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.

The Condensin Complex Is Essential for Amitotic Segregation of Bulk Chromosomes, but Not Nucleoli, in the Ciliate Tetrahymena thermophila

“…The cell division cleavage furrow cut into the macronucleus before segregation of DNA was complete, and many cells had extranuclear DNA bodies (data not shown). These "extrusion bodies" resembled those reported for mutants of a class I histone deacetylase (49). Micronuclear mitosis appeared normal (data not shown).…”

Class I Histone Deacetylase Thd1p Promotes Global Chromatin Condensation in Tetrahymena thermophila

“…These events require linker histone H1 for the regulation of specific genes and an HP1-like protein (Hhp1p) (25,26,46,47). Previously, cells at least fivefold reduced in Thd1p protein levels ("⌬THD1" cells) were characterized during logarithmic growth in nutrient-rich medium (66). In the present study, the effect of Thd1p was examined under starvation conditions.…”

“…Unless otherwise noted, for all experiments CU428 cells were grown at 30°C with shaking in 1% (wt/vol) enriched proteose peptone (SPP) (20) liquid medium to mid-logarithmic phase (cell density of 2 ϫ 10 5 to 5 ϫ 10 5 cells/ml). The mutant ⌬THD1 cells otherwise possessed the same genetic background as CU428 (66). ⌬THD1 cells have a partial gene replacement in the polyploid amitotic macronucleus that must be maintained under selection with paromomycin.…”

“…Cells deficient in Thd1p contain higher amounts of macronuclear DNA, large extrusion bodies, and enlarged nucleoli (66). In the present study, we addressed whether Thd1p plays a role in normal global chromatin condensation during cell starvation.…”

“…We previously described Tetrahymena Thd1p, a class I HDAC that is selectively recruited to developing new macronuclei (64) and is important for the integrity of macronuclear chromatin in logarithmically dividing cells (66). Cells deficient in Thd1p contain higher amounts of macronuclear DNA, large extrusion bodies, and enlarged nucleoli (66).…”