Class I histone deacetylase Thd1p promotes global chromatin condensation in Tetrahymena thermophila

Abstract

Class I histone deacetylases (HDACs) regulate DNA-templated processes such as transcription. They act both at specific loci and more generally across global chromatin, contributing to acetylation patterns that may underlie large-scale chromatin dynamics. Although hypoacetylation is correlated with highly condensed chromatin, little is known about the contribution of individual HDACs to chromatin condensation mechanisms. Using the ciliated protozoan Tetrahymena thermophila, we investigated the role of a specific class I HDAC, Tauhd1p, in the reversible condensation of global chromatin. In this system, the normal physiological response to cell starvation includes the widespread condensation of the macronuclear chromatin and general repression of gene transcription. We show that the chromatin in Thd1p-deficient cells failed to condense during starvation. The condensation failure correlated with aberrant hyperphosphorylation of histone H1 and the overexpression of CDC2, encoding the major histone H1 kinase. Changes in the rate of acetate turnover on core histones and in the distribution of acetylated lysines 9 and 23/27 on histone H3 isoforms that were found to correlate with normal chromatin condensation were absent from Thd1p mutant cells. These results point to a role for a class I HDAC in the formation of reversible higher-order chromatin structures and global genome compaction through mechanisms involving the regulation of H1 phosphorylation and core histone acetylation/deacetylation kinetics.

FIG. 1.

Macronuclear chromatin fails to condense in starved ΔTHD1 cells. (A) Vegetatively growing or starved wild-type (WT) or ΔTHD1 cells were fixed in paraformaldehyde, stained with the DNA-specific dye DAPI, and visualized by fluorescence microscopy. M, macronucleus; m, micronucleus. Bars, 5 μm. Arrow points to an extrusion body commonly observed in mutant cells (66). (B) A conjugating pair of wild-type (WT) and ΔTHD1 (Δ) cells. Cells were fixed, stained with DAPI, and visualized by fluorescence microscopy. The wild-type cell was distinguished by the presence of ingested fluorescent beads. Cell borders are enhanced by a gray line. Bar, 5 μm. (C) The DAPI-stained areas of 150 macronuclei in unmated cells from each strain were calculated. Bars represent the average area; standard error bars are shown. WT, wild-type.

FIG. 2.

Macronuclear chromatin bodies fail to enlarge in starved ΔTHD1 cells. (A) Growing and starved wild-type (WT) and ΔTHD1 cells were fixed and processed for ultrastructural analysis by transmission electron microscopy. A representative macronucleus is shown for each strain and condition. nu, nucleolus; cb, chromatin body; mic, micronucleus. Black arrow in bottom right panel indicates a putative nucleolus. (B) ΔTHD1 cells respond to starvation conditions in ways similar to wild-type cells. Nuclear proteins resolved by acid-urea-PAGE and stained with Coomassie brilliant blue revealed similar starvation-induced protein expression changes in wild-type and ΔTHD1 cells. WT, wild-type; Gr, growing; St, starved.

FIG. 3.

(A) Steady-state acetylation is unchanged in starved ΔTHD1 cells. Comparison of differentially acetylated isoforms. Core histones extracted from purified nuclei were resolved by acid-urea- PAGE and stained with Coomassie brilliant blue. The number of acetyl groups on the core histone isoforms is indicated by the numbers on the left. (B) Acetate turnover on core histones is affected in ΔTHD1 cells. Isolated macronuclei were incubated with [³H]acetyl-CoA for 30 min. Histones were then extracted, resolved by acid-urea-PAGE, stained with Coomassie brilliant blue, and subjected to autoradiography. The region of each core histone ladder is indicated. The position of the unacetylated species in each ladder is indicated by “0.” The left panel shows the Coomassie-stained and dried gel that was subjected to autoradiography. The right panel shows the autoradiograph showing the incorporation of [³H]acetate. (C) Thd1p regulates expression of HATs and HDACs. Reverse transcriptase PCR was used to assess transcript levels of several HAT and HDAC genes. The genomic lane for THD3 was relocated to the right-most position on the gel. G, genomic template; WT, wild-type; Δ, ΔTHD1.

FIG. 4.

Distribution of acetyl moieties on histone H3 Lys9 and Lys27 is altered in ΔTHD1 cells. (A) Histones extracted from purified nuclei were resolved by acid-urea-PAGE, transferred to nitrocellulose membrane, and probed first with anti-H4AcLys16 antiserum. The blot was then stripped and reprobed with anti-panH4 to detect the positions of all H4 molecules. Numbers indicate the number of acetyl modifications on the H4 molecules at each position. (B) Histones extracted from purified nuclei were resolved in wide lanes by acid-urea-PAGE and transferred to nitrocellulose membrane. Multiple strips were cut from each lane and probed with the indicated antibodies. Numbers indicate the number of acetyl modifications on the H3 molecules at each position. The starved ΔTHD1 strip probed with anti-H3AcLys9 was shifted down slightly in comparison to the others. WT, wild-type; Δ, ΔTHD1; Gr, growing; St, starved; α, anti.

FIG. 5.

Histone H3 lysine 27 methylation increases with cell starvation. Histones extracted from purified nuclei were resolved by acid-urea-PAGE, transferred to nitrocellulose membrane, and probed first with anti-H3Me₃27 antiserum. The blot was then stripped and reprobed with anti-panH3 to show the positions of all H3 isoforms. The numbers indicate the number of acetyl modifications on the H3 molecules at each position. WT, wild-type; Gr, growing; St, starved; α, anti.

FIG. 6.

Histone H1 is hyperphosphorylated in starved ΔTHD1 cells. (A) H1 histones were isolated from core histones, resolved by acid-urea-PAGE, and stained with Coomassie brilliant blue. The numbers represent the number of phosphoryl modifications on the isoforms in each band. (B) CYP1 is not induced by starvation in ΔTHD1 cells. Total RNA from growing and starved wild-type and ΔTHD1 cells was resolved by formaldehyde-agarose gel electrophoresis and visualized by staining with ethidium bromide. RNA transferred to a nylon membrane was probed with a fragment from the CYP1 gene. The 26s rRNA stained with ethidium bromide was used as a loading control. (C) Total cDNA from each strain was used as template in PCRs with CDC2 primers or RAD51 primers as an internal control for template concentration. The amount of template used was previously determined, by dilution experiment, to yield CDC2 or RAD51 amplification products in the linear range (data not shown). G, genomic DNA used as template; WT, wild-type; Δ, THD1.