Exploring the Mechanism of Action of Curaxins: Chromatin Dynamics and the Role of FACT

Novel DNA binding small molecules, curaxins, have potent anti-cancer activity in the absence of DNA damage. This activity has been linked to the histone chaperone dimer Facilitates Chromatin Transcription (FACT), which strongly binds chromatin as a result of drug treatment. This strong binding has been termed c-trapping. The specific mechanism of c-trapping however is unknown. This work therefore examined the effect of curaxins on DNA, chromatin, and FACT. Our work reveals that intercalation of curaxins in DNA induces loss of nucleosomes from chromatin, chromatin decompaction, negative supercoiling, and inhibition of FACT histone chaperone function. Next we demonstrate that c-trapping occurs as a result of FACT subunit Structure Specific Recognition Protein 1 (SSRP1) binding to chromatin upon curaxin treatment. This binding is mediated via the extreme c-terminal intrinsically disordered domain (CID) of SSRP1. We've also observe that SSRP1 CID domain is important in curaxin induced p53 response. Analysis of SSRP1 bound genomic regions in curaxin treated cells identified using ChIP-sequencing reveals a binding preference of SSRP1 for non-coding regions in the genome enriched for repetitive stretches of dinucleotide (ApC/ TpG) repeats. These repeats are prone to forming non-B DNA or alternative DNA structures (ADS). We then verify the formation of Z-DNA with CBL0137 and cruciform DNA with CBL0100 treatment, and finally that SSRP1 binds these structures in vivo and in vitro. Thus, we believe that c-trapping is binding of SSRP1 to these ADS.;This study demonstrates that curaxin treatment causes changes in cellular chromatin which results in the accumulation of alternative DNA structures, c-trapping of FACT via the CID domain of SSRP1, and p53 activation. Our observations concerning the role of FACT in c-trapping indicate that FACT may play a role in the recognition of non-B DNA in the absence of DNA damage, to prevent DNA damage and ultimately the development of cancer.