Loss of Sin3/Rpd3 Histone Deacetylase Restores the DNA Damage Response in Checkpoint-Deficient Strains of Saccharomyces cerevisiae

Eukaryotes contain large networks of chromatin remodeling machinery that coordinately regulate gene transcription (19). Chromatin remodeling complexes are commonly grouped into two classes based on their activity: (i) ATP-dependent chromatin remodeling complexes (45, 49) and (ii) histone acetylase and histone deacetylase (HDAC) complexes (23, 43). The second class is made up of protein complexes that modulate the status of histone acetylation, which occurs posttranslationally at lysine residues located at the amino-terminal ends of histones. Acetylation of the lysine residues neutralizes their positive charge, which interferes with the histone-DNA electrostatic interaction (15) and thereby loosens chromatin structure, allowing for a more transcriptionally competent DNA template. Conversely, removal of acetyl groups by HDAC activity restores the positive charge on the histone lysine residue and thereby reestablishes a tight interaction between the histone and DNA. Increased histone acetylation is generally associated with transcriptional activity, whereas decreased acetylation is associated with transcriptional repression. Saccharomyces cerevisiae expresses multiple HDACs, which are encoded by SIR2, HOS1, HOS2, HOS3, HDA1, HST1 to -4, and RPD3. Rpd3 is known to associate into a large ≥2-MDa multiprotein complex that includes Sin3 (21). The Sin3/Rpd3 HDAC complex does not directly bind to DNA but interacts with other factors that target the complex to specific genes. For example, Sin3 mediates interactions with site-specific DNA-binding proteins such as Ume6 to target HDAC activity to specific promoter regions (20, 35). Microarray transcription profiles generated from rpd3Δ and sin3Δ mutants indicate that in each mutant a significant number of genes are similarly up- or down-regulated compared to wild type (3). These data, combined with the genetic evidence that Rpd3 and Sin3 regulate genes in a nonadditive manner, indicate that the proteins function together in the same regulatory pathway (41, 47). Yeast Rpd3 most closely resembles enzymes in the human class I HDAC category, including HDAC1, HDAC2, HDAC3, and HDAC8 (5, 9, 17, 46). Human HDACs are also directed to specific promoter targets through association with an assortment of proteins, including the human homolog of the yeast Sin3 protein. These complexes in turn interact with other proteins that include several tumor suppressor genes and oncogenes involved in cell cycle progression and cancer development (9, 50). For example, HDAC1, -2, and -3 are thought to be important for regulating the G1/S transition in human cells through interaction with Rb, a tumor suppressor gene that represses transcription of E2F-regulated genes. The relationship between HDACs and Rb, along with other examples of HDAC influence on cell proliferation, clearly demonstrates the significance of histone deacetylation in the regulation of the cell cycle in human cells. Eukaryotes also possess well-conserved pathways to monitor genomic stability and respond to DNA damage. These pathways are termed checkpoints and function to delay transitions between the different cell cycle phases in response to damaged DNA and defects in replication and chromosome segregation (14, 53). S. cerevisiae contains at least three conserved checkpoint pathways that elicit cell cycle arrest in response to genotoxic stress, and these pathways differ in both the genes required for their activity and the stimuli to which they respond. While the DNA damage checkpoint responds to insults caused by DNA-damaging agents, the replication and spindle checkpoints react to problems associated with replication and bipolar spindle-kinetochore attachment, respectively. Though these pathways respond to fundamentally different stimuli, recent studies indicate that checkpoint pathways may overlap in their response to damage (12, 26, 29). It is likely that the machinery participating in the different checkpoint responses must localize to DNA, and this localization is likely to be influenced by chromatin structure. Therefore, chromatin status may also play a key role in coordinating checkpoint responses to genotoxic stress. We previously reported the identification of a human cDNA encoding a member of the forkhead/winged helix family of transcription factors, CHES1 (checkpoint suppressor 1; FOXN3), which can function as a high-copy suppressor of the S. cerevisiae G2/M checkpoint mutants rad9, mec1, rad24, rad53, and dun1 (33). Specifically, Ches1 confers increased survival after exposure to UV irradiation, ionizing irradiation, and methylmethane sulfonate (MMS). Furthermore, Ches1 is able to reconstitute a DNA damage-induced G2/M arrest absent in checkpoint-deficient strains, even in a mec1Δ mutant strain, which is normally nonviable. This present study was initiated to identify Ches1 in vivo binding partners. We found that a functional glutathione S-transferase (GST)-Ches1 fusion protein interacts with Sin3, a component of the Sin3/Rpd3 HDAC complex in S. cerevisiae. This led to investigation of the requirement for Sin3 in Ches1-mediated responses. Expression of CHES1 in a sin3Δ strain had no additional effect on sensitivity to DNA damage, and overexpression of SIN3 in cells with the RAD9 checkpoint gene deleted attenuated the Ches1-mediated cell cycle delay in response to DNA damage, which suggests that Ches1 functions to inhibit Sin3 activity. Deletion of either SIN3 or its associated HDAC, RPD3, in rad9 and mec1 DNA damage checkpoint mutant strains restores a DNA damage-inducible cell cycle arrest and increases survival after DNA damage. Restoration of the damage-induced arrest in the HDAC-deficient mutants requires an intact spindle checkpoint pathway, which suggests that the spindle checkpoint can act as a redundant pathway in response to DNA damage to delay cell cycle progression.