TonEBP/OREBP is part of a large protein complex 
. Many proteins in this complex are known contribute to regulation of TonEBP/OREBP, including PKAcs 
, PARP1 
, Jun, Fos 
, RHA 
and ATM 
. In the present studies we find that MDC1 also is physically associated with TonEBP/OREBP (, and ) and contributes to its activation by high NaCl (). The complex containing TonEBP/OREBP and MDC1 apparently is preassembled since MDC1 coimmunoprecipitates with TonEBP/OREBP whether NaCl is elevated or not (), as do other TonEBP/OREBP-associated proteins 
. Evidently, high NaCl is not required for assembly of the large complex containing TonEBP/OREBP and the proteins that contribute to its osmotic regulation.
MDC1 works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks 
. High NaCl increases DNA breaks 
and alters chromosome structure 
. Several other DNA damage response proteins also physically associate with TonEBP/OREBP, including DNA-PKcs (PRKDC) 
, Ku86 (XRCC5) 
, ATM 
, and PARP1 
. Further, ATM 
, and MDC1 (present studies) all contribute to regulation of high NaCl-induced activation of TonEBP/OREBP. ATM is activated by high NaCl (determined from increased phosphorylation at S1981) and contributes to the high NaCl-induced increases of TonEBP/OREBP transcriptional activity 
, transactivating activity 
, and nuclear localization 
. Although MDCI contributes to high NaCl-induced increase in TonEBP/OREBP transcriptional () and transactivating activity (), it does not contribute to the nuclear localization (). ATM is a critical DNA repair protein, consistent with its activation in direct response to DNA damage. However, it may not be the DNA damage, itself, but the associated changes in chromatin that activate ATM in response to IR 
and high NaCl.
High NaCl inhibits repair of DNA breaks caused by ultraviolet radiation (UV) 
, as well as the breaks caused by the high NaCl, itself 
. Although those breaks are not repaired as long as NaCl remains high, they are rapidly repaired when the NaCl is lowered 
. Further, the response of many damage response proteins to high NaCl-induced DNA breaks differs from their response to IR or UV. High NaCl, like IR or UV, activates ATM 
, but it does not induce γH2AX (phosphorylated histone H2AX) 
unless the level of salt is raised enough to kill the cells by apoptosis 
. Also, high NaCl does not induce formation of MRN foci, composed of M
AD50 and N
, and it reversibly inhibits induction of γH2AX by UV or IR 
MDC1 becomes partially immobilized to chromatin and recruits other DNA damage response proteins to the sites of DNA damage during repair of DNA breaks caused by IR 
. In contrast, although high NaCl increases DNA breaks, it reduces the fraction of MDC1 bound to chromatin (). A critical difference is that high NaCl inhibits DNA repair 
, inhibiting activation of DNA repair proteins, like γH2AX and reducing recruitment of the MRN complex to foci at the breaks 
. We propose that exclusion of MDC1 from chromatin contributes to the inhibition by high NaCl of repair of DNA breaks. On the other hand, high NaCl-induced DNA breaks are rapidly repaired when NaCl is lowered 
. The repair is accompanied by activation of DNA damage response proteins, like γH2AX, MRE11, and Chk1
. Accompanying that repair, the fraction of MDC1 bound to chromatin also increases dramatically (). We suggest that much of the increased binding of MDC1 occurs at the sites of DNA damage and contributes to their repair.
In so far as osmotic regulation of TonEBP/OREBP by MDC1 depends on their physical association and the transcriptional activity of TonEBP/OREBP depends on its binding to specific DNA elements 
the high NaCl-induced reduction of the fraction of MDC1 that is bound to chromatin () appears paradoxical. However, recall that MDC1 has at least two roles; it is both a DNA damage response protein and an osmotic regulator of TonEBP/OREBP. High NaCl inhibits DNA repair and reduces binding to DNA of repair proteins, including MDC1 (see above). Nevertheless, that leaves a substantial amount of MDC1 still bound to chromatin (), and the association of MDC1 with TonEBP/OREBP does remain intact (). We suggest that high NaCl-induced solubilization of chromatin-bound MDC1 could occur from pools not associated with TonEBP/OREBP, leaving MDC1 still associated with the TonEBP/OREBP bound to its specific DNA elements. On the other hand, regulation of TonEBP/OREBP by MDC1 does not necessarily require that they are bound together to DNA. TonEBP and MDC1 interact in the soluble fraction from the cells (), and TonEBP/OREBP that is activated in the soluble fraction could subsequently bind to chromatin where it could activate transcription. An additional consideration is that release of MDC1 from chromatin near TonEBP/OREBP DNA elements could promote access of TonEBP/OREBP to those elements. The elements could be masked by proteins like MDC1, MRE11, Nbs1, and Rad51, that are released from chromatin when NaCl is elevated 
. Also, release of MDC1 from chromatin could change chromatin conformation in a way specific for high NaCl.
In so far as DNA damage and/or changes in chromatin promote the role of ATM 
and MDC1 in high NaCl-induced activation of TonEBP/OREBP, those changes might serve as sensors for hypertonicity. In this context it would be interesting to know whether DNA-PK and Ku86, which also physically associate with TonEBP/OREBP 
, have a similar role. Expression of Ku86, is already known to provide osmoprotection by an additional mechanism. It reduces high NaCl-induced chromosomal breakage, presumably by bridging broken DNA ends 
. IR and UV do not directly activate TonEBP 
, so, any role of DNA damage in activating TonEBP/OREBP requires the context of hypertonicity.
In conclusion, we find the hypothesis attractive that high NaCl-induced increase in DNA breaks or alteration of chromatin structure provides a signal for the activation of TonEBP/OREBP through DNA damage response proteins, but we recognize that direct link between changes in DNA integrity and/or chromatin structure induced by high NaCl and activation of TonEBP/OREBP remains conjectural.