Finish the unfinished: Chd1 resolving hexasome-nucleosome complex with FACT.

In this issue of Molecular Cell, Engeholm et al.1 present cryo-EM structures of the chromatin remodeler Chd1 bound to a hexasome-nucleosome complex, an intermediate state during transcription either with or without FACT to restore the missing H2A-H2B dimer. These two binding modes reveal how Chd1 and FACT cooperate in nucleosome re-establishment during transcription.

Complexes of HMO1 with DNA: Structure and Affinity.

Saccharomyces cerevisiae HMO1 is an architectural nuclear DNA-binding protein that stimulates the activity of some remodelers and regulates the transcription of ribosomal protein genes, often binding to a DNA motif called IFHL. However, the molecular mechanism dictating this sequence specificity is unclear. Our circular dichroism spectroscopy studies show that the HMO1:DNA complex forms without noticeable changes in the structure of DNA and HMO1. Molecular modeling/molecular dynamics studies of the DNA complex with HMO1 Box B reveal two extended sites at the N-termini of helices I and II of Box B that are involved in the formation of the complex and stabilize the DNA bend induced by intercalation of the F114 side chain between base pairs. A comparison of the affinities of HMO1 for 24 bp DNA fragments containing either randomized or IFHL sequences reveals a twofold increase in the stability of the complex in the latter case, which may explain the selectivity in the recognition of the IFHL-containing promoter regions.

Resolution of transcription-induced hexasome-nucleosome complexes by Chd1 and FACT.

To maintain the nucleosome organization of transcribed genes, ATP-dependent chromatin remodelers collaborate with histone chaperones. Here, we show that at the 5' ends of yeast genes, RNA polymerase II (RNAPII) generates hexasomes that occur directly adjacent to nucleosomes. The resulting hexasome-nucleosome complexes are then resolved by Chd1. We present two cryoelectron microscopy (cryo-EM) structures of Chd1 bound to a hexasome-nucleosome complex before and after restoration of the missing inner H2A/H2B dimer by FACT. Chd1 uniquely interacts with the complex, positioning its ATPase domain to shift the hexasome away from the nucleosome. In the absence of the inner H2A/H2B dimer, its DNA-binding domain (DBD) packs against the ATPase domain, suggesting an inhibited state. Restoration of the dimer by FACT triggers a rearrangement that displaces the DBD and stimulates Chd1 remodeling. Our results demonstrate how chromatin remodelers interact with a complex nucleosome assembly and suggest how Chd1 and FACT jointly support transcription by RNAPII.

LAG-3 sustains TOX expression and regulates the CD94/NKG2-Qa-1b axis to govern exhausted CD8 T cell NK receptor expression and cytotoxicity.

Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.

FACT mediates the depletion of macroH2A1.2 to expedite gene transcription.

The histone variant macroH2A is generally linked to transcriptionally inactive chromatin, but how macroH2A regulates chromatin structure and functions in the transcriptional process remains elusive. This study reveals that while the integration of human macroH2A1.2 into nucleosomes does not affect their stability or folding dynamics, it notably hinders the maintenance of facilitates chromatin transcription's (FACT's) function. We show that FACT effectively diminishes the stability of macroH2A1.2-nucleosomes and expedites their depletion subsequent to the initial unfolding process. Furthermore, we identify the residue S139 in macroH2A1.2 as a critical switch to modulate FACT's function in nucleosome maintenance. Genome-wide analyses demonstrate that FACT-mediated depletion of macroH2A-nucleosomes allows the correct localization of macroH2A, while the S139 mutation reshapes macroH2A distribution and influences stimulation-induced transcription and cellular response in macrophages. Our findings provide mechanistic insights into the intricate interplay between macroH2A and FACT at the nucleosome level and elucidate their collective role in transcriptional regulation and immune response of macrophages.

A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance.

Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone-binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.

The HMG-box module in FACT is critical for suppressing epigenetic variegation of heterochromatin in fission yeast.

Chromatin condensation state is the key for retrieving genetic information. High-mobility group protein (HMG) proteins exhibit DNA-binding and bending activities, playing an important role in the regulation of chromatin structure. We have shown that nucleosomes tightly packaged into heterochromatin undergo considerable dynamic histone H2A-H2B maintenance via the direct interaction between HP1/Swi6 and facilitate chromatin transcription (FACT), which is composed of the Spt16/Pob3 heterodimer and Nhp6. In this study, we analyzed the role of Nhp6, an HMG box protein, in the FACT at heterochromatin. Pob3 mutant strains showed derepressed heterochromatin-dependent gene silencing, whereas Nhp6 mutant strains did not show significant defects in chromatin regulation or gene expression, suggesting that these two modules play different roles in chromatin regulation. We expressed a protein fusing Nhp6 to the C-terminus of Pob3, which mimics the multicellular FACT component Ssrp1. The chromatin-binding activity of FACT increased with the number of Nhp6 fused to Pob3, and the heterochromatin formation rate was promoted more strongly. Furthermore, we demonstrated that this promotion of heterochromatinization inhibited the heterochromatic variegation caused by epe1+ disruption. Heterochromatic variegation can be observed in a variety of regulatory steps; however, when it is caused by fluctuations in chromatin arrangement, it can be eliminated through the strong recruitment of the FACT complex.

The TOX-RAGE axis mediates inflammatory activation and lung injury in severe pulmonary infectious diseases.

Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.

FACT maintains chromatin architecture and thereby stimulates RNA polymerase II pausing during transcription in vivo.

Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT.

A novel mouse model carrying a gene trap insertion into the Hmgxb4 gene locus to examine Hmgxb4 expression in vivo.

HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the ß-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of ß-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.

Yeast Crf1p is an activator with different roles in regulation of target genes.

Under stress conditions, ribosome biogenesis is downregulated. This process requires that expression of ribosomal RNA, ribosomal protein, and ribosome biogenesis genes be controlled in a coordinated fashion. The mechanistic Target of Rapamycin Complex 1 (mTORC1) participates in sensing unfavorable conditions to effect the requisite change in gene expression. In Saccharomyces cerevisiae, downregulation of ribosomal protein genes involves dissociation of the activator Ifh1p in a process that depends on Utp22p, a protein that also functions in pre-rRNA processing. Ifh1p has a paralog, Crf1p, which was implicated in communicating mTORC1 inhibition and hence was perceived as a repressor. We focus here on two ribosomal biogenesis genes, encoding Utp22p and the high mobility group protein Hmo1p, both of which are required for communication of mTORC1 inhibition to target genes. Crf1p functions as an activator on these genes as evidenced by reduced mRNA abundance and RNA polymerase II occupancy in a crf1Δ strain. Inhibition of mTORC1 has distinct effects on expression of HMO1 and UTP22; for example, on UTP22, but not on HMO1, the presence of Crf1p promotes the stable depletion of Ifh1p. Our data suggest that Crf1p functions as a weak activator, and that it may be required to prevent re-binding of Ifh1p to some gene promoters after mTORC1 inhibition in situations when Ifh1p is available. We propose that the inclusion of genes encoding proteins required for mTORC1-mediated downregulation of ribosomal protein genes in the same regulatory circuit as the ribosomal protein genes serves to optimize transcriptional responses during mTORC1 inhibition.

Chromatin damage generated by DNA intercalators leads to degradation of RNA Polymerase II.

In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.

Hmo1 Promotes Efficient Transcription Elongation by RNA Polymerase I in Saccharomyces cerevisiae.

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.

Transcriptional regulation of FACT involves Coordination of chromatin accessibility and CTCF binding.

Histone chaperone FACT (facilitates chromatin transcription) is well known to promote chromatin recovery during transcription. However, the mechanism how FACT regulates genome-wide chromatin accessibility and transcription factor binding has not been fully elucidated. Through loss-of-function studies, we show here that FACT component Ssrp1 is required for DNA replication and DNA damage repair and is also essential for progression of cell phase transition and cell proliferation in mouse embryonic fibroblast cells. On the molecular level, absence of the Ssrp1 leads to increased chromatin accessibility, enhanced CTCF binding, and a remarkable change in dynamic range of gene expression. Our study thus unequivocally uncovers a unique mechanism by which FACT complex regulates transcription by coordinating genome-wide chromatin accessibility and CTCF binding.

Genome-wide regulation of Pol II, FACT, and Spt6 occupancies by RSC in Saccharomyces cerevisiae.

RSC (remodels the structure of chromatin) is an essential ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae. RSC utilizes its ATPase subunit, Sth1, to slide or remove nucleosomes. RSC has been shown to regulate the width of the nucleosome-depleted regions (NDRs) by sliding the flanking nucleosomes away from NDRs. As such, when RSC is depleted, nucleosomes encroach NDRs, leading to transcription initiation defects. In this study, we examined the effects of the catalytic-dead Sth1 on transcription and compared them to those observed during acute and rapid Sth1 depletion by auxin-induced degron strategy. We found that rapid depletion of Sth1 reduces recruitment of TBP and Pol II in highly transcribed genes, as would be expected considering its role in regulating chromatin structure at promoters. In contrast, cells harboring the catalytic-dead Sth1 (sth1-K501R) exhibited a severe reduction in TBP binding, but, surprisingly, also displayed a substantial accumulation in Pol II occupancies within coding regions. The Pol II occupancies further increased upon depleting endogenous Sth1 in the catalytic-dead mutant, suggesting that the inactive Sth1 contributes to Pol II accumulation in coding regions. Notwithstanding the Pol II increase, the ORF occupancies of histone chaperones, FACT and Spt6 were significantly reduced in the mutant. These results suggest a potential role for RSC in recruiting/retaining these chaperones in coding regions. Pol II accumulation despite substantial reductions in TBP, FACT, and Spt6 occupancies in the catalytic-dead mutant could indicate severe transcription elongation and termination defects. Such defects would be consistent with studies showing that RSC is recruited to coding regions in a transcription-dependent manner. Thus, these findings imply a role for RSC in transcription elongation and termination processes, in addition to its established role in transcription initiation.

Targeting of HBP1/TIMP3 axis as a novel strategy against breast cancer.

Malignant proliferation and metastasis are the main causes of breast cancer death. The transcription factor high mobility group (HMG) box-containing protein 1 (HBP1) is an important tumor suppressor whose deletion or mutation is closely related to the appearance of tumors. Here, we investigated the role of HBP1 in breast cancer suppression. HBP1 enhances the activity of the tissue inhibitors of metalloproteinases 3 (TIMP3) promoter, thereby increasing protein and mRNA levels of TIMP3. TIMP3 increases the phosphatase and tensin homolog (PTEN) protein level by inhibiting its degradation and acts as a metalloproteinase inhibitor to inhibit the protein levels of MMP2/9. In this study, we demonstrated that the HBP1/TIMP3 axis plays a crucial role in inhibiting the tumorigenesis of breast cancer. HBP1 deletion interferes with the regulation of the axis and induces the occurrence and malignant progression of breast cancer. In addition, the HBP1/TIMP3 axis promotes the sensitivity of breast cancer to radiation therapy and hormone therapy. Our study opens new perspectives on the treatment and prognosis of breast cancer.

TFAM deficiency in dendritic cells leads to mitochondrial dysfunction and enhanced antitumor immunity through cGAS-STING pathway.

BACKGROUND: Mitochondrial transcription factor A (TFAM) is a transcription factor that maintains mitochondrial DNA (mtDNA) stabilization and initiates mtDNA replication. However, little is known about the immune regulation function and TFAM expression in immune cells in the tumors. METHODS: Mouse tumor models were applied to analyze the effect of TFAM deficiency in myeloid cell lineage on tumor progression and tumor microenvironment (TME) modification. In vitro, primary mouse bone marrow-derived dendritic cells (BMDCs) were used in the investigation of the altered function and the activated pathway. OVA was used as the model antigen to validate the activation of immune responses in vivo. STING inhibitors were used to confirm the STING activation provoked by Tfam deficient in DCs. RESULTS: The deletion of TFAM in DCs led to mitochondrial dysfunction and mtDNA cytosolic leakage resulting in the cGAS-STING pathway activation in DCs, which contributed to the enhanced antigen presentation. The deletion of TFAM in DCs has interestingly reversed the immune suppressive TME and inhibited tumor growth and metastasis in tumor models. CONCLUSIONS: We have revealed that TFAM knockout in DCs ameliorated immune-suppressive microenvironment in tumors through STING pathway. Our work suggests that specific TFAM knockout in DCs might be a compelling strategy for designing novel immunotherapy methods in the future.

Opposing Roles of FACT for Euchromatin and Heterochromatin in Yeast.

DNA is stored in the nucleus of a cell in a folded state; however, only the necessary genetic information is extracted from the required group of genes. The key to extracting genetic information is chromatin ambivalence. Depending on the chromosomal region, chromatin is characterized into low-density "euchromatin" and high-density "heterochromatin", with various factors being involved in its regulation. Here, we focus on chromatin regulation and gene expression by the yeast FACT complex, which functions in both euchromatin and heterochromatin. FACT is known as a histone H2A/H2B chaperone and was initially reported as an elongation factor associated with RNA polymerase II. In budding yeast, FACT activates promoter chromatin by interacting with the transcriptional activators SBF/MBF via the regulation of G1/S cell cycle genes. In fission yeast, FACT plays an important role in the formation of higher-order chromatin structures and transcriptional repression by binding to Swi6, an HP1 family protein, at heterochromatin. This FACT property, which refers to the alternate chromatin-regulation depending on the binding partner, is an interesting phenomenon. Further analysis of nucleosome regulation within heterochromatin is expected in future studies.

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.

Epigenomic and transcriptomic landscaping unraveled candidate repositioned therapeutics for non-functioning pituitary neuroendocrine tumors.

PURPOSE: Non-functioning pituitary neuroendocrine tumors are challengingly diagnosed tumors in the clinic. Transsphenoidal surgery remains the first-line treatment. Despite the development of state-of-the-art techniques, no drug therapy is currently approved for the treatment. There are also no randomized controlled trials comparing therapeutic strategies or drug therapy for the management after surgery. Therefore, novel therapeutic interventions for the therapeutically challenging NF-PitNETs are urgently needed. METHODS: We integrated epigenome and transcriptome data (both coding and non-coding) that elucidate disease-specific signatures, in addition to biological and pharmacological data, to utilize rational pathway and drug prioritization in NF-PitNETs. We constructed an epigenome- and transcriptome-based PPI network and proposed hub genes. The signature-based drug repositioning based on the integration of multi-omics data was performed. RESULTS: The construction of a disease-specific network based on three different biological levels revealed DCC, DLG5, ETS2, FOXO1, HBP1, HMGA2, PCGF3, PSME4, RBPMS, RREB1, SMAD1, SOCS1, SOX2, YAP1, ZFHX3 as hub proteins. Signature-based drug repositioning using hub proteins yielded repositioned drug candidates that were confirmed in silico via molecular docking. As a result of molecular docking simulations, palbociclib, linifanib, trametinib, eplerenone, niguldipine, and zuclopenthixol showed higher binding affinities with hub genes compared to their inhibitors and were proposed as potential repositioned therapeutics for the management of NF-PitNETs. CONCLUSION: The proposed systems' biomedicine-oriented multi-omics data integration for drug repurposing to provide promising results for the construction of effective clinical therapeutics. To the best of our knowledge, this is the first study reporting epigenome- and transcriptome-based drug repositioning for NF-PitNETs using in silico confirmations.