FACT is a sensor of DNA torsional stress in eukaryotic cells

Abstract

Transitions of B-DNA to alternative DNA structures (ADS) can be triggered by negative torsional strain, which occurs during replication and transcription, and may lead to genomic instability. However, how ADS are recognized in cells is unclear. We found that the binding of candidate anticancer drug, curaxin, to cellular DNA results in uncoiling of nucleosomal DNA, accumulation of negative supercoiling and conversion of multiple regions of genomic DNA into left-handed Z-form. Histone chaperone FACT binds rapidly to the same regions via the SSRP1 subunit in curaxin-treated cells. In vitro binding of purified SSRP1 or its isolated CID domain to a methylated DNA fragment containing alternating purine/pyrimidines, which is prone to Z-DNA transition, is much stronger than to other types of DNA. We propose that FACT can recognize and bind Z-DNA or DNA in transition from a B to Z form. Binding of FACT to these genomic regions triggers a p53 response. Furthermore, FACT has been shown to bind to other types of ADS through a different structural domain, which also leads to p53 activation. Thus, we propose that FACT acts as a sensor of ADS formation in cells. Recognition of ADS by FACT followed by a p53 response may explain the role of FACT in DNA damage prevention.

Potent antileukemic activity of curaxin CBL0137 against MLL‐rearranged leukemia

“…10 Despite many clinical trials, over the past 20 years, no real improvements have been made regarding outcome or prognosis for patients with MLL-r leukemia. 13,[20][21][22] Here, we confirm that CBL0137 induced p53 pathway activation and an IFN response in MLL-r leukemia models in vivo. In our study, we demonstrated that the curaxin CBL0137 has antileukemic effects in preclinical in vitro and in vivo models of MLL-r leukemia at doses that are well tolerated in mice.…”

“…[45][46][47] In addition, as CBL0137 can act through a number of anticancer mechanisms, including inhibition of the NFκB pathway, activation of the p53 pathway, c-trapping of FACT and induction of an IFN response, 13,[20][21][22] it is possible that the CBL0137-induced inhibition of cancer cell growth is mediated by different mechanisms in different cancer cells. It is possible that in vitro, the anticancer action of CBL0137 is predominantly mediated through its FACT-dependent action of NFκB pathway inhibition, and not through induction of an IFN response.…”

“…[13][14][15][16][17][18][19] CBL0137 acts through a novel, nongenotoxic anticancer mechanism that is based on inducing chromatin disassembly in cells without causing DNA damage, thereby triggering several pathways with anticancer effects. 20 The CBL0137-induced nucleosome disassembly triggers increased binding of the histone chaperone Facilitates Chromatin Transcription (FACT) to chromatin through a process termed chromatin-trapping (C-trapping). 13,21,22 FACT is involved in chromatin remodeling during transcription, replication and DNA damage repair and has been shown to drive in vitro cell transformation.…”

Evidence for the nuclear import of histones H3.1 and H4 as monomers

“…Fixing and imaging cells 30 min after rapamycin addition showed that nuclei positive for PCNA foci were also positive for H3.1‐EGFP foci, whereas nuclei negative for PCNA foci were also negative for H3.1‐EGFP foci (Fig B). Cells absent of PCNA foci, and by deduction not replicating their genomes, demonstrated a nucleolar enrichment of H3.1‐EGFP, which has previously been shown to be an artefact of excess soluble histones in the nucleus (Musinova et al , ; Safina et al , ).…”

Getting down to the FACT: therapeutic targeting of MYC-dependent tumors

“…CBL0137 binds to DNA but, unlike conventional DNA intercalating agents, it is non-mutagenic and hence was shown not to induce a DNA damage response (3). Instead, CBL0137 binding to DNA causes a prevalence of alternative DNA structures and destabilized nucleosomes which FACT recognizes and consequentially becomes trapped at (4). This leads to cell death via FACT-dependent activation of p53 as well as downregulation of a subset of NF-κB-dependent genes (3).…”