Evolutionary Landscape of SOX Genes to Inform Genotype-to-Phenotype Relationships.

The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype-phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG (High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood-brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box associated with cardiovascular complications within European populations, and SOX7 A379V (rs143587868) within an SOXF conserved far C-terminal domain heterozygous in 0.716% of African individuals with associated eye phenotypes. This SOX data compilation builds a robust genotype-to-phenotype association for a gene family through more robust ortholog data integration.

Hmo1 Protein Affects the Nucleosome Structure and Supports the Nucleosome Reorganization Activity of Yeast FACT.

Yeast Hmo1 is a high mobility group B (HMGB) protein that participates in the transcription of ribosomal protein genes and rDNA, and also stimulates the activities of some ATP-dependent remodelers. Hmo1 binds both DNA and nucleosomes and has been proposed to be a functional yeast analog of mammalian linker histones. We used EMSA and single particle Förster resonance energy transfer (spFRET) microscopy to characterize the effects of Hmo1 on nucleosomes alone and with the histone chaperone FACT. Hmo1 induced a significant increase in the distance between the DNA gyres across the nucleosomal core, and also caused the separation of linker segments. This was opposite to the effect of the linker histone H1, which enhanced the proximity of linkers. Similar to Nhp6, another HMGB factor, Hmo1, was able to support large-scale, ATP-independent, reversible unfolding of nucleosomes by FACT in the spFRET assay and partially support FACT function in vivo. However, unlike Hmo1, Nhp6 alone does not affect nucleosome structure. These results suggest physiological roles for Hmo1 that are distinct from Nhp6 and possibly from other HMGB factors and linker histones, such as H1.

The histone chaperone FACT: a guardian of chromatin structure integrity.

The identification of FACT as a histone chaperone enabling transcription through chromatin in vitro has strongly shaped how its roles are envisioned. However, FACT has been implicated in essentially all aspects of chromatin biology, from transcription to DNA replication, DNA repair, and chromosome segregation. In this review, we focus on recent literature describing the role and mechanisms of FACT during transcription. We highlight the prime importance of FACT in preserving chromatin integrity during transcription and challenge its role as an elongation factor. We also review evidence for FACT's role as a cell-type/gene-specific regulator of gene expression and briefly summarize current efforts at using FACT inhibition as an anti-cancer strategy.

Electron microscopy analysis of ATP-independent nucleosome unfolding by FACT.

FACT is a histone chaperone that participates in nucleosome removal and reassembly during transcription and replication. We used electron microscopy to study FACT, FACT:Nhp6 and FACT:Nhp6:nucleosome complexes, and found that all complexes adopt broad ranges of configurations, indicating high flexibility. We found unexpectedly that the DNA binding protein Nhp6 also binds to the C-terminal tails of FACT subunits, inducing more open geometries of FACT even in the absence of nucleosomes. Nhp6 therefore supports nucleosome unfolding by altering both the structure of FACT and the properties of nucleosomes. Complexes formed with FACT, Nhp6, and nucleosomes also produced a broad range of structures, revealing a large number of potential intermediates along a proposed unfolding pathway. The data suggest that Nhp6 has multiple roles before and during nucleosome unfolding by FACT, and that the process proceeds through a series of energetically similar intermediate structures, ultimately leading to an extensively unfolded form.

Structural insights into multifunctionality of human FACT complex subunit hSSRP1.

Human structure-specific recognition protein 1 (hSSRP1) is an essential component of the facilitates chromatin transcription complex, which participates in nucleosome disassembly and reassembly during gene transcription and DNA replication and repair. Many functions, including nuclear localization, histone chaperone activity, DNA binding, and interaction with cellular proteins, are attributed to hSSRP1, which contains multiple well-defined domains, including four pleckstrin homology (PH) domains and a high-mobility group domain with two flanking disordered regions. However, little is known about the mechanisms by which these domains cooperate to carry out hSSRP1's functions. Here, we report the biochemical characterization and structure of each functional domain of hSSRP1, including the N-terminal PH1, PH2, PH3/4 tandem PH, and DNA-binding high-mobility group domains. Furthermore, two casein kinase II binding sites in hSSRP1 were identified in the PH3/4 domain and in a disordered region (Gly617-Glu709) located in the C-terminus of hSSRP1. In addition, a histone H2A-H2B binding motif and a nuclear localization signal (Lys677‒Asp687) of hSSRP1 are reported for the first time. Taken together, these studies provide novel insights into the structural basis for hSSRP1 functionality.

HMGs as rheostats of chromosomal structure and cell proliferation.

High mobility group proteins (HMGs) are the most abundant nuclear proteins next to histones and are robustly expressed across tissues and organs. HMGs can uniquely bend or bind distorted DNA, and are central to such processes as transcription, recombination, and DNA repair. However, their dynamic association with chromatin renders capturing HMGs on chromosomes challenging. Recent work has changed this and now implicates these factors in spatial genome organization. Here, I revisit older and review recent literature to describe how HMGs rewire spatial chromatin interactions to sustain homeostasis or promote cellular aging. I propose a 'rheostat' model to explain how HMG-box proteins (HMGBs), and to some extent HMG A proteins (HMGAs), may control cellular aging and, likely, cancer progression.

Analysis of the P53N a Novel Protein Encoded on Chromosome 22q12.1-12.3 in Glioblastomas and Ependymomas Specimens.

The P53N gene maps precisely to human chromosome sub-band 22q12.1-12.3, a region where loss of heterozygosity has been reported in 30% of astrocytic tumors and associated with progression to anaplasia. Moreover, a putative tumor suppressor gene has been indicated on 22q11 region involved in pathogenesis of ependymal tumors. Our objectives to examine the expression level of novel membrane-associated protein (termed P53N) encoded by a novel human gene on chromosome 22q12.1-12.3 in glioblastomas and ependymomas. Serial analysis of gene expression (SAGE) and immunofluorescence analysis of the P53N in the brain tumor tissues were performed. Our analysis revealed that there was high expression of the P53N mRNA in brain ependymoma and brain well-differentiated astrocytoma libraries. The P53N protein. P53N protein contains a high mobility group (HMG) domain at amino acid positions 301 to 360 expressed highly in glioblastoma and ependymoma specimens. Anti-P53N carboxyl-terminal peptide antibody localized the P53N protein to the cytoplasmic membranes of protoplasmic astrocytes in the glioblastoma and ependymoma specimens. These results are in good agreement with the SAGE analysis and the predicted transmembrane topology for the P53N protein and support a possible transmembrane model in which the P53N contains a predicted transmembrane region with its amino terminus localized to the inside of the cytoplasmic membrane.

Regulation of chromatin structure and function: insights into the histone chaperone FACT.

In eukaryotic cells, changes in chromatin accessibility are necessary for chromatin to maintain its highly dynamic nature at different times during the cell cycle. Histone chaperones interact with histones and regulate chromatin dynamics. Facilitates chromatin transcription (FACT) is an important histone chaperone that plays crucial roles during various cellular processes. Here, we analyze the structural characteristics of FACT, discuss how FACT regulates nucleosome/chromatin reorganization and summarize possible functions of FACT in transcription, replication, and DNA repair. The possible involvement of FACT in cell fate determination is also discussed.Abbreviations: FACT: facilitates chromatin transcription, Spt16: suppressor of Ty16, SSRP1: structure-specific recognition protein-1, NTD: N-terminal domain, DD: dimerization domain, MD: middle domain, CTD: C-terminus domain, IDD: internal intrinsically disordered domain, HMG: high mobility group, CID: C-terminal intrinsically disordered domain, Nhp6: non-histone chromosomal protein 6, RNAPII: RNA polymerase II, CK2: casein kinase 2, AID: acidic inner disorder, PIC: pre-initiation complex, IR: ionizing radiation, DDSB: DNA double-strand break, PARlation: poly ADP-ribosylation, BER: base-excision repair, UVSSA: UV-stimulated scaffold protein A, HR: homologous recombination, CAF-1: chromatin assembly factor 1, Asf1: anti-silencing factor 1, Rtt106: regulator of Ty1 transposition protein 106, H3K56ac: H3K56 acetylation, KD: knock down, SETD2: SET domain containing 2, H3K36me3: trimethylation of lysine36 in histone H3, H2Bub: H2B ubiquitination, iPSCs: induced pluripotent stem cells, ESC: embryonic stem cell, H3K4me3: trimethylation of lysine 4 on histone H3 protein subunit, CHD1: chromodomain protein.

The role of FACT in managing chromatin: disruption, assembly, or repair?

FACT (FAcilitates Chromatin Transcription) has long been considered to be a transcription elongation factor whose ability to destabilize nucleosomes promotes RNAPII progression on chromatin templates. However, this is just one function of this histone chaperone, as FACT also functions in DNA replication. While broadly conserved among eukaryotes and essential for viability in many organisms, dependence on FACT varies widely, with some differentiated cells proliferating normally in its absence. It is therefore unclear what the core functions of FACT are, whether they differ in different circumstances, and what makes FACT essential in some situations but not others. Here, we review recent advances and propose a unifying model for FACT activity. By analogy to DNA repair, we propose that the ability of FACT to both destabilize and assemble nucleosomes allows it to monitor and restore nucleosome integrity as part of a system of chromatin repair, in which disruptions in the packaging of DNA are sensed and returned to their normal state. The requirement for FACT then depends on the level of chromatin disruption occurring in the cell, and the cell's ability to tolerate packaging defects. The role of FACT in transcription would then be just one facet of a broader system for maintaining chromatin integrity.

In vitro studies on non-canonical DNA binding specificities of KAP6 and HMO1 and mechanistic insights into DNA bound and unbinding dynamics of KAP6.

High mobility group box (HMGB) members are DNA binding proteins with varied functions present across kingdoms. The mechanism by which HMGBs with varying number of HMG boxes are able to carry out similar functions, are poorly understood. Moreover, how non-canonical DNAs are recognized by HMGB proteins is not clear. To address these, we carried out detailed biochemical and computational studies to characterize two HMGB members- Kinetoplast associated protein (KAP6) of Trypanosoma and High mobility group protein 1 (HMO1) from yeast. Here, we report that KAP6 binds non-canonical DNAs tighter than B-form DNA. Among non-canonical DNAs, KAP6 has the highest affinity for splayed and flap structures, but least for Holliday Junction (HJ). In contrast, HMO1 binds tighter to HJ. Computational analysis show that the secondary structural elements involved in DNA interaction are conserved in HMGB members KAP6 and mitochondrial transcription factor A. Simulation analyses revealed that the ~90° bend in DNA induced by KAP6 HMG box is a result of two ~45° bends, by Helix 1 and Helix 2 of the protein. Our data also suggests that the orthologs of HMO1 and KAP6 are oligomers in solution, which could be necessary for their functioning such as DNA bending and looping.

Histone chaperone FACT FAcilitates Chromatin Transcription: mechanistic and structural insights.

The histone chaperone FACT (FAcilitates Chromatin Transcription) modulates nucleosome structure during many processes requiring access to chromatinized DNA. Here, we review recent biochemical and structural insight into how FACT acts in both nucleosome assembly and disassembly, to maintain chromatin structure while DNA is being worked on by polymerases.

Transcription-facilitating histone chaperons interact with genomic and synthetic G4 structures.

Affinity for G-quadruplex (G4) structures may be a common feature of transcription-facilitating histone chaperons (HCs). This assumption is based on previous unmatched studies of HCs FACT, nucleolin (NCL), BRD3, and ATRX. We verified this assumption and considered its implications for the therapeutic applications of synthetic (exogenous) G4s and the biological significance of genomic G4s. First, we questioned whether exogenous G4s that recognize cell-surface NCL and could trap other HCs in the nucleus are usable as anticancer agents. We performed in vitro binding assays and selected leading multi-targeted G4s. They exhibited minor effects on cell viability. The presumed NCL-regulated intracellular transport of G4s was inefficient or insufficient for tumor-specific G4 delivery. Next, to clarify whether G4s in the human genome could recruit HCs, we compared available HC ChIP-seq data with G4-seq/G4-ChIP-seq data. Several G4s, including the well-known c-Myc quadruplex structure, were found to be colocalized with HC occupancy sites in cancer cell lines. As evidenced by our molecular modeling data, c-Myc G4 might interfere with the HC function of BRD3 but is unlikely to prevent the BRD3-driven assembly of the chromatin remodeling complex. The c-Myc case illustrates the intricate role of genomic G4s in chromatin remodeling, nucleosome remodeling, and transcription.

The "adductome": A limited repertoire of adducted proteins in human cells.

Proteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that "RADAR-MS" can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCRF-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r = 0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins - the "adductome" - is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer.

FACT caught in the act of manipulating the nucleosome.

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone known as 'facilitates chromatin transcription' (FACT1) (consisting of subunits SPT16 and SSRP1) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication and DNA repair2. However, the mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-electron-microscopic structures of human FACT in complex with partially assembled subnucleosomes, with supporting biochemical and hydrogen-deuterium exchange data. We find that FACT is engaged in extensive interactions with nucleosomal DNA and all histone variants. The large DNA-binding surface on FACT appears to be protected by the carboxy-terminal domains of both of its subunits, and this inhibition is released by interaction with H2A-H2B, allowing FACT-H2A-H2B to dock onto a complex containing DNA and histones H3 and H4 (ref. 3). SPT16 binds nucleosomal DNA and tethers H2A-H2B through its carboxy-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A-H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating removal of the H2A-H2B dimer, stabilizing intermediate subnucleosomal states and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.

Computer-Aided Discovery of Small Molecule Inhibitors of Thymocyte Selection-Associated High Mobility Group Box Protein (TOX) as Potential Therapeutics for Cutaneous T-Cell Lymphomas.

Cutaneous T-cell lymphomas (CTCL) are the most common primary lymphomas of the skin. We have previously identified thymocyte selection-associated high mobility group (HMG) box protein (TOX) as a promising drug target in CTCL; however, there are currently no small molecules able to directly inhibit TOX. We aimed to address this unmet opportunity by developing anti-TOX therapeutics with the use of computer-aided drug discovery methods. The available NMR-resolved structure of the TOX protein was used to model its DNA-binding HMG-box domain. To investigate the druggability of the corresponding protein-DNA interface on TOX, we performed a pilot virtual screening of 200,000 small molecules using in silico docking and identified 'hot spots' for drug-binding on the HMG-box domain. We then performed a large-scale virtual screening of 7.6 million drug-like compounds that were available from the ZINC15 database. As a result, a total of 140 top candidate compounds were selected for subsequent in vitro validation. Of those, 18 small molecules have been characterized as selective TOX inhibitors.

The nucleosome acidic patch directly interacts with subunits of the Paf1 and FACT complexes and controls chromatin architecture in vivo.

The nucleosome core regulates DNA-templated processes through the highly conserved nucleosome acidic patch. While structural and biochemical studies have shown that the acidic patch controls chromatin factor binding and activity, few studies have elucidated its functions in vivo. We employed site-specific crosslinking to identify proteins that directly bind the acidic patch in Saccharomyces cerevisiae and demonstrated crosslinking of histone H2A to Paf1 complex subunit Rtf1 and FACT subunit Spt16. Rtf1 bound to nucleosomes through its histone modification domain, supporting its role as a cofactor in H2B K123 ubiquitylation. An acidic patch mutant showed defects in nucleosome positioning and occupancy genome-wide. Our results provide new information on the chromatin engagement of two central players in transcription elongation and emphasize the importance of the nucleosome core as a hub for proteins that regulate chromatin during transcription.

[HMGB Proteins as DNA Chaperones That Modulate Chromatin Activity].

HMGB proteins are involved in structural rearrangements caused by regulatory chromatin remodeling factors. Particular interest is attracted to a DNA chaperone mechanism, suggesting that the HMGB proteins introduce bends into the double helix, thus rendering DNA accessible to effector proteins and facilitating their activity. The review discusses the role that the HMBG proteins play in key intranuclear processes, including assembly of the preinitiation complex during transcription of ribosomal genes; transcription by RNA polymerases I, II, and III; recruitment of the SWI/SNF complex during transcription of nonribosomal genes; DNA repair; etc. The functions of the HMGB proteins are considered in detail with the examples of yeast HMO1 and NHP6. The two proteins possess unique features in adition to properties characteristic of the HMGB proteins. Thus, NHP6 stimulates a large-scale ATP-independent unwrapping of nucleosomal DNA by the FACT complex, while in its absence FACT stabilizes the nucleosome. HMO1 acts as an alternative linker histone. Both HMO1 and NHP6 are of applied interest primarly because they are homologs of human HMGB1, an important therapeutic target of anticancer and anti-inflammatory treatments.

Structure-specific recognition protein-1 (SSRP1) is an elongated homodimer that binds histones.

The histone chaperone complex facilitates chromatin transcription (FACT) plays important roles in DNA repair, replication, and transcription. In the formation of this complex, structure-specific recognition protein-1 (SSRP1) heterodimerizes with suppressor of Ty 16 (SPT16). SSRP1 also has SPT16-independent functions, but how SSRP1 functions alone remains elusive. Here, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) techniques, we characterized human SSRP1 and that from the amoeba Dictyostelium discoideum and show that both orthologs form an elongated homodimer in solution. We found that substitutions in the SSRP1 pleckstrin homology domain known to bind SPT16 also disrupt SSRP1 homodimerization. Moreover, AUC and SAXS analyses revealed that SSRP1 homodimerization and heterodimerization with SPT16 (resulting in FACT) involve the same SSRP1 surface, namely the PH2 region, and that the FACT complex contains only one molecule of SSRP1. These observations suggest that SSRP1 homo- and heterodimerization might be mutually exclusive. Moreover, isothermal titration calorimetry analyses disclosed that SSRP1 binds both histones H2A-H2B and H3-H4 and that disruption of SSRP1 homodimerization decreases its histone-binding affinity. Together, our results provide evidence for regulation of SSRP1 by homodimerization and suggest a potential role for homodimerization in facilitating SPT16-independent functions of SSRP1.

Acetylation-Dependent Recruitment of the FACT Complex and Its Role in Regulating Pol II Occupancy Genome-Wide in Saccharomyces cerevisiae.

Histone chaperones, chromatin remodelers, and histone modifying complexes play a critical role in alleviating the nucleosomal barrier for DNA-dependent processes. Here, we have examined the role of two highly conserved yeast (Saccharomyces cerevisiae) histone chaperones, facilitates chromatin transcription (FACT) and Spt6, in regulating transcription. We show that the H3 tail contributes to the recruitment of FACT to coding sequences in a manner dependent on acetylation. We found that deleting a H3 histone acetyltransferase Gcn5 or mutating lysines on the H3 tail impairs FACT recruitment at ADH1 and ARG1 genes. However, deleting the H4 tail or mutating the H4 lysines failed to dampen FACT occupancy in coding regions. Additionally, we show that FACT depletion reduces RNA polymerase II (Pol II) occupancy genome-wide. Spt6 depletion leads to a reduction in Pol II occupancy toward the 3'-end, in a manner dependent on the gene length. Severe transcription and histone-eviction defects were also observed in a strain that was impaired for Spt6 recruitment (spt6Δ202) and depleted of FACT. Importantly, the severity of the defect strongly correlated with wild-type Pol II occupancies at these genes, indicating critical roles for Spt6 and Spt16 in promoting high-level transcription. Collectively, our results show that both FACT and Spt6 are important for transcription globally and may participate during different stages of transcription.

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.