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Page 2 of 11 Chang et al. J Cancer Metastasis Treat 2019;5:78 I http://dx.doi.org/10.20517/2394-4722.2019.31

and centromere function [18,19] . FACT is a heterodimer of the suppressor of Ty 16 (Spt16) protein and the
structure-specific recognition protein 1 (SSRP1) in mammals or the polymerase one binding protein 3
(Pob3) in yeast [1,8,9,20,21] . Unlike human SSRP1, the yeast Pob3 lacks the high mobility group B1 (HMGB1)
DNA-binding domain; however, yeast FACT functionally interacts with the non-histone protein 6 (Nhp6)
that consists of a single HMG box [22-24] . Both Spt16 and SSRP1 are able to interact with H2A/H2B dimers
and H3/H4 tetramers [20,25-29] . FACT is involved in both assembly and disassembly of nucleosomes, as well
as in nucleosome unfolding that is dependent on its interactions with other factors. Single-particle Förster
resonance energy transfer (spFRET) microscopy studies demonstrated that in the presence of Nhp6
protein yeast FACT can induce large-scale, reversible, and ATP-independent unfolding (reorganization)
of individual nucleosomes that involves uncoiling of nucleosomal DNA [30,31] . In contrast, in the absence
[32]
of Nhp6 protein, FACT can stabilize nucleosomes lacking the unstructured histone tails . The Spt16
and SSRP1 subunits of FACT also contain additional intrinsic DNA-binding sites [33,34] . SSRP1 can bind
[35]
the left-handed Z-form of DNA . Notably, SSRP1 is usually phosphorylated by casein kinase 2, which
inhibits its DNA binding activity [36,37] ; the phosphorylated FACT can only bind destabilized nucleosomes or
hexasomes [22,38] .

Curaxins are carbazole-based compounds that intercalate into DNA and alter the physical properties of
both DNA and chromatin without causing structural damage (i.e., phosphodiester bond breaks or chemical
modifications) [34,39,40] . These drugs inhibit the activities of FACT, which has particularly severe consequences
[35]
in cancer cells . The curaxins CBL0137 and CBL0100 have been demonstrated to have broad anticancer
activity in many different models [35,41-45] . Clinical candidate CBL0137 has higher metabolic stability and
[35]
water solubility, and efficiently inhibits growth of various cancers in preclinical models . CBL0100 is
more biologically active but is rarely used in animal studies due to its lower solubility and higher toxicity.
Curaxins were first identified in a screen for small molecule compounds capable of simultaneously
[35]
inhibiting NF-κB, activating p53, and preferentially killing tumor cells . Curaxins induce chromatin
trapping (c-trapping) of FACT in less than 1 min after addition to cells, and strongly inhibit normal human
[35]
FACT activities in vivo . This c-trapping of FACT involves rapid formation of Z-DNA and binding of the
[34]
SSRP1 subunit (z-trapping) , as well as curaxin-dependent nucleosome unfolding resulting in binding of
[31]
FACT to the disrupted nucleosomes (n-trapping) . The anticancer activity of curaxins is highly dependent
on c-trapping of FACT [31,34,35] . However, in addition to the c-trapping and activation of p53, curaxins also
[46]
induce dramatic changes in chromatin structure (e.g., disruption of long-range chromatin interactions ,
see below) and dysregulation of multiple cellular responses, including changes in transcription profiles [31,40]
[47]
and induction of transcription of repeats activates interferon (TRAIN) .

This review focuses on recent studies of the roles of FACT and curaxins in gene expression and genome
structure; other topics have been extensively covered in several excellent recent reviews [3,39,48] .

ROLES OF FACT IN TRANSCRIPTION, REPLICATION, AND DNA DAMAGE REPAIR
FACT is detected at transcribed regions of genes in multiple eukaryotes [40,49-51] . FACT is associated with
both promoters and coding regions of transcribed genes [31,49,52,53] . Insertion of transposons (Ty elements)
[54]
often interferes with the expression of nearby genes in Saccharomyces cerevisiae . Depletion of yeast SPT16
causes cell cycle arrest in early G1 phase due to failure to transcribe cyclin genes, and less severe mutations
cause the Spt- (suppressor of Ty) phenotype due to inappropriate activation of a cryptic promoter in
[54]
Ty1 . Yeast FACT and Nhp6 are also involved in maintaining nucleosome-depleted regions near many
[52]
yeast promoters . The nucleosome unfolding activity of yeast FACT working together with Nhp6 protein
that has been detected in vitro likely plays a role in nucleosome destabilization and/or histone removal at
promoter regions [2,30] . The in vitro studies of chromatin transcription by yeast RNA polymerase II (Pol II)
have determined that FACT can facilitate Pol II transcript elongation through chromatin and nucleosome
survival during this process by transiently interacting with the DNA binding surface of the H2A/H2B