Background: CHAC1 is associated with the stress response in atherosclerosis. Results: ATF4, ATF3, and CEBP␤ regulate CHAC1 transcription. Human CHAC1 protein overexpression depletes glutathione. Conclusion: CHAC1 is induced following multiple cell stress signals and leads to depletion of glutathione. Significance: CHAC1 may be an essential link between stress signaling and the oxidative status of the cell, contributing to multiple diseases.

Extended heterochromatin domains, which are repressive to transcription and help define centromeres and telomeres, are formed through specific interactions between silencing proteins and nucleosomes. This study reveals that in Saccharomyces cerevisiae, the same nucleosomal surface is critical for the formation of multiple types of heterochromatin, but not for local repression mediated by a related transcriptional repressor. Thus, this region of the nucleosome may be generally important to long-range silencing. In S. cerevisiae, the Sir proteins perform long-range silencing, whereas the Sum1 complex acts locally to repress specific genes. A mutant form of Sum1p, Sum1-1p, achieves silencing in the absence of Sir proteins. A genetic screen identified mutations in histones H3 and H4 that disrupt Sum1-1 silencing and fall in regions of the nucleosome previously known to disrupt Sir silencing and rDNA silencing. In contrast, no mutations were identified that disrupt wild-type Sum1 repression. Mutations that disrupt silencing fall in two regions of the nucleosome, the tip of the H3 tail and a surface of the nucleosomal core (LRS domain) and the adjacent base of the H4 tail. The LRS/H4 tail region interacts with the Sir3p bromo-adjacent homology (BAH) domain to facilitate Sir silencing. By analogy, this study is consistent with the LRS/H4 tail region interacting with Orc1p, a paralog of Sir3p, to facilitate Sum1-1 silencing. Thus, the LRS/H4 tail region of the nucleosome may be relatively accessible and facilitate interactions between silencing proteins and nucleosomes to stabilize long-range silencing.T HE formation of silenced chromatin, or heterochromatin, in eukaryotes is important for proper gene regulation and chromosome stability and helps define centromeres and telomeres. One interesting property of heterochromatin is its capacity to spread along a chromosome to form an extended, repressive domain. This spreading is enabled by specific interactions between silencing proteins and nucleosomes. Consequently, particular surfaces on the nucleosome can be critical to the assembly of heterochromatin. This study reveals that in the budding yeast Saccharomyces cerevisiae, the same nucleosomal surface is critical for the formation of three types of silenced chromatin, mediated by the Sir, RENT, or Sum1-1 complexes, suggesting that this region of the nucleosome may be generally important to long-range silencing.In S. cerevisiae, silenced chromatin domains associated with Sir proteins are found at the cryptic mating-type loci HMLa and HMRa and subtelomeric regions (Rusche et al. 2003). A distinct form of silencing occurs in the rDNA repeats and is mediated by the RENT complex. Strains lacking Sir2p, Sir3p, or Sir4p lose silencing at the mating-type loci and consequently express both a and a mating-type information, resulting in the loss of cell-type identity and an inability to mate. Interestingly, silencing and mating can be restored by the SUM1-1 mutation, which was originally identified as a suppressor of a sir2...

Acetaminophen (APAP) is a commonly used pain reliever and a well‐established, dose‐related hepatotoxin. Hepatotoxicity occurs when metabolism of excessive APAP generates high concentrations of the reactive metabolite N‐acetyl‐p‐benzoquinone imine (NAPQI). NAPQI depletes cellular glutathione (GSH) and forms protein adducts leading to cellular stress, mitochondrial dysfunction, and, ultimately, oncotic necrosis of Zone 3 hepatocytes. The endoplasmic reticulum (ER) stress pathway is important to maintain cellular protein folding abilities in times of stress. However, extended or aberrant ER stress signaling can lead to apoptosis through the PERK‐ATF4‐CHOP pathway. Our research explores the regulation and contribution of CHAC1, an enzyme within this pathway, in response to APAP overdose.As purified mouse and yeast CHAC1 was shown to degrade GSH, the consequences of CHAC1 overexpression on GSH levels in human liver was explored in a human hepatoma cell line (HepG2). Overexpression of CHAC1 by transient transfection leads to a ~50% reduction in total GSH levels. As depletion of GSH is an important step in the pathology of APAP hepatotoxicity, the expression of CHAC1 mRNA in HepG2 cells was examined following 24‐hour treatment with 30mM APAP. CHAC1 mRNA was induced five‐fold by APAP, along with other components of the PERK‐ATF4‐CHOP pathway. Interestingly, overexpression of CHAC1 in HepG2 cells treated with APAP led to increased toxicity versus APAP alone, implicating a role for CHAC1 in response to APAP. Overall, these data implicate a new role for CHAC1 in the pathology of drug‐induced liver injury.

CHAC1 is a gamma‐glutamylcyclotransferase downstream of ATF4 in the Unfolded Protein Response. CHAC1 degrades glutathione, the primary, intracellular antioxidant, and decreases Notch signaling. Notch is the receptor for a well‐conserved signaling pathway that is essential for development and proper maintenance of adult tissues, such as mucle and bone. Due to the importance of glutathione and Notch, it is likely that CHAC1 is critical for cell function and development. Chac1−/− mice were generated to elucidate the effects of Chac1 in vivo; however, knockout of Chac1 is lethal. Interestingly, Chac1+/− mice have decreased body weight, suggesting Chac1 is important in body composition regulation.To dissect the underlying cause for reduced body mass in Chac1+/− mice, four metrics were analyzed: food intake, fat mass, muscle mass, and bone function. Analysis of food intake illustrates that there is no change in food intake; thus, food intake does not account for the decrease in body weight, but does suggest a misregulation of energy balance. Abdominal fat pad weight is not consistently changed at any 6, 13, or 33 weeks of age ages, suggesting no changes in fat mass. Chac1+/− mice of both sexes have decreased gastrocnemius weights at 6 weeks and 33 weeks of age, suggesting decreased muscle mass as the cause of the reduced body weight. Finally, markers of osteoblasts were analyzed in the femur of male and female Chac1+/− mice versus control. Markers for osteoblasts, such as Runx2 and Col1a1, are decreased in Chac1+/− mice at 6 weeks. These data suggest that there is a decrease in osteoblast differentiation and decreased bone density.Overall, these data illustrate that genetic inhibition of Chac1 in mice leads to multiple changes in body composition, including lower muscle mass and changes in bone development, demonstrating the importance of Chac1 in the proper regulation of muscle and bone. Furthermore, Chac1 may play an important role in the progression of muscle and bone pathology in conditions such as osteoporosis, aging, and muscle wasting.Support or Funding InformationNIH #HL09470; COBRE P30GM106392; Louisiana Board of Regents Fellowship #LESQSF(2012‐17)‐GF‐08 to RRC

CHAC1 is a pro‐apoptotic member of the endoplasmic reticulum stress pathway. The yeast homologue (YER163c) is the first known cytosolic protein to actively degrade glutathione (GSH), the most abundant antioxidant in the cell. However, the enzymatic activity of CHAC1 has not been fully characterized in mammalian cells. Herein, we investigate the effects of modulating CHAC1 in vitro on levels of cellular GSH and components of the GSH metabolic pathway in mammalian cells. Additionally, we define a role for CHAC1 in sensitizing cells to oxidative damage following heme treatment. Overexpression of CHAC1 in human and mouse cells leads to a large decrease in total GSH, comparable to treatment with 250uM buthionine sulphoximine, a chemical inhibitor of GSH synthesis. We probed the effects of co‐administration of excess heme, an inducer of intracellular oxidative stress. CHAC1 overexpression increased sensitivity to heme‐mediated toxicity and decreased total GSH. Supplementation with N‐acetyl cysteine, a GSH precursor, alleviated this sensitivity. CHAC1 overexpression and knockdown leads to modulation of the mRNA for the GSH anabolic enzymes as measured by qPCR. Collectively, this evidence suggests that CHAC1 sensitizes cells to oxidative stress due to degradation of GSH and potential feedback in the GSH anabolic pathway. Grant Funding Source: RRC supported by Louisiana Board of Regents Fellowship #LESQSF; INM supported by NIH grant #HL094709