One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently dephosphorylates gammaH2AX in vitro. gammaH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets gammaH2AX after its displacement from DNA. The dephosphorylation of gammaH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.

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The yeast Mec1/Tel1 kinases, ATM/ATR in mammals, coordinate the DNA damage response by phosphorylating proteins involved in DNA repair and checkpoint pathways. Recently, ATP-dependent chromatin remodeling complexes, such as the INO80 complex, have also been implicated in DNA damage responses, although regulatory mechanisms that direct their function remain unknown. Here, we show that the Ies4 subunit of the INO80 complex is phosphorylated by the Mec1/Tel1 kinases during exposure to DNA-damaging agents. Mutation of Ies4's phosphorylation sites does not significantly affect DNA repair processes, but does influence DNA damage checkpoint responses. Additionally, ies4 phosphorylation mutants are linked to the function of checkpoint regulators, such as the replication checkpoint factors Tof1 and Rad53. These findings establish a chromatin remodeling complex as a functional component in the Mec1/Tel1 DNA damage signaling pathway that modulates checkpoint responses and suggest that posttranslational modification of chromatin remodeling complexes regulates their involvement in distinct processes.

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Epithelial-mesenchymal transition (EMT) is an underlying mechanism of tissue fibrosis by generating myofibroblasts, which serve as the primary source of extracellular matrix production from tissue epithelial cells. Recently, it has been suggested that EMT is implicated in immunosuppressive cyclosporine A (CsA)-induced renal fibrosis. In the present study, the potential role of NRF2, which is the master regulator of genes associated with the cellular antioxidant defense system, in CsA-induced EMT-renal fibrosis has been investigated. Pre-treatment of rat tubular epithelial NRK-52E cells with sulforaphane, an activator of NRF2, could prevent EMT gene changes such as the loss of E-cadherin and the increase of α-smooth muscle actin (α-SMA) expression. Conversely, genetic inhibition of NRF2 in these cells aggravated changes in CsA-induced EMT markers. These in vitro observations could be confirmed in vivo: CsA-treatment developed severe renal damage and fibrosis with increased expression of α-SMA in NRF2-deficient mice compared to wild-type mice. NRF2-mediated amelioration of CsA-EMT changes could be accounted in part by the regulation of heme oxygenase-1 (HO-1). CsA treatment increased HO-1 expression in an NRF2-dependent manner in NRK cells as well as murine fibroblasts. Induction of HO-1 by CsA appears to be advantageous by counteracting EMT gene changes: specific increase of HO-1 expression by cobalt protoporphyrin prevented CsA-mediated α-SMA induction, while genetic inhibition of HO-1 by siRNA substantially enhanced α-SMA induction compared to control cells. Collectively, our current results suggest that the NRF2-HO-1 system plays a protective role against CsA-induced renal fibrosis by modulating EMT gene changes.