Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine-and tobaccospecific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17␤-hydroxy-11␤-[4-dimethylamino phenyl]-17␣-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a Ϫ95 to ϩ12 region containing an hepatic nuclear factor 4 (HNF4) ␣ response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4␣-and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4␣ increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4␣ mRNA levels by 4-fold; however, the amount of HNF4␣ nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4␣ to the HNF4-RE and that an interaction of GR and HNF4␣ occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4␣ to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4␣ with an HNF4-RE rather than a glucocorticoid response element.

BackgroundDNA double-strand breaks (DSBs) caused by ionizing radiation or by the stalling of DNA replication forks are among the most deleterious forms of DNA damage. The ability of cells to recognize and repair DSBs requires post-translational modifications to histones and other proteins that facilitate access to lesions in compacted chromatin, however our understanding of these processes remains incomplete. UHRF1 is an E3 ubiquitin ligase that has previously been linked to events that regulate chromatin remodeling and epigenetic maintenance. Previous studies have demonstrated that loss of UHRF1 increases the sensitivity of cells to DNA damage however the role of UHRF1 in this response is unclear.ResultsWe demonstrate that UHRF1 plays a critical role for facilitating the response to DSB damage caused by γ-irradiation. UHRF1-depleted cells exhibit increased sensitivity to γ-irradiation, suggesting a compromised cellular response to DSBs. UHRF1-depleted cells show impaired cell cycle arrest and an impaired accumulation of histone H2AX phosphorylation (γH2AX) in response to γ-irradiation compared to control cells. We also demonstrate that UHRF1 is required for genome integrity, in that UHRF1-depleted cells displayed an increased frequency of chromosomal aberrations compared to control cells.ConclusionsOur findings indicate a critical role for UHRF1 in maintenance of chromosome integrity and an optimal response to DSB damage.

Highlights d MutSb, but not MutSa, interacts directly with SLX4 d MutSb stimulates the resolution of recombination intermediates by SMX d Defects in MutSb lead to the accumulation of unresolved anaphase bridges at mitosis d MutSb is required for efficient Holliday junction resolution and genome stability

Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair. The relationship of this repair defect to disease traits remains unclear, given that crosslink sensitivity is recapitulated in FA mouse models without most of the other disease-related features. Mice deficient in Mus81 are also defective in crosslink repair, yet MUS81 mutations have not been linked to FA. Using mice deficient in both Mus81 and the FA pathway protein FancC, we show both proteins cooperate in parallel pathways, as concomitant loss of FancC and Mus81 triggered cell-type-specific proliferation arrest, apoptosis and DNA damage accumulation in utero. Mice deficient in both FancC and Mus81 that survived to birth exhibited growth defects and an increased incidence of congenital abnormalities. This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity. Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.

No abstract