Histone Chaperone Facilitates Chromatin Transcription Research Articles

H2B ubiquitination recruits FACT to maintain a stable altered nucleosome state for transcriptional activation

Histone H2B mono-ubiquitination at lysine 120 (ubH2B) has been found to regulate transcriptional elongation by collaborating with the histone chaperone FACT (Facilitates Chromatin Transcription) and plays essential roles in chromatin-based transcriptional processes. However, the mechanism of how ubH2B directly collaborates with FACT at the nucleosome level still remains elusive. In this study, we demonstrate that ubH2B impairs the mechanical stability of the nucleosome and helps to recruit FACT by enhancing the binding of FACT on the nucleosome. FACT prefers to bind and deposit H2A-ubH2B dimers to form an intact nucleosome. Strikingly, the preferable binding of FACT on ubH2B-nucleosome greatly enhances nucleosome stability and maintains its integrity. The stable altered nucleosome state obtained by ubH2B and FACT provides a key platform for gene transcription, as revealed by genome-wide and time-course ChIP-qPCR analyses. Our findings provide mechanistic insights of how ubH2B directly collaborates with FACT to regulate nucleosome dynamics for gene transcription.

Visualizing both nucleosome disassembly and reassembly during FACT chaperone activity.

The histone chaperone FACT (FAcilitates Chromatin Transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (strong site) interactions, altering the energy landscape of unwrapping and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling and DNA unwinding. Single molecule multi-channel fluorescence observes component histones as they disassemble under tension and are lost. Yet if tension is released, reassembly is also observed and surprisingly both loss and reassembly are accelerated by key domains of FACT. We resolve this ambiguity, noting that all histone-DNA kinetics are faster with FACT. Of the individual domains we studied, two were shown to influence nucleosome structure and dynamics with distinctly contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones, and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate how key FACT domains combine to catalyze nucleosome disassembly and reassembly.

Human FACT subunits coordinate to catalyze both disassembly and reassembly of nucleosomes

The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly.

Structural basis of nucleosome disassembly and reassembly by RNAPII elongation complex with FACT.

During gene transcription, RNA polymerase II (RNAPII) traverses nucleosomes in chromatin, but the mechanism has remained elusive. Using cryo-electron microscopy, we obtained structures of the RNAPII elongation complex (EC) passing through a nucleosome in the presence of the transcription elongation factors Spt6, Spn1, Elf1, Spt4/5, and Paf1C and the histone chaperone FACT (facilitates chromatin transcription). The structures show snapshots of EC progression on DNA mediating downstream nucleosome disassembly, followed by its reassembly upstream of the EC, which is facilitated by FACT. FACT dynamically adapts to successively occurring subnucleosome intermediates, forming an interface with the EC. Spt6, Spt4/5, and Paf1C form a "cradle" at the EC DNA-exit site and support the upstream nucleosome reassembly. These structures explain the mechanism by which the EC traverses nucleosomes while maintaining the chromatin structure and epigenetic information.

Chaperoning heterochromatin: new roles of FACT in chromatin silencing

The multitasking histone chaperone FACT (FAcilitates Chromatin Transcription) contributes to actively transcribed euchromatin and repressed heterochromatin. However, its precise role in gene silencing has remained obscure. Here, we discuss new insights into the silent chromatin functions and recruitment mechanisms of FACT, and their possible implications in cell identity and cancer.

Histone chaperone FACT complex coordinates with HIF to mediate an expeditious transcription program to adapt to poorly oxygenated cancers.

Cancer cells adapt to hypoxia through HIFs (hypoxia-inducible factors), which initiate the transcription of numerous genes for cancer cell survival in the hypoxia microenvironment. In this study, we find that the FACT (facilitates chromatin transcription) complex works cooperatively with HIFs to facilitate the expeditious expression of HIF targets for hypoxia adaptation. Knockout (KO) of the FACT complex abolishes HIF-mediated transcription by impeding transcription elongation in hypoxic cancer cells. Interestingly, the FACT complex is post-translationally regulated by PHD/VHL-mediated hydroxylation and proteasomal degradation, in similar fashion to HIF-1/2α. Metabolic tracing confirms that FACT KO suppresses glycolytic flux and impairs lactate extrusion, leading to intracellular acidification and apoptosis in cancer cells. Therapeutically, hepatic artery ligation and anti-angiogenic inhibitors adversely induce intratumoral hypoxia, while co-treatment with FACT inhibitor curaxin remarkably hinders the growth of hypoxic tumors. In summary, our findings suggest that the FACT complex is a critical component of hypoxia adaptation and a therapeutic target for hypoxic tumors.

H2A mono-ubiquitination differentiates FACT’s functions in nucleosome assembly and disassembly

The histone chaperone FACT (FAcilitates Chromatin Transcription) plays an essential role in transcription and DNA replication by its dual functions on nucleosome assembly to maintain chromatin integrity and nucleosome disassembly to destabilize nucleosome and facilitate its accessibility simultaneously. Mono-ubiquitination at Lysine 119 of H2A (ubH2A) has been suggested to repress transcription by preventing the recruitment of FACT at early elongation process. However, up to date, how ubH2A directly affects FACT on nucleosome assembly and disassembly remains elusive. In this study, we demonstrated that the dual functions of FACT are differently regulated by ubH2A. The H2A ubiquitination does not affect FACT’s chaperone function in nucleosome assembly and FACT can deposit ubH2A–H2B dimer on tetrasome to form intact nucleosome. However, ubH2A greatly restricts FACT binding on nucleosome and inhibits its activity of nucleosome disassembly. Interestingly, deubiquitination of ubH2A rescues the nucleosome disassembly function of FACT to activate gene transcription. Our findings provide mechanistic insights of how H2A ubiquitination affects FACT in breaking nucleosome and maintaining its integrity, which sheds light on the biological function of ubH2A and various FACT’s activity under different chromatin states.

The histone chaperone FACT facilitates heterochromatin spreading by regulating histone turnover and H3K9 methylation states.

SUMMARYHeterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks.

Histone chaperone FACT complex inhibitor CBL0137 interferes with DNA damage repair and enhances sensitivity of medulloblastoma to chemotherapy and radiation.

Medulloblastoma (MB) is a malignant pediatric brain tumor with a poor prognosis. Post-surgical radiation and cisplatin-based chemotherapy have been a mainstay of treatment, which often leads to substantial neurocognitive impairments and morbidity, highlighting the need for a novel therapeutic target to enhance the sensitivity of MB tumors to cytotoxic therapies. We performed a comprehensive study using a cohort of 71MB patients' samples and pediatric MB cell lines and found that MB tumors have elevated levels of nucleosome remodeling FACT (FAcilitates Chromatin Transcription) complex and DNA repair enzyme AP-endonuclease1 (APE1). FACT interacts with APE1 and facilitates recruitment and acetylation of APE1 to promote repair of radiation and cisplatin-induced DNA damage. Further, levels of FACT and acetylated APE1 both are correlate strongly with MB patients' survival. Targeting FACT complex with CBL0137 inhibits DNA repair and alters expression of a subset of genes, and significantly improves the potency of cisplatin and radiation in vitro and in MB xenograft. Notably, combination of CBL0137 and cisplatin significantly suppressed MB tumor growth in an intracranial orthotopic xenograft model. We conclude that FACT complex promotes chemo-radiation resistance in MB, and FACT inhibitor CBL0137 can be used as a chemo-radiation sensitizer to augment treatment efficacy and reduce therapy-related toxicity in high-risk pediatric patients.

CBL0137 increases the targeting efficacy of Rovalpituzumab tesirine against tumour-initiating cells in small cell lung cancer

Small cell lung cancer (SCLC) is characterised by high relapse rates. Tumour-initiating cells (TICs) are responsible for drug resistance and recurrence of cancer. Rovalpituzumab tesirine (Rova-T), a potent humanised antibody–drug conjugate, selectively targets delta-like protein 3, which is highly expressed in SCLC TICs. The experimental drug CBL0137 (CBL) inhibits the histone chaperone FACT (facilitates chromatin transcription), which is required for the expression of transcription factors that are essential for TIC maintenance. Rova-T and CBL each target SCLC TICs as single agents. However, acquired or intrinsic resistance to single agents is a major problem in cancer. Therefore, we investigated the potential effect of combining Rova-T and CBL in SCLC to eradicate TICs more effectively. Our preclinical studies report a novel and highly translatable therapeutic strategy of dual targeting TICs using Rova-T in combination with CBL to potentially increase survival of SCLC patients.

The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly invitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

Ultrasensitive Change in Nucleosome Binding by Multiple Phosphorylations to the Intrinsically Disordered Region of the Histone Chaperone FACT

Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation (“ultrasensitive response”). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434–624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.

Targeting Histone Chaperone FACT Complex Overcomes 5-Fluorouracil Resistance in Colon Cancer.

Fluorouracil (5-FU) remains a first-line chemotherapeutic agent for colorectal cancer. However, a subset of colorectal cancer patients who have defective mismatch-repair (dMMR) pathway show resistance to 5-FU. Here, we demonstrate that the efficacy of 5-FU in dMMR colorectal cancer cells is largely dependent on the DNA base excision repair (BER) pathway. Downregulation of APE1, a key enzyme in the BER pathway, decreases IC50 of 5-FU in dMMR colorectal cancer cells by 10-fold. Furthermore, we discover that the facilitates chromatin transcription (FACT) complex facilitates 5-FU repair in DNA via promoting the recruitment and acetylation of APE1 (AcAPE1) to damage sites in chromatin. Downregulation of FACT affects 5-FU damage repair in DNA and sensitizes dMMR colorectal cancer cells to 5-FU. Targeting the FACT complex with curaxins, a class of small molecules, significantly improves the 5-FU efficacy in dMMR colorectal cancer in vitro (∼50-fold decrease in IC50) and in vivo xenograft models. We show that primary tumor tissues of colorectal cancer patients have higher FACT and AcAPE1 levels compared with adjacent nontumor tissues. Additionally, there is a strong clinical correlation of FACT and AcAPE1 levels with colorectal cancer patients' response to chemotherapy. Together, our study demonstrates that targeting FACT with curaxins is a promising strategy to overcome 5-FU resistance in dMMR colorectal cancer patients.

Abstract 1755: Chromatin trapping is a key factor for anticancer cytotoxicity of DNA-binding small molecule drugs

Abstract DNA-targeting small molecules have been widely used for cancer treatment due to their ability to induce DNA damage. The DNA-damaging effect of small molecules depends on chromatin organization, however, the exact mechanism by which small molecules affect the chromatin, as well as the role of chromatin alterations in their biological activity is poorly understood. We previously identified the curaxin CBL0137 which showed a broad anti-cancer activity. CBL0137 is a small molecule which binds DNA but does not cause DNA damage. Rather, the curaxin alters DNA/histone interactions and causes nucleosome disassembly in vitro and in cells, leading to the phenomenon we named "chromatin damage." We have previously shown, that disassembled nucleosomes are bound by the histone chaperone FACT (FAcilitates Chromatin Transcription. We named this massive binding of FACT to different components of disassembled chromatin in curaxin-treated cells “c-trapping”. This finding led us to believe that other small DNA-binding molecules may also have the ability to induce c-trapping and chromatin damage. We selected a set of DNA-targeting agents, representing different modes of DNA binding and inhibition of topoisomerases: inhibitors of topoisomerase cleavage activity, inhibitors of topoisomerases re-ligation, compounds that do not bind DNA directly while form a cleavable complex with DNA and topoisomerases I or II, as well as topoisomerases II catalytic inhibitors that do not bind DNA. We investigated this set of compounds examining their cytotoxicity, and their ability to induce c-trapping, which we detected with western blotting, fluorescent imaging and immunofluorescent staining. We examined their effect on nucleosome stability and chromatin destabilization via micrococcal nuclease assays and recombinant mononucleosomes. DNA - damaging activity of tested compounds was also evaluated. We found that only compounds that directly bind DNA are able to induce c-trapping, which in turn correlates with nucleosome destabilization of tested compounds and their cytotoxicity. We showed that this effect was not dependent on DNA damage. Additionally, we showed that FACT can bind DNA at different stages of nucleosome disassembly, including the linker histone removal followed by core histone's eviction, and negative supercoiling accumulation. This work suggests that cytotoxicity of DNA binding molecules is based not on their direct DNA-damage but on the ability of these compounds to induce the chromatin disruption. Citation Format: Elimelech Nesher, Alfiya Safina, Ieman Aljahdali, Scott Portwood, Eunice Wang, Jianmin Wang, Igor Koman, Katerina Gurova. Chromatin trapping is a key factor for anticancer cytotoxicity of DNA-binding small molecule drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1755.

Abstract 5193: Conditional knockout of histone chaperone FACT in mice: Consequences for normal tissues

Abstract Histone chaperone FAcilitates Chromatin Transcription (FACT) is, a complex of SSRP1 and SPT16, involved in chromatin remodeling during transcription, replication, and DNA repair. FACT is selectively expressed during early stages of development and later exclusively present in the stem cell niches in adult normal tissues. It is expressed in undifferentiated aggressive tumors with poor prognosis.¹ FACT inhibition is not tolerated by most tumor, unlike normal cells. This vulnerability of tumor cells upon FACT inactivation suggests evaluating FACT inhibition as an antitumor therapeutic approach.¹ Since FACT knockout (KO) is embryonically lethal (E3.5),² study of FACT’s function in different non-tumor cells of mammalian organisms was not possible and was mostly studied in single cell organisms and in tumor cell lines. Therefore, to assess the safety of FACT inhibition as an anticancer approach, by understanding its role in normal physiology, we generated a conditional KO of Ssrp1 mouse model using CreERT2-LoxP system.³ We harvested stem cells of different lineages to better understand the role of FACT in normal mouse physiology. Using the hematopoietic stem cells (HSCs) from the bone marrow (BM), we performed BM colony formation assay. We found that FACT inactivation significantly decreased the number of colonies of HSCs. Furthermore, inhibiting FACT significantly increased caspase 3 expression in mouse adipose stem cells (ASCs), resulting in apoptosis and decreased survival. These results prompted us to comprehensively evaluate roles of FACT in stem cell development and survival. To this end, fibroblasts in a conditional knockout mouse were reprogrammed by retroviral vector encoding four pluripotency transcription factors (Oct3/4, Klf4, Sox2 and c-Myc). We have found successful generation of induced pluripotent stem (iPS) cells 14 days after reprogramming. In conclusion, FACT associates with development and/or survival of mouse HSCs and ASCs. Further evaluation using iPS cells is expected to delineate roles of FACT in various stages of stem cell development and thus normal physiology. 1. Garcia H, et al. Cell Rep, 2013 4(1) 2. Cao S, et al. Mol Cel Bio. 2003 23(15) 3. Sandlesh P et al. PLOS ONE. 2018 13(6) Citation Format: Masaki A. Ito, Poorva Sandlesh, Imon Goswami, Laura A. Prendergast, Katerina Gurova. Conditional knockout of histone chaperone FACT in mice: Consequences for normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5193.

The Histone Chaperone FACT Coordinates H2A.X-Dependent Signaling and Repair of DNA Damage

SummarySafeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression.

Abstract 348: Targeting histone chaperone FACT complex and APE1 sensitizes colon cancer cells to chemotherapy

Abstract Apurinic/apyrimidinic (AP) sites are one of the most common types of DNA damage that occur spontaneously and following chemotherapy. Human AP-endonuclease (APE1) is primarily responsible for initiating the repair of AP sites via the highly coordinated DNA base excision repair (BER) pathway. Although the repair of AP sites by APE1 utilizing naked DNA or nucleosomal DNA substrate has been extensively investigated in vitro, till to date, how APE1 repairs AP sites in chromatin in cells via BER pathway remains largely unknown. Chromatin remodeling histone chaperone FACT (facilitates chromatin transcription) complex has been previously shown to mediate nucleosome disruption which allows DNA repair proteins to gain access to damage sites. Its involvement in nucleotide excision repair (NER) pathway has been demonstrated. However, the role of FACT in BER remains unclear. Our lab has recently found that APE1 is acetylated at AP sites damage in chromatin and acetylation enhances the DNA repair activity of APE1. Here, we show that APE1 interacts with histone chaperone complex FACT. Subsequently, we confirmed the interaction of APE1 with FACT complex by Co-IP and immunofluorescence (IF) and found that both subunit of FACT complex, SPT16 and SSRP1, interact with APE1 in the nucleus and in chromatin. By Western blot (WB), IF and co-IP, we found enhanced interaction upon induction of DNA damage. To understand the functional importance of FACT, we downregulated its levels using siRNA and noted increased cell sensitivity upon damage by using MTT and colony formation. The number of AP sites in the genome increased significantly upon FACT knockdown. FRAP data demonstrated increased APE1 turnover time in damage sites after FACT knock-down suggesting the role of FACT in nucleosome disruption in facilitating the APE1 function in BER pathway. Decreasing FACT level led to reduced acetylation level of APE1 at damage sites and impaired DNA repair as evident by comet assay. Additionally, we found elevated levels of AcAPE1 and FACT in colon cancer tissues and multiple cell lines as compared to normal adjacent tissues and cells. We used curaxin which blocks the function of FACT and found increased cell sensitivity to various chemotherapeutic agents in multiple colon cancer cell lines. In nude mice xenograft, combination of curaxin and 5-FU group slowed the growth of tumor to the largest extent as compared to 5-FU and curaxin groups. Together our study suggests that FACT is required for APE1 to gain access to AP sites in the context of chromatin. Interruption of APE1 and FACT interaction exhibits synergistic effect on cell viability and proliferation upon DNA damage with chemotherapeutic drugs. We demonstrate targeting both FACT and BER pathway together as a novel strategy for chemosensitization of colon cancer cells. Citation Format: Heyu Song, Shrabasti Roychoudhury, Pranjal Biswas, Jiping Zeng, Kishor Bhakat. Targeting histone chaperone FACT complex and APE1 sensitizes colon cancer cells to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 348.

The FACT inhibitor CBL0137 Synergizes with Cisplatin in Small-Cell Lung Cancer by Increasing NOTCH1 Expression and Targeting Tumor-Initiating Cells.

Traditional treatments of small-cell lung cancer (SCLC) with cisplatin, a standard-of-care therapy, spare the tumor-initiating cells (TIC) that mediate drug resistance. Here we report a novel therapeutic strategy that preferentially targets TICs in SCLC, in which cisplatin is combined with CBL0137, an inhibitor of the histone chaperone facilitates chromatin transcription (FACT), which is highly expressed in TICs. Combination of cisplatin and CBL0137 killed patient-derived and murine SCLC cell lines synergistically. In response to CBL0137 alone, TICs were more sensitive than non-TICs, in part, because CBL0137 increased expression of the tumor suppressor NOTCH1 by abrogating the binding of negative regulator SP3 to the NOTCH1 promoter, and in part because treatment decreased the high expression of stem cell transcription factors. The combination of cisplatin and CBL0137 greatly reduced the growth of a patient-derived xenograft in mice and also the growth of a syngeneic mouse SCLC tumor. Thus, CBL0137 can be a highly effective drug against SCLC, especially in combination with cisplatin.Significance: These findings reveal a novel therapeutic regimen for SCLC, combining cisplatin with an inhibitor that preferentially targets tumor-initiating cells. Cancer Res; 78(9); 2396-406. ©2018 AACR.

Functional roles of the DNA-binding HMGB domain in the histone chaperone FACT in nucleosome reorganization

The essential histone chaperone FACT (facilitates chromatin transcription) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB-domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA-binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization in vitro, indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic in vivo Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone-binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA-bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.

Prognostic value of histone chaperone FACT subunits expression in breast cancer.

Understanding the underlying reasons for tumor aggressiveness, such as why some tumors grow slowly and locally, while others rapidly progress to a lethal metastatic disease, is still limited. This is especially critical in breast cancer (BrCa) due to its high prevalence and also due to the possibility that it can be detected early. Several oncogenes and tumor suppressors have been identified and are used in the prognosis and treatment of BrCa. However, even with these markers, the outcome within BrCa subtypes is highly variable. Chromatin organization has long been acknowledged as a factor that plays an important role in tumor progression, but molecular mechanisms defining chromatin dynamics are largely missing. We have recently found that histone chaperone FACT (facilitates chromatin transcription) is overexpressed in ~18–20% of BrCa cases. FACT is elevated upon transformation of mammary epithelial cells and is essential for viability of tumor cells. BrCa cells with high FACT have a more aggressive transcriptional program than those with low FACT cells. Based on this we propose that FACT may be a marker of aggressive BrCa. In this study, we aimed to comprehensively characterize the pattern of FACT expression in BrCa in relation to other molecular and clinical prognostic markers. We developed and tested an assay for the detection and quantitation of protein levels of both FACT subunits, SSRP1, and SPT16, in clinical samples. We compared the value of mRNA and protein as potential markers of disease aggressiveness using a large cohort of patients (n=1092). We demonstrated that only SSRP1 immunohistochemical staining is a reliable indicator of FACT levels in tumor samples. High SSRP1 correlated with known markers of poor prognosis, such as negative hormone receptor status, presence of Her2, high-grade tumors, and tumors of later clinical stage. At the same time, no strong correlation between SSRP1 expression and survival was detected when all samples were analyzed together. Clear trend toward longer survival of patients with low or no SSRP1 expression in tumor samples was seen in several subgroups of patients, and most importantly significant association of high SSRP1 expression with shorter disease-free survival was detected in patients with early-stage and low-grade BrCa, the category of patients with the highest demand in predictive marker of disease progression.