Class I histone deacetylase Thd1p affects nuclear integrity in Tetrahymena thermophila

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.

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 Neo^r 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 ΔTHD1 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 ΔTHD1 1.5 cells. Primers were complementary to the Neo^r 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 ΔTHD1 DNA.

FIG. 2.

(A) Total nuclear proteins from wild type (WT), ΔTHD1, and ΔTHD1 revertants grown nonselectively for 60 generations (ΔTHD1 cured) were resolved by SDS-PAGE, transferred to nitrocellulose, and cut, and the pieces were immunoblotted with antibodies against Thd1p (α-Thd1p) or histone H4 (α-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; Δ, mutant (ΔTHD1); R, revertants. Samples treated with TSA are indicated.

FIG. 3.

Acetylation state of bulk core histones in ΔTHD1 cells is unchanged. (A) The acetylated state of bulk histones was compared between wild-type (WT) and ΔTHD1 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.

FIG. 4.

The doubling time of ΔTHD1 cultures is slower than the control (ΔHHP1) culture. Growth curves were initiated at a cell density of 2 × 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.

FIG. 5.

ΔTHD1 cells contain extranuclear bodies of macronucleus-derived chromatin and variable DAPI-staining areas. (A) Wild-type (WT), control, and ΔTHD1 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 (Neo^r). 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 ΔTHD1 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. ΔTHD1 cells were fixed and immunostained with antibodies detecting acetylated histone H4 (α-AcH4). Immunostained cells were also stained with DAPI. m, micronucleus; M, macronucleus; bars, 5 μm. (D) Extrusion bodies from ΔTHD1 cells lack micronuclear linker histone. Cells were processed as described for panel C, but immunostaining was performed with an antibody detecting micronuclear linker histone (α-micLH). Bars, 5 μm.

FIG. 6.

Extrusion bodies and macronuclei contain fragmented DNA. Wild-type (WT) and ΔTHD1 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.

FIG. 7.

Macronuclear DNA content is more variable in ΔTHD1 cells. Wild-type (WT) and ΔTHD1 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.

FIG. 8.

(A) Regions of condensed chromatin (chromatin bodies) are similar, but nucleolus size is greater in ΔTHD1 cells. Wild-type (WT) and ΔTHD1 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 ΔTHD1 cells was measured. The average area (av) of the chromatin bodies and the number (n) of measurements are indicated.