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Page 4 of 11 Chang et al. J Cancer Metastasis Treat 2019;5:78 I http://dx.doi.org/10.20517/2394-4722.2019.31

Figure 1. Nhp6 or curaxins can initiate uncoiling of nucleosomal DNA to promote reorganization by FACT. Either Nhp6/HMGB or curaxins
can weaken the histone: DNA contacts near the entry/exit points of the nucleosomal DNA, exposing the binding site in H2A-H2B dimers
[1]
for the acidic/hydrophobic anchor sequences found in the C-terminal domains of both subunits of FACT . The DNA distortion initiates
a stepwise series of binding events by different domains of FACT that ultimately leads to uncoiling of the DNA to make the reorganized
form of the nucleosome. Each step is reversible, providing a pathway for assembly of nucleosomes. Structures of the domains of FACT
[2]
are shown, with the HMGB domain of SSRP1 represented by Nhp6 (modified from Figure 8 ). FACT: facilitates chromatin transcription;
Nhp6: non-histone protein 6; HMGB: high mobility group B; Spt16: suppressor of ty 16; Pob3: polymerase one binding protein 3

[34]
in unwinding of the DNA helix and a change in the DNA topology . In the presence of curaxins, the
nucleosome structure is destabilized in a dose- and time-dependent manner [31,34,39,40] . In cells, CBL0137
does not cause histone loss or nucleosome disassembly at concentrations lower than 3 mmol/L during
a short time period (< 20 min in cancer cells). However, histone loss is detectable in cells treated with
[34]
≥ 3 mmol/L CBL0137, and chromatin structure is destabilized when the concentration is > 5 mmol/L .
Similarly, CBL0137 and CBL0100 cause only minimal disruption of nucleosomes in cell free conditions at
concentrations ≤ 2.5 mmol/L after incubation for several minutes [31,34] , while nucleosomes become unstable
[31]
and are partially disassembled after incubation for a longer time period (> 1 h) .
Although at concentrations ≤ 2.5 mmol/L curaxins only minimally affect nucleosome structure, they
significantly alter the structure of the internucleosomal linker DNA, increasing the average distance
[46]
between the DNA regions entering and exiting a nucleosome . On defined nucleosomal arrays the
change in the structure of linker DNA is translated into a considerable decrease in the probability of
long-range interactions, impacting enhancer-promoter communication (EPC) in vitro. As a result,
enhancer-dependent transcription is strongly inhibited by curaxins; no direct effect on the activity of the
[46]
transcriptional machinery itself was observed . In agreement with the in vitro data, enhancer-dependent
[46]
transcription is preferentially inhibited by curaxins in cells .
Curaxin treatment has been shown to cause FACT (detected by fluorescence microscopy and chromatin
immunoprecipitation) in cancer cells to disperse from transcribed regions and become trapped in other
regions within 1 min [31,34] . This FACT redistribution results in genome-wide changes in FACT-dependent
[31]
transcription profiles . This phenomenon was recapitulated using a highly purified Pol II transcription
[31]
system and positioned mononucleosomes in vitro . Curaxins inhibit human (hFACT) action during
chromatin transcription in vitro by trapping FACT complexes on competitor nucleosomes. Under
conditions where hFACT and curaxins alone had minimal effects on nucleosomes in spFRET assay, they
induced extensive, reversible uncoiling of nucleosomal DNA (nucleosome unfolding or reorganization)
[31]
when combined, demonstrating highly synergistic action . The non-transcribed regions of the genome
are in excess over transcribed regions in a human cell, so FACT trapping by unfolded nucleosomes
[31]
(n-trapping) causes depletion of hFACT from active regions of chromatin . Thus, FACT n-trapping is an
important mechanism behind the short-term curaxin action in cancer cells.