation, phosphorylation, ADP-ribosylation, and ubiquitination (25, 31, 50). Such modifications work singly or in combination to influence various DNA-templated processes such as transcription, replication, and DNA repair, likely through modification of chromatin structures (25).

The reversible acetylation of specific lysine residues has been well characterized. Steady-state acetylation levels are influenced by the opposing actions of two types of enzymes: histone acetyltransferases catalyze the transfer of acetyl moieties to specific lysine residues, while histone deacetylases (HDACs) remove them (4). Histone acetylation, which is positively correlated with transcription, is mechanistically linked to the regulation of gene expression; HDACs and histone acetyltransferases are common components of repression and activation complexes, respectively. Most of the HDACs identified to date fall into three phylogenetic classes depending on their homology to the Saccharomyces cerevisiae deacetylases Rpd3p (class I), Hda1 (class II), or NAD-dependent Sir2p that differ in localization, tissue-expression patterns, and general activities (11). Class I HDACs, which are present in all eukaryotes examined so far, are commonly found in corepressor complexes and mediate repression by a variety of transcription factors (35).

Although much is now known of requirements for class I HDAC-dependent gene repression, less is known about the contributions of these enzymes to global chromatin integrity and organization. There is evidence that histone deacetylation promotes folding of nucleosomal arrays into more complex structures (21, 49, 53), and it is well known that hypoacetylation is associated with regions of highly condensed chromatin (reviewed in reference 19). However, defining mechanistic relationships between individual class I HDAC activities and chromatin organization in the nucleus requires further study. Genetic approaches to studying these enzymes have been challenged by the essential functions of HDAC genes. For example, in mice, disruption of a single HDAC1 allele caused cell proliferation defects, and disruption of both gene copies caused embryonic lethality (30). Null alleles of a class I HDAC in Arabidopsis (AtHD1) caused pleiotropic developmental abnormalities (48). In yeast, where HDAC genes are nonessential, the class I homolog Rpd3p was shown to act both locally at specific sites and on global chromatin (41, 52), raising the possibility that these enzymes are important for more general chromatin architectures.

The ciliated protozoan Tetrahymena thermophila provides a unique opportunity to study the role of HDACs in chromatin integrity and organization. Each cell has a transcriptionally active, highly acetylated macronucleus and a transcriptionally inert micronucleus containing unacetylated, highly condensed chromatin for most of the cell cycle (1, 9, 51). Within the macronucleus are bodies of highly condensed chromatin whose size and number are affected by mutations in chromatin-associated proteins such as histone H1 and Hhp1 (23, 46). Moreover, due to polyploidy of the macronucleus, it is possible to create partial deletion mutants in which expression of essential genes is only reduced instead of eliminated (16). We previously described Tetrahymena Thd1p, a class I HDAC (Rpd3p homolog) that is selectively recruited to developing new macronuclei at a time when these nuclei become transcriptionally active (54). This suggested that Thd1p might help to establish a baseline acetylation state and appropriate chromatin structures necessary for the integrity of active chromatin in macronuclei. In this study, to address whether Thd1p influences nuclear structures, we created a cell line with reduced Thd1p expression and analyzed several resulting nuclear phenotypes. The phenotypes described suggest roles for Thd1p in macronuclear chromatin integrity including regulation of DNA content and nucleolar structure.


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MATERIALS AND METHODS
 
Strains and cell culture. A genetically marked strain of Tetrahymena thermophila, CU428 (Chx/Chx[cy-s]VII) was used as the wild-type strain in the experiments reported. Unless indicated otherwise, CU428 cells were grown at 30°C with shaking in 1% (wt/vol) enriched proteose peptone (SPP) (17) liquid medium to mid-logarithmic phase and a cell density of 2 x 105 to 5 x 105 cells/ml for all experiments. {Delta}THD1 cells were pregrown to mid-logarithmic phase in SPP containing 300 µg/ml paromomycin (Sigma Chemical Co.) and then transferred to medium lacking paromomycin and grown for an additional 5 to 10 population doublings prior to use in experiments at a cell density of 2 x 105 to 5 x 105 cells/ml.

Plasmid construction. The THD1 3' region was obtained by PCR on genomic DNA using the primers 5'-TGAAGTTTTGTCTGGTG-3' and 5'-TCCTTAAGATCTTTAACAGC-3'. The resulting ~7-kb product was ligated to EcoRV-digested plasmid p4T2-1, which contained a 1.5-kb drug resistance marker driven by the HHF1 promoter, followed by the Neor gene and the BTU2 terminator (15). We refer to the resulting plasmid as p3'THD-Neo. The THD1 5' region was obtained by PCR on genomic DNA using the primers 5'-GCATATAGATAAATGAAGG-3' and 5'-ACACAATCGTTTATATAGC-3'. The resulting ~8-kb product was filled in with T4 polymerase and blunt-end ligated to pBluescript that was digested with SmaI. The resulting plasmid, called p5'THD1, was double digested with BamHI and SpeI, and a ~7.0-kb 5'THD1 fragment was gel purified and directionally cloned into SpeI/BamHI-digested p3'THD-Neo to make pTHD-Neo. For transformation, pTHD1-Neo was double digested with XmnI and XhoI to produce a linear fragment containing the THD1 5' and 3' sequences interrupted by the drug resistance cassette that was subsequently used to coat gold beads for transformation.

Macronuclear THD1 replacement. CU428 cells were grown in SPP medium to a cell density of 5 x 105 cells/ml, collected by centrifugation, and starved by resuspending in 10 mM Tris-HCl (pH 7.5) at a cell density of 2.0 x 105 cells/ml and incubating the suspension at 30°C in a stationary incubator for 20 h. Biolistic transformation following a published protocol (5) was performed on 50-ml aliquots of the starved cells, except that 0.1-µm gold particles were used instead of tungsten particles. A Bio-Rad Biolistic PDS-1000/He particle delivery system was used for transformation by nuclear bombardment. To recover transformants, the target cells were transferred to flasks and grown with shaking in 50 ml of SPP medium. Paromomycin (80 µg/ml) was added after 5 h of incubation at 30°C with shaking, and cells were plated on six 96-well microtiter plates. Six transformants were obtained after growth under selection with paromomycin. Single cells from two of the transformants were serially transferred and grown in increasing concentrations of paromomycin. The transformants were able to divide in drug concentrations up to 1.4 mg/ml, at which point cell culture density decreased.

Southern hybridization. Total genomic DNA was isolated as previously described (15) from CU428 and {Delta}THD1 subclones, and 12 µg of each was digested with SpeI and XmaI. Resulting fragments were resolved on a 1% agarose gel, transferred to nylon membrane, and probed according to a standard procedure (44). To make the probe, a 0.5-kb fragment from THD1 was amplified by PCR on genomic DNA using the primers 5'-TGGCAATGACAGATACAC-3' and 5'-ACACAATCGTTTATATAGC-3'. The resulting product was labeled with [{alpha}-P32]dATP by the random prime method (44). Hybridization was carried out at 42°C in hybridization buffer containing 50% formamide. This same procedure was also used on DNA from macronuclei purified on a sucrose gradient as previously described (2). The purity of isolated macronuclei was assessed by methyl green staining of nuclei and counting by light microscopy. By this analysis, a 14:1 purification of macronuclei from micronuclei was demonstrated.

Immunoblot analysis. CU428 and {Delta}THD1 cells were grown to a density of 2 x 105 cells/ml. Cells (105) were collected by centrifugation and lysed by incubation in 30 µl of sodium dodecyl sulfate (SDS) gel loading buffer [50 mM Tris-Cl, pH 6.8, 100 mM dithiothreitol, 2% (wt/vol) SDS, 0.1% bromophenol blue, 10% (vol/vol) glycerol] and heated in a boiling water bath for 5 min. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on an 8% polyacrylamide gel, which was then transferred to nitrocellulose and probed with a 1:800 dilution of {alpha}-Thd1p polyclonal antiserum as described previously (54). Immunoreactivity was detected by chemiluminescence using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5,000 dilution; Amersham) following the manufacturer's protocol.

Histone deacetylase activity assays. Histone deacetylase activity from 106 macronuclei was extracted by DNaseI digestion as described (37). One-tenth (105 nucleus equivalent) of the extract was analyzed for deacetylase activity using Tetrahymena histones that were acetylated in vivo in the presence of [3H]acetate (2 x 104 dpm per assay) as described and used in previous work (54).

Growth analyses. Vegetatively growing CU428 and {Delta}THD1 cells were used to inoculate 100 ml of SPP medium at a starting density of 2 x 104 cells/ml. Cultures were grown at 30°C with shaking (200 rpm). Cells in aliquots of the cultures (200 µl) were counted with a hemocytometer at intervals of 2.5 to 3 h. Similar results were obtained in growth experiments using a Beckman Coulter particle counter to obtain cell counts (data not included).

Indirect immunofluorescence and DAPI staining of cells. CU428 and {Delta}THD1 cells in mid-logarithmic growth (2 x 105 to 5 x 105 cells/ml) were fixed in paraformaldehyde and processed for indirect immunofluorescence as previously described (47), except that Triton X-100 (0.2%) was added to the fixative solution prior to use, and cells were dropped onto coverslips lacking polylysine coating. For detection of acetylated histone H4, cells were incubated with {alpha}-acetylated histone H4 antiserum at a 1:800 dilution; {alpha}-micLH antiserum (antiserum against the micronuclear-specific linker histone) was used at a 1:200 dilution to detect micronuclear histone H1. Primary antibodies were detected with rhodamine-conjugated goat anti-rabbit immunoglobulin G (1:1,000 dilution; Jackson Laboratories). Cells were additionally stained with 0.1 µg/ml 4',6 diamino-2-phenylindole dihydrochloride (DAPI; Sigma Chemical Co.) according to a common protocol (47).

DNA fragmentation assay. Logarithmically growing {Delta}THD1 and CU428 cells were end labeled in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) according to the manufacturer's recommendations (Roche Biochemical) except for modifications described in previous studies (32). In addition, cells were fixed in 2% paraformaldehyde and 0.2% Triton X-100 as previously described (47) prior to treatment with TUNEL reagents. Cells were counterstained with DAPI (2 µg/ml) prior to fluorescence microscopy.

Fluorometric quantitation of nuclear DNA. A previously published method (34) was modified for use with Tetrahymena cells. One milliliter of logarithmically growing {Delta}THD1 or CU428 cells was counted using a Beckman Coulter particle counter, collected by centrifugation, and lysed by incubation in 1 ml of TNE-XDS buffer (10 mM Tris-HCl, 1 mM EDTA, 0.2 M NaCl, 0.01% Triton-X, and 0.001% SDS, pH 7.0) for 10 min at room temperature. Aliquots (50 µl) of each lysate were pipetted in triplicate into clear 96-well Falcon microtiter plates. A standard curve was constructed using herring sperm DNA in the following final concentrations in TNE-XDS: 0 µg/ml, 15 µg/ml, 20 µg/ml, 25 µg/ml, 30 µg/ml, 35 µg/ml, and 40 µg/ml. To each well, including experimental samples, was added 50 µl of 10 µg/ml Hoechst 33258 in TNE buffer (10 mM Tris, 1 mM EDTA, 0.2 M NaCl), for a final dye concentration of 5 µg/ml. Fluorescence intensities were obtained using a Molecular Devices Gemini EM fluorescence plate reader at an excitation of 356 nm and an emission of 458 nm. Background fluorescence intensity calculated from the standard curve was subtracted from the experimental values. To obtain the fluorescence intensity per cell, the fluorescence intensity of each lysate sample was divided by the number of cells in the sample. Differences between the CU428 and {Delta}THD1 samples were statistically significant using a t test for independent means (P < 0.0001).

Flow cytometry. Logarithmically growing cells were lysed in 0.25 M sucrose, 10 mM MgCl2, and 0.5% NP-40 at a concentration of 1.5 x 105 cells/ml. Propidium iodide was added to a final concentration of 50 µg/ml, and the nuclei were incubated for 1 h at room temperature and then dounce homogenized prior to flow cytometry analysis using a Becton Dickinson FACScan Cytometer.

Histone isoform analysis. Total histones or only H1 histones were extracted from 5 x 106 isolated nuclei, resolved by acid-urea PAGE, and visualized by staining with Coomassie brilliant blue R-250, as previously described (55). The amount of protein loaded from each strain was equalized according to protein quantitation by A280 using a Beckman DU530 UV/visible spectrophotometer.

Ultrastructural analyses. Logarithmically growing cells (5 x 105 cells/ml) were washed in 40 mM HEPES buffer (pH 7.5) and fixed for 1 h at room temperature in glutaraldehyde (2.5% in 0.1 M sodium cacodylate, pH 7.2). The samples were dehydrated by three incubations in 100% ethanol, slowly infiltrated and embedded in Spurr's resin, and polymerized at 70°C for 8 h. Sections were visualized using a JEOL 1010 transmission electron microscope at 80 kV. For chromatin body analysis, images captured on negatives were digitized using a Hewlett-Packard ScanJet scanner. The size and number of chromatin bodies per unit area in multiple macronuclei were determined using National Institutes of Health Image 1.61 software.


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RESULTS
 
Thd1p levels are reduced in cells carrying somatic gene replacements of THD1. To disrupt the THD1 gene in macronuclei, a disruption construct was made and used to transform Tetrahymena cells by a standard method involving homologous recombination (8). The construct included a disruption cassette (15) containing a neomycin-resistance (Neor) gene conferring resistance to paromomycin flanked by a 0.7-kb 5' and a 0.7-kb 3' THD1 sequence. The Neor gene replaced ~200 bp of THD1 gene coding sequence, including sequence encoding critical active site residues in the deacetylation motif (27). This construct was used to replace the macronuclear gene copies encoding Thd1p (Fig. 1A). Multiple transformants were obtained after growth under selection with increasing concentrations of paromomycin. Following growth in the presence of 300 µg/ml of paromomycin, multiple subclones of two of the transformants (referred to as {Delta}THD1.1 and {Delta}THD1.5) were analyzed by Southern hybridization to confirm integration of the disruption construct at the THD1 locus. Genomic DNA from wild-type and {Delta}THD1 cells was double digested with SpeI/XmaI and probed with a fragment corresponding to part of the 5' flanking region (Fig. 1A). A band of 3.2 kb representing the intact THD1 gene was detected in wild-type cells, whereas an additional band of 0.7 kb representing the disruption fragment was detected in the transformant subclones (called {Delta}THD1.1 and {Delta}THD1.5) (Fig. 1B, left), indicating correct integration of the Neor disruption construct. Integration at the THD1 locus was further confirmed by PCR with primers to the Neor gene and sequence flanking the THD1 target site. Product of the expected size (1.7 kb) was generated only in the {Delta}THD1 clones (Fig. 1C).



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  		FIG. 1. Thd1p is reduced in cells containing disrupted THD1 (A) A 3.2-kb SpeI fragment of the genomic THD1 gene (top) and the gene disruption cassette (2.9-kb fragment from pTHD1-Neo) integrated at the THD1 locus (bottom) are shown. The gene disruption cassette contains a 5' 0.7-kb and a 3' 0.7-kb THD1 fragment, both containing THD1 coding and coding flanking sequences (shown as gray boxes) and the Neor gene driven by an HHF1 promoter (shown as a thick black line) flanked by the BTU2 terminator (shown as a thin black line). A 0.5-kb fragment from the 5' THD1 sequence was used as a probe for the Southern hybridization. Arrows represent primers used for PCR to confirm integration of the disruption cassette. (B) Total genomic DNA from wild type (WT) and two {Delta}THD1 transformants (1.1 and 1.5) was double digested with SpeI and XmaI and analyzed by Southern hybridization with a 5' THD1 probe. The 3.2-kb band is derived from the undisrupted copies of THD1; the 0.7-kb band is derived from the disrupted copies. Signals were quantitated by densitometry. In both transformants the fraction of THD1 copies that remained undisrupted was more than the expected 5% from the undisrupted micronuclear copies (16). The right panel shows data from Southern hybridization using DNA from macronuclei partially purified from micronuclei (~14 macronuclei:1 micronucleus). (C) PCR was performed on genomic DNA isolated from wild-type and {Delta}THD1 1.5 cells. Primers were complementary to the Neor gene and to genomic DNA flanking the site of disruption cassette integration at the THD1 locus (as diagrammed in panel A). As expected, a 1.7-kb fragment (only band shown) was amplified only from {Delta}THD1 DNA.

The presence of the 3.2-kb band in the transformant samples indicated that a fraction (estimated 50%) of the ~45 gene copies remained undisrupted (Fig. 1B, left). Single transformed cells were then additionally grown in 500 µg/ml up to 1.4 mg/ml paromomycin to increase the fraction of disrupted gene copies. The Southern hybridization analysis revealed that even in these higher concentrations, cells still retained a fraction of the undisrupted somatic gene copies after each additional growth period, although the fraction was smaller (20 to 30%) at the higher concentrations of drug (Fig. 1B, middle and right). The fraction of undisrupted alleles did not decrease any further when cells were grown in paromomycin at concentrations higher than 500 µg/ml (Fig. 1B, right). Together, these results indicated that the majority of THD1 alleles were disrupted in the mutant cells, which will hereafter be referred to as THD1 knockdown or {Delta}THD1 cells since a portion of the THD1 gene was replaced in the disrupted alleles. The inability to completely replace all of the alleles with the disruption construct, even under selection with higher concentrations of paromomycin in which cells failed to grow (1.4 mg/ml) (Fig. 1B, right), indicates that THD1 is probably an essential gene (16).

To test whether Thd1p expression was reduced in the THD1 knockdown cells, an immunoblot was performed on total macronuclear proteins from wild-type and {Delta}THD1 cells grown for six population doublings in medium lacking paromomycin to help control for any effects from growth in the drug. Probing with {alpha}-Thd1p antiserum revealed that Thd1p production was reduced in {Delta}THD1 cells compared with wild-type cells (Fig. 2A). Similar immunoblotting experiments on serial dilutions of total macronuclear proteins revealed that the {Delta}THD1 cells were at least fivefold reduced in Thd1p expression compared with wild-type cells (data not shown). Furthermore, while the reduction in Thd1p synthesis was stable during growth of transformants in the presence of 300 µg/ml paromomycin, approximately 60 generations of growth (by successive transfers) in medium lacking paromomycin produced revertant cells that synthesized levels of Thd1p similar to wild-type cells (Fig. 2A, third lane). This same result was obtained for cells pregrown in the highest concentration of paromomycin allowing cell survival (1.4 mg/ml), further demonstrating that mutant cells still retained wild-type alleles when grown at this drug concentration.



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  		FIG. 2. (A) Total nuclear proteins from wild type (WT), {Delta}THD1, and {Delta}THD1 revertants grown nonselectively for 60 generations ({Delta}THD1 cured) were resolved by SDS-PAGE, transferred to nitrocellulose, and cut, and the pieces were immunoblotted with antibodies against Thd1p ({alpha}-Thd1p) or histone H4 ({alpha}-H4) as a loading control. As an additional loading control, samples were run on the gel in duplicate, and one set was stained with Coomassie brilliant blue (Coom). The bottom panel shows two Coomassie brilliant blue-stained bands migrating at approximately 50 kDa. (B) HDAC activity in different strains was assessed by measuring the amount of [3H]acetate released when [3H]-acetylated histones were incubated with nuclear extract. Bars represent the average of two experiments. H, histones alone (no extract); WT, wild-type; {Delta}, mutant ({Delta}THD1); R, revertants. Samples treated with TSA are indicated.

The total amount of histone deacetylase activity in isolated macronuclei was assessed using in vivo labeled Tetrahymena histones as substrate (labeled in the presence of [3H]acetate) and measuring the amount of radioactivity released as previously described (54). The amount of HDAC activity correlated with the amount of Thd1p expressed; mutant cells reduced in Thd1p expression had less HDAC activity (20 to 25% less) compared to wild-type or revertant cells (Fig. 2B). In all cases, the addition of 50 nM trichostatin A (TSA), a potent histone deacetylase inhibitor, greatly reduced the amount of released [3H]acetyl moieties, indicating that the majority of acetate released in the different strains was due to HDAC activity.

The amount of bulk histone acetylation is unaffected, but histone H1 is less phosphorylated in {Delta}THD1 cells. Thd1p deacetylates all four core histones in vitro (54). To test whether the ~20% reduction of total HDAC activity in {Delta}THD1 cells affected the overall acetylation state of bulk histones, total histones were partially purified based on their acid solubility and analyzed on an acid-urea polyacrylamide gel, which effectively separates Tetrahymena acetylated histone isoforms (55). No difference in the acetylation state of bulk histones H2B, H3, and H4 was detected (Fig. 3). The distribution of histone H2A isoforms in this gel system is more complicated and thus was not analyzed. One difference in histone modifications was the amount of phosphorylation of linker histone H1. The isoforms of H1 resolved in this gel system arise from differences in the number of phosphoryl groups. A greater proportion of unphosphorylated isoforms relative to more highly phosphorylated forms (revealed by darker staining) indicated that the total H1 population was less phosphorylated overall in {Delta}THD1 cells (Fig. 3A and B; compare bands marked by asterisks).



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  		FIG. 3. Acetylation state of bulk core histones in {Delta}THD1 cells is unchanged. (A) The acetylated state of bulk histones was compared between wild-type (WT) and {Delta}THD1 cells. Histones extracted from purified nuclei were resolved by acid-urea polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. The number of acetyl groups on the core histone isoforms is indicated by the numbers on the left. Histone H1 bands represent isoforms that differ in the number of phosphoryl groups. The asterisk indicates unphosphorylated histone H1. (B) H1 histones were isolated from core histones, resolved by acid-urea polyacrylamide gel electrophoresis, and stained with Coomassie brilliant blue. The faster-migrating bands represent histone H1 isoforms with fewer phosphoryl groups; unphosphorylated H1 is indicated with an asterisk.

{Delta}THD1 cell cultures have an increased doubling time and higher cell densities in stationary phase. A difference in growth behavior in {Delta}THD1 cells was revealed by a comparison of growth curves. Cells were pregrown under paromomycin selection and then transferred to medium lacking paromomycin and grown for an additional five population doublings before growth rate data were collected. A strain carrying a Neor disruption cassette integrated at the HHP1 locus was used as a control for effects of pregrowth in paromomycin. Hhp1 is a protein that localizes to heterochromatin, and {Delta}HHP1 cells were previously shown to have no vegetative growth phenotype (22). These cells were treated identically to {Delta}THD1 cells and were referred to as control cells. Growth curves revealed that the average population doubling time for {Delta}THD1 cells in mid-logarithmic growth was ~1.4 times that of wild-type and control cells (3.4 h, 2.4 h, and 2.6 h, respectively) (Fig. 4 and data not shown). Despite the reduced growth rate of {Delta}THD1 cells, the population grew past the normal stationary cell density (1 x 106 to 2 x 106 cells/ml) to reach about a twofold higher cell density at stationary phase (Fig. 4).



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  		FIG. 4. The doubling time of {Delta}THD1 cultures is slower than the control ({Delta}HHP1) culture. Growth curves were initiated at a cell density of 2 x 104 cells/ml. Cell counts and density calculations were made every 2.5 h for 40 h as described in the Materials and Methods and plotted on a logarithmic scale. Doubling time is listed. The curve for growth of wild-type cultures is nearly identical to the control curve and, thus, is not shown. Similar results were obtained using a particle counter instead of a hemocytometer for cell counts.

{Delta}THD1 cells contain extranuclear bodies of degrading chromatin derived from the macronucleus. To begin to address whether reduced levels of Thd1p affect chromatin organization, we examined the area occupied by DNA in the macronucleus. Wild-type, control ({Delta}HHP1), and {Delta}THD1 cells were fixed, stained with the DNA-specific dye DAPI, and examined by fluorescence microscopy (Fig. 5A). The DAPI-stained area in {Delta}THD1 cells was more variable in shape (less uniformly round) and unevenly distributed compared to wild-type and control macronuclei, and the majority of macronuclei contained large pockets devoid of DAPI staining (Fig. 5A, compare frames i to iii). Macronucleus sizes were estimated by calculating the DAPI-stained area based on radius measurements in 100 cells from each strain. This analysis revealed a wider range of nucleus sizes in {Delta}THD1 cells, although the average size was similar to wild-type and control cells (Fig. 5B).



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  		FIG. 5. {Delta}THD1 cells contain extranuclear bodies of macronucleus-derived chromatin and variable DAPI-staining areas. (A) Wild-type (WT), control, and {Delta}THD1 cells were fixed, stained with the DNA-specific dye DAPI, and visualized by fluorescence microscopy. m, micronucleus; M, macronucleus. Control cells are resistant to paromomycin due to complete replacement of the heterochromatin-associated protein gene HHP1 with a disruption cassette containing the neomycin resistance gene (Neor). These cells were previously shown to have no cytological phenotypes during vegetative growth (22) and are thus used as a control for any effects due to pregrowth of {Delta}THD1 cells in 300 µg/ml of paromomycin. In frame iii of panel A, arrows point to macronuclear "pockets" that stain less intensely with DAPI. In frame v, the arrow points to a "bridge" of DNA between the macronucleus and the extrusion body. Bars, 5 µm. (B) The DAPI-stained area of 100 macronuclei from each strain was calculated. Bars represent the average area. Vertical lines represent the range of areas calculated. WT, wild type. (C) Extrusion bodies contain acetylated chromatin. {Delta}THD1 cells were fixed and immunostained with antibodies detecting acetylated histone H4 ({alpha}-AcH4). Immunostained cells were also stained with DAPI. m, micronucleus; M, macronucleus; bars, 5 µm. (D) Extrusion bodies from {Delta}THD1 cells lack micronuclear linker histone. Cells were processed as described for panel C, but immunostaining was performed with an antibody detecting micronuclear linker histone ({alpha}-micLH). Bars, 5 µm.

Staining of {Delta}THD1 cells with DAPI also revealed round extranuclear bodies of chromatin (named extrusion bodies, or EBs, consistent with previously published descriptions [10]) that were variable in size and were present in cells in different stages of the cell cycle (Fig. 5A, frames iii to v). These bodies were larger toward the interior of the cell, smaller at the periphery of the cell, and appeared to bud directly from the macronucleus (Fig. 5A, frame v). Similar bodies were not detected in cells carrying partial deletions of another essential gene grown in the same amount of paromomycin (M. Cervantes, unpublished), indicating that this characteristic was not caused by partial deletion of any essential gene (data not shown). To determine whether the extrusion bodies originated from the macronucleus or the micronucleus, {Delta}THD1 cells were fixed, immunostained with antiserum against acetylated histone H4, and examined by fluorescence microscopy. The positive staining of extrusion bodies (Fig. 5C) suggested that these bodies were derived from the macronucleus since only macronuclear, not micronuclear, chromatin is acetylated (18, 51). To confirm that the EBs were not micronucleus derived, {Delta}THD1 cells were fixed and immunostained with antiserum against the micronuclear-specific linker histone, micLH. As shown in Fig. 5D, extrusion bodies failed to stain with the micLH antiserum, indicating that they lacked micronuclear chromatin. From cytological results, we concluded that the extrusion bodies in {Delta}THD1 cells were derived from the macronucleus.

Extrusion of fragmented DNA in membrane-bound bodies is a characteristic of apoptotic cells and is mechanistically linked to DNA fragmentation (14, 28). To determine if macronuclear chromatin was undergoing fragmentation in {Delta}THD1 cells and whether extrusion bodies contained degrading DNA, a TUNEL assay was performed. This assay was used previously in Tetrahymena to detect degrading DNA in apoptotic nuclei (32). DNA 3' ends were labeled with digoxigenin-dUTP and detected by immunofluorescence microscopy using anti-digoxigenin-dUTP. As shown in Fig. 6, fragmented DNAs (3' DNA ends) were detected in both the extrusion bodies and macronuclei of {Delta}THD1 cells. The fluorescence in extrusion bodies was more intense, indicating higher concentrations of fragmented DNA compared to that in the macronucleus. In contrast, fragmented DNA was not detected in the macronuclei of wild-type cells by this assay. These results raised the possibility that DNA in extrusion bodies was actively undergoing degradation.



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  		FIG. 6. Extrusion bodies and macronuclei contain fragmented DNA. Wild-type (WT) and {Delta}THD1 cells were fixed and stained with DAPI, and 3' DNA termini were detected using a TUNEL assay (see Materials and Methods). M, macronucleus; m, micronucleus; bars, 5 µm.

DNA content is greater in {Delta}THD1 cells. The presence of extra bodies of chromatin prompted us to test whether total DNA content was higher in {Delta}THD1 cells. Relative DNA content was determined by fluorescence of cell lysates treated with Hoescht, a DNA-intercalating fluorescent dye. This method was used previously for DNA quantification in cell lysates (6, 34). Comparison of the amount of DNA per cell revealed that {Delta}THD1 cells had, on average, approximately 50% more DNA than wild-type cells (wild type, 8.8 ± 0.5 relative fluorescence units; {Delta}THD1, 13.1 ± 0.3 relative fluorescence units [P < 0.0001]). To determine the distribution of DNA to the macronucleus, micronucleus, and extrusion bodies, the DNA content of each was measured directly by flow cytometry following cell lysis with NP-40 and DNA staining with propidium iodide. In {Delta}THD1 cells, macronuclear DNA content was greater compared to wild-type cells, while that in the micronucleus was the same (Fig. 7). Extra peaks of fluorescence between the micronuclear and macronuclear peaks in {Delta}THD1 samples indicated the presence of smaller bodies containing intermediate amounts of DNA. Although these were thought to represent the extrusion bodies, the possibility that they also represent macronuclei with less DNA could not be ruled out. Thus, the 50% increase in total DNA content in {Delta}THD1 cells may be attributed to both a higher amount of DNA in the macronuclei and the presence of extrusion bodies that contain variable amounts of DNA. Together, these results suggest a role for Thd1p in regulating macronuclear DNA content.



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  		FIG. 7. Macronuclear DNA content is more variable in {Delta}THD1 cells. Wild-type (WT) and {Delta}THD1 cells were lysed and stained with the DNA-specific dye propidium iodide. DNA contents of the micronucleus (m), macronucleus (M), and extrusion bodies were measured by flow cytometry. The bracketed peak may represent the extrusion bodies or macronuclei with less DNA.

Regions of heterochromatin appear unaffected, but nucleoli are enlarged in {Delta}THD1 cells. We predicted that if class I HDACs promote more global chromatin condensation and heterochromatin formation, then {Delta}THD1 cells would have fewer or smaller regions of macronuclear heterochromatin. To test this possibility, the number and size of chromatin bodies (regions of highly condensed chromatin) in mid-logarithmic cultures of {Delta}THD1 and wild-type cells were compared by transmission electron microscopy (TEM). Surprisingly, there were no detectable differences in chromatin body numbers or sizes (Fig. 8A and B). TEM images also unexpectedly revealed that the nucleoli in mutant cells were grossly enlarged compared to wild-type nucleoli (Fig. 8A). In Tetrahymena, the ~9,000 rRNA gene repeats are organized into multiple nucleoli that reside around the nuclear periphery. Multiple enlarged nucleoli were evident in greater than 80% of the cell sections examined, whereas nucleolar structures of comparable size were never detected in the wild-type sections. This finding suggests that Thd1p has an additional role in nucleolar structure.



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  		FIG. 8. (A) Regions of condensed chromatin (chromatin bodies) are similar, but nucleolus size is greater in {Delta}THD1 cells. Wild-type (WT) and {Delta}THD1 cells in mid-logarithmic growth were fixed and processed for ultrastructural analysis by transmission electron microscopy. No consistent differences in the area or numbers of chromatin bodies between the strains was detected, but enlargement of the multiple, peripherally located nucleoli was observed; N, nucleolus; m,micronucleus; cb, chromatin body. (B) Distribution of chromatin body area. The area of chromatin bodies from wild-type (WT) or {Delta}THD1 cells was measured. The average area (av) of the chromatin bodies and the number (n) of measurements are indicated.


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DISCUSSION
 
In this study we describe nuclear phenotypes of Tetrahymena cells reduced in expression of Thd1p, a developmentally regulated homolog of class I HDACs found in eukaryotes from yeast to human. Due to its polyploid macronucleus, Tetrahymena afforded the opportunity to create a Thd1p knockdown cell line to address the role of Thd1p in global chromatin integrity. These cells had several phenotypes (extrusion bodies, DNA degradation, and increased DNA content) that demonstrated the importance of Thd1p for aspects of nuclear integrity. In the course of this study, the inability to completely replace all somatic THD1 gene copies with a Neor disruption cassette was taken as evidence that THD1 is an essential gene. This is not surprising since class I HDACs are typically involved in gene regulation and are essential in other organisms (30, 48). Although previous fractionation of Tetrahymena cellular extracts identified only two major peaks of HDAC activity, with Thd1p being present in one (54), genome sequence and expression analyses have since revealed several other HDAC homologs including two others that are most similar to the class I family. Since Thd1p appears to be an essential enzyme, at least some of its functions are not redundant to the other HDAC enzymes. In our study, however, it remains a possibility that the decreased Thd1p activity in knockdown cells altered the activity of other HDACs and consequently influenced the nuclear phenotypes observed.

Although total HDAC activity was reduced (Fig. 2B), an increase in the level of bulk core histone acetylation was not detected (Fig. 3) in {Delta}THD1 cells. We acknowledge that this could have resulted from increased activity of other HDACs or insufficient reduction of Thd1p activity to produce a detectable difference. Nevertheless, considering this result, we speculate that the observed nuclear phenotypes were probably caused indirectly from acetylation changes at specific gene loci instead of directly from changes in global histone acetylation levels. Changes in combinations of posttranslational modifications, which occur interdependently, create patterns that regulate transcription and other DNA-templated processes at specific sites (25). For example, some HDACs in other organisms regulate specific gene activity by providing modification-specific binding surfaces for the recruitment of repressors (7, 20, 26). In our study, acetylation changes at specific sites or a redistribution of acetyl moieties on bulk chromatin in {Delta}THD1 cells would not have been detected by the gel analysis, which instead revealed overall levels. One surprising difference detected in levels of bulk histone modifications was the decreased phosphorylation of linker histone H1 (Fig. 3A and B). This result suggests that Thd1p might normally promote H1 phosphorylation, a novel role for a histone deacetylase. Although it is known that one type of histone modification can influence other modifications (25), there is no precedent for core histone acetylation affecting the phosphorylation of histone H1. In our study, the reduced H1 phosphorylation in {Delta}THD1 cells is probably not caused by more highly acetylated bulk histones, since the latter were not detected (Fig. 2). Instead, the intriguing possibility that Thd1p affects the expression or activity of specific H1 phosphatases and/or kinases, like Cdc2 kinase (42), will be addressed in future studies. Given the role of linker histones in chromatin dynamics, it would be easy to speculate that Thd1p might affect chromatin integrity indirectly through modifying H1 phosphorylation. However, no difference in macronuclear morphology was previously detected in cells lacking H1 phosphorylation (13), making this possibility less likely.

Cytological examination of the knockdown mutants revealed the presence of EBs (extranuclear bodies of chromatin) that appeared round and variable in size and that were present in all stages of the cell cycle (Fig. 5A). EBs are normally generated in wild-type cells (typically no more than one per cell) during the amitotic division of the macronucleus as a means of regulating macronuclear DNA content in daughter cells (10, 12). However, these previously described bodies are smaller and degrade quickly (typically within 20 min) following macronuclear division. In contrast, the larger extrusion bodies observed in {Delta}THD1 knockdown cells were present (typically, 1 to 3 per cell) in cells that were not undergoing macronuclear division. Furthermore, approximately 65% of logarithmically growing {Delta}THD1 cells contained extrusion bodies, compared to 10% of wild-type cells (38a). Similar to results with normal extrusion bodies in wild-type cells, immunostaining experiments demonstrated that those in knockdown cells were derived from the macronucleus (Fig. 5C and D) and contained degrading DNA (Fig. 6). However, unlike those in wild-type cells, the extrusion bodies appeared to bud directly from nondividing macronuclei (Fig. 5A, frame v) instead of originating during division. Although it is tempting to speculate that the large pockets lacking DAPI staining in the macronuclei of {Delta}THD1 cells (Fig. 3A, frame iii) result from loss of DNA in extrusion bodies, they may also result from other phenomena such as enlarged nucleoli (Fig. 8A), which typically stain less intensely with DAPI. In addition, it is unlikely that extrusion bodies contain nucleolar chromatin as they failed to stain with antiserum against the nucleolus-enriched protein Nopp52 (data not shown). Considering our data, we favor a couple of explanations for extrusion body formation. First, similar to nuclear outpocketing, or "blebbing" in apoptotic cells (28), it may be functionally coupled with the degrading chromatin observed in the macronucleus (Fig. 6). Second, although extrusion bodies appear to form directly from a nondividing macronucleus, they might be generated to regulate DNA content, similar to that in wild-type cells. This model is consistent with there being more macronuclear DNA (Fig. 7) and 50% more total cellular DNA (including that in the micronucleus and extrusion bodies) in {Delta}THD1 cells.

One striking phenotype of {Delta}THD1 cells was the presence of fragmented chromatin, primarily in extrusion bodies. Nuclear chromatin degradation has been observed in mammalian cells treated with the general HDAC inhibitor TSA due to apoptosis (33). It is unlikely that decreasing the expression of Thd1p induced a similar apoptotic response in Tetrahymena, since preliminary work failed to detect other hallmarks of apoptosis, such as a nucleosomal ladder and increased nuclear acidity (34a). Alternatively, we speculate that increased DNA fragmentation could result from inefficient repair of DNA damage, a process that appears to involve class I HDACs (3, 24, 39).

Our data also suggest that Thd1p normally plays a role in regulating macronuclear DNA content, a previously undescribed role for class I HDACs. A fraction of macronuclei in {Delta}THD1 cells contained more DNA, and there was an increase in total cellular DNA content, some of it contained in extrusion bodies (Fig. 7). The possibility that Thd1p regulates replication will be addressed in future studies. Our data could also result from unequal partitioning of DNA during the amitotic division of the macronucleus. However, there was no cytological evidence supporting this possibility; dividing macronuclei in {Delta}THD1 cells consistently appeared evenly distributed between both daughter cells (data not shown).

One unexpected phenotype of the knockdown cells was gross enlargement of the nucleoli. Similar enlargement, characteristic of nutrient-starved Tetrahymena cells, is coincident with decreased ribosome biogenesis and gene expression (36). In contrast to wild-type cells, the enlarged nucleoli were present during mid-logarithmic growth, a time when maximum protein synthesis is expected. It is thought that enlargement in starved cells results from nucleolar aggregation, something we could not discern in the mutant cells. Enlarged nucleoli have previously been detected in transformed mammalian cells, resulting from increased ribosome biogenesis and production of rRNA (43). In preliminary studies on {Delta}THD1 cells, rRNA was increased at least twofold in the knockdown cells, and the copy number of the rRNA genes examined by hybridization was only modestly increased (~1.5-fold); both of these facts may partially account for the nucleolar enlargement (data not shown). It is tempting to speculate that the enlarged nucleoli phenotype and the ability of {Delta}THD1 cultures to grow past the normal stationary phase cell density (Fig. 4) are related, since enlarged nucleoli were previously observed in highly proliferating transformed cells (43). Finally, there is precedent for class I HDAC influence on rRNA chromatin structure (40, 45), and in Tetrahymena, nucleosomes are precisely positioned along the rRNA gene repeats flanking the replication origins (38). It will be interesting to test whether Thd1p acts at these sites to regulate rRNA gene replication and transcription.

The various phenotypes described in this study indicate several roles for Thd1p in the maintenance of nuclear integrity. We speculate that these Thd1p functions may be necessary for the correct development and maintenance of the transcriptionally active macronucleus, which may explain why expression of this enzyme is induced early in macronuclear development (54).


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ACKNOWLEDGMENTS
 
We are grateful to Douglas Chalker for his technical advice and helpful discussions, to Sue Ellen Gruber for supervising TEM work, and to C. David Allis for guidance on work supportive to this study. We also gratefully acknowledge Jennifer Armstrong and James Davie for their critical reading of the manuscript, Kathryn Naumes and Katrina Pelekanakis for providing preliminary results, and Kersey Black for graphics assistance.

This research was supported by grants from the National Institutes of Health to M.-C.Y. (R01GM26210) and to E.A.W. (GM18785-04).


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FOOTNOTES
 
* Corresponding author. Mailing address for Emily A. Wiley: W. M. Keck Joint Science Department, Claremont Colleges, 925 N. Mills Ave., Claremont, CA 91711. Phone: (909) 607-9698. Fax: (909) 621-8588. E-mail: ewiley{at}jsd.claremont.edu. Present address for Meng-Chao Yao: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan. Phone: 886-2-2782-1436. E-mail: mcyao{at}imb.sinica.edu.tw. Back


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Eukaryotic Cell, May 2005, p. 981-990, Vol. 4, No. 5
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