DNA Polymerase-Parental DNA Interaction Is Essential for Helicase-Polymerase Coupling during Bacteriophage T7 DNA Replication.

DNA helicase and polymerase work cooperatively at the replication fork to perform leading-strand DNA synthesis. It was believed that the helicase migrates to the forefront of the replication fork where it unwinds the duplex to provide templates for DNA polymerases. However, the molecular basis of the helicase-polymerase coupling is not fully understood. The recently elucidated T7 replisome structure suggests that the helicase and polymerase sandwich parental DNA and each enzyme pulls a daughter strand in opposite directions. Interestingly, the T7 polymerase, but not the helicase, carries the parental DNA with a positively charged cleft and stacks at the fork opening using a ß-hairpin loop. Here, we created and characterized T7 polymerases each with a perturbed ß-hairpin loop and positively charged cleft. Mutations on both structural elements significantly reduced the strand-displacement synthesis by T7 polymerase but had only a minor effect on DNA synthesis performed against a linear DNA substrate. Moreover, the aforementioned mutations eliminated synergistic helicase-polymerase binding and unwinding at the DNA fork and processive fork progressions. Thus, our data suggested that T7 polymerase plays a dominant role in helicase-polymerase coupling and replisome progression.

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.

Long non-coding RNA MEG8 induced by PLAG1 promotes clear cell renal cell carcinoma through the miR-495-3p/G3BP1 axis.

Clear cell renal cell carcinoma (ccRCC) is recognized as one of the most lethal malignancies among the urological system, with constantly increasing mortality. While the molecular mechanisms underlying ccRCC progression are still poorly understood, the molecular and functional role of lncRNA in multiple diseases has been well demonstrated. In this study, we hypothesized that lncRNA MEG8 might participate in ccRCC development. At first, we found that MEG8 expression was increased in ccRCC tumor tissues and cells. Next, we demonstrated that MEG8 knockdown suppressed cell viability, migration, and invasion in vitro and inhibited tumor growth in vivo. Subsequently, we utilized bioinformatics analysis, ChIP, and luciferase assays, and we found that PLAG1 could transcriptionally regulate MEG8 in ccRCC cells. Furthermore, MEG8 promoted G3BP1 expression to aggravate ccRCC tumorigenic properties through sponging miR-495-3p. Our study identified a novel PLAG1/MEG8/miR-495-3p/G3BP1 network in ccRCC development, which might be a promising direction for developing new diagnoses or therapeutic agents for ccRCC.

Role of stress granules in modulating senescence and promoting cancer progression: Special emphasis on glioma.

Stress granules (SGs) contain mRNAs and proteins stalled in translation during stress; these are increasingly being implicated in diseases, including neurological disorders and cancer. The dysregulated assembly, persistence, disassembly and clearance of SGs contribute to the process of senescence. Senescence has long been a mysterious player in cellular physiology and associated diseases. The systemic process of aging has been pivotal in the development of various neurological disorders like age-related neuropathy, Alzheimer's disease and Parkinson's disease. Glioma is a cancer of neurological origin with a very poor prognosis and high rate of recurrence, SGs have only recently been implicated in its pathogenesis. Senescence has long been established to play an antitumorigenic role, however, relatively less studied is its protumorigenic importance. Here, we have evaluated the existing literature to assess the crosstalk of the two biological phenomena of senescence and SG formation in the context of tumorigenesis. In this review, we have attempted to analyze the contribution of senescence in regulating diverse cellular processes, like, senescence associated secretory phenotype (SASP), microtubular reorganization, telomeric alteration, autophagic clearance and how intricately these phenomena are tied with the formation of SGs. Finally, we propose that interplay between senescence, its contributing factors and the genesis of SGs can drive tumorigenicity of gliomas, which can potentially be utilized for therapeutic intervention.

Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance.

BACKGROUND: Eya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown. METHODS: We engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo. RESULTS: Eya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death-inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor-specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor-specific expression in response to Six2 activity. CONCLUSIONS: Our results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

Neuroblastoma Cells Depend on CSB for Faithful Execution of Cytokinesis and Survival.

Neuroblastoma, the most common extra-cranial solid tumor of early childhood, is one of the major therapeutic challenges in child oncology: it is highly heterogenic at a genetic, biological, and clinical level. The high-risk cases have one of the least favorable outcomes amongst pediatric tumors, and the mortality rate is still high, regardless of the use of intensive multimodality therapies. Here, we observed that neuroblastoma cells display an increased expression of Cockayne Syndrome group B (CSB), a pleiotropic protein involved in multiple functions such as DNA repair, transcription, mitochondrial homeostasis, and cell division, and were recently found to confer cell robustness when they are up-regulated. In this study, we demonstrated that RNAi-mediated suppression of CSB drastically impairs tumorigenicity of neuroblastoma cells by hampering their proliferative, clonogenic, and invasive capabilities. In particular, we observed that CSB ablation induces cytokinesis failure, leading to caspases 9 and 3 activation and, subsequently, to massive apoptotic cell death. Worthy of note, a new frontier in cancer treatment, already proved to be successful, is cytokinesis-failure-induced cell death. In this context, CSB ablation seems to be a new and promising anticancer strategy for neuroblastoma therapy.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple ßα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one ß-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Current and emerging roles of Cockayne syndrome group B (CSB) protein.

Cockayne syndrome (CS) is a segmental premature aging syndrome caused primarily by defects in the CSA or CSB genes. In addition to premature aging, CS patients typically exhibit microcephaly, progressive mental and sensorial retardation and cutaneous photosensitivity. Defects in the CSB gene were initially thought to primarily impair transcription-coupled nucleotide excision repair (TC-NER), predicting a relatively consistent phenotype among CS patients. In contrast, the phenotypes of CS patients are pleiotropic and variable. The latter is consistent with recent work that implicates CSB in multiple cellular systems and pathways, including DNA base excision repair, interstrand cross-link repair, transcription, chromatin remodeling, RNAPII processing, nucleolin regulation, rDNA transcription, redox homeostasis, and mitochondrial function. The discovery of additional functions for CSB could potentially explain the many clinical phenotypes of CSB patients. This review focuses on the diverse roles played by CSB in cellular pathways that enhance genome stability, providing insight into the molecular features of this complex premature aging disease.

CDCA7 and HELLS suppress DNA:RNA hybrid-associated DNA damage at pericentromeric repeats.

Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that is caused by mutations in either DNMT3B, ZBTB24, CDCA7, HELLS, or yet unidentified gene(s). Previously, we reported that the CDCA7/HELLS chromatin remodeling complex facilitates non-homologous end-joining. Here, we show that the same complex is required for the accumulation of proteins on nascent DNA, including the DNMT1/UHRF1 maintenance DNA methylation complex as well as proteins involved in the resolution or prevention of R-loops composed of DNA:RNA hybrids and ssDNA. Consistent with the hypomethylation state of pericentromeric repeats, the transcription and formation of aberrant DNA:RNA hybrids at the repeats were increased in ICF mutant cells. Furthermore, the ectopic expression of RNASEH1 reduced the accumulation of DNA damage at a broad range of genomic regions including pericentromeric repeats in these cells. Hence, we propose that hypomethylation due to inefficient DNMT1/UHRF1 recruitment at pericentromeric repeats by defects in the CDCA7/HELLS complex could induce pericentromeric instability, which may explain a part of the molecular pathogenesis of ICF syndrome.

Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication.

G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

SWI/SNF inactivation in the endometrial epithelium leads to loss of epithelial integrity.

Although ARID1A mutations are a hallmark feature, mutations in other SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling subunits are also observed in endometrial neoplasms. Here, we interrogated the roles of Brahma/SWI2-related gene 1 (BRG1, SMARCA4), the SWI/SNF catalytic subunit, in the endometrial epithelium. BRG1 loss affects more than one-third of all active genes and highly overlaps with the ARID1A gene regulatory network. Chromatin immunoprecipitation studies revealed widespread subunit-specific differences in transcriptional regulation, as BRG1 promoter interactions are associated with gene activation, while ARID1A binding is associated with gene repression. However, we identified a physiologically relevant subset of BRG1 and ARID1A co-regulated epithelial identity genes. Mice were genetically engineered to inactivate BRG1 specifically in the endometrial epithelium. Endometrial glands were observed embedded in uterine myometrium, indicating adenomyosis-like phenotypes. Molecular similarities were observed between BRG1 and ARID1A mutant endometrial cells in vivo, including loss of epithelial cell adhesion and junction genes. Collectively, these studies illustrate overlapping contributions of multiple SWI/SNF subunit mutations in the translocation of endometrium to distal sites, with loss of cell integrity being a common feature in SWI/SNF mutant endometrial epithelia.

PICH regulates the abundance and localization of SUMOylated proteins on mitotic chromosomes.

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by the abnormal distribution of genetic information. Polo-like kinase 1-interacting checkpoint helicase (PICH) is a DNA translocase essential for chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by the small ubiquitin-like modifier (SUMO). However, it is unclear how these activities cooperate to resolve chromosome bridges. Here, we show that PICH specifically disperses SUMO2/3 foci on mitotic chromosomes. This PICH function is apparent toward SUMOylated topoisomerase IIα (TopoIIα) after inhibition of TopoIIα by ICRF-193. Conditional depletion of PICH using the auxin-inducible degron (AID) system resulted in the retention of SUMO2/3-modified chromosomal proteins, including TopoIIα, indicating that PICH functions to reduce the association of these proteins with chromosomes. Replacement of PICH with its translocase-deficient mutants led to increased SUMO2/3 foci on chromosomes, suggesting that the reduction of SUMO2/3 foci requires the remodeling activity of PICH. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taking the results together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.

SMARCAL1, the annealing helicase and the transcriptional co-regulator.

The ATP-dependent chromatin remodeling proteins play an important role in DNA repair. The energy released by ATP hydrolysis is used for myriad functions ranging from nucleosome repositioning and nucleosome eviction to histone variant exchange. In addition, the distant member of the family, SMARCAL1, uses the energy to reanneal stalled replication forks in response to DNA damage. Biophysical studies have shown that this protein has the unique ability to recognize and bind specifically to DNA structures possessing double-strand to single-strand transition regions. Mutations in SMARCAL1 have been linked to Schimke immuno-osseous dysplasia, an autosomal recessive disorder that exhibits variable penetrance and expressivity. It has long been hypothesized that the variable expressivity and pleiotropic phenotypes observed in the patients might be due to the ability of SMARCAL1 to co-regulate the expression of a subset of genes within the genome. Recently, the role of SMARCAL1 in regulating transcription has been delineated. In this review, we discuss the biophysical and functional properties of the protein that help it to transcriptionally co-regulate DNA damage response as well as to bind to the stalled replication fork and stabilize it, thus ensuring genomic stability. We also discuss the role of SMARCAL1 in cancer and the possibility of using this protein as a chemotherapeutic target.

Mgs1 protein supports genome stability via recognition of G-quadruplex DNA structures.

The integrity of the genetic material is crucial for every organism. One intrinsic attack to genome stability is stalling of the replication fork which can result in DNA breakage. Several factors, such as DNA lesions or the formation of stable secondary structures (eg, G-quadruplexes) can lead to replication fork stalling. G-quadruplexes (G4s) are well-characterized stable secondary DNA structures that can form within specific single-stranded DNA sequence motifs and have been shown to block/pause the replication machinery. In most genomes several helicases have been described to regulate G4 unfolding to preserve genome integrity, however, different experiments raise the hypothesis that processing of G4s during DNA replication is more complex and requires additional, so far unknown, proteins. Here, we show that the Saccharomyces cerevisiae Mgs1 protein robustly binds to G4 structures in vitro and preferentially acts at regions with a strong potential to form G4 structures in vivo. Our results suggest that Mgs1 binds to G4-forming sites and has a role in the maintenance of genome integrity.

LncRNA DDX11-AS1: a novel oncogene in human cancer.

Long noncoding RNA (lncRNA) is a newly identified type of noncoding RNA with a length of more than 200 nucleotides. The latest research shows that lncRNAs play important roles in the occurrence and development of human tumours by acting both as carcinogenic genes and as tumour suppressor genes. LncRNAs plays a role in various biological processes, such as cell growth, apoptosis, migration and invasion. The newly discovered lncRNA DDX11-AS1 is abnormally highly expressed in various malignant tumours, such as hepatocellular carcinoma, colorectal cancer, osteosarcoma, bladder cancer, NSCLC and gastric cancer. DDX11-AS1 mainly regulates the expression of related genes through direct or indirect ways to perform its functions in carcinogenicity. These results indicate that DDX11-AS1 may be a marker or therapeutic target of tumours. This review summarizes the biological function and mechanism of DDX11-AS1 in the process of tumour development.

First-in-Class Inhibitors of Oncogenic CHD1L with Preclinical Activity against Colorectal Cancer.

Since the discovery of CHD1L in 2008, it has emerged as an oncogene implicated in the pathology and poor prognosis of a variety of cancers, including gastrointestinal cancers. However, a mechanistic understanding of CHD1L as a driver of colorectal cancer has been limited. Until now, there have been no reported inhibitors of CHD1L, also limiting its development as a molecular target. We sought to characterize the clinicopathologic link between CHD1L and colorectal cancer, determine the mechanism(s) by which CHD1L drives malignant colorectal cancer, and discover the first inhibitors with potential for novel treatments for colorectal cancer. The clinicopathologic characteristics associated with CHD1L expression were evaluated using microarray data from 585 patients with colorectal cancer. Further analysis of microarray data indicated that CHD1L may function through the Wnt/TCF pathway. Thus, we conducted knockdown and overexpression studies with CHD1L to determine its role in Wnt/TCF-driven epithelial-to-mesenchymal transition (EMT). We performed high-throughput screening (HTS) to identify the first CHD1L inhibitors. The mechanism of action, antitumor efficacy, and drug-like properties of lead CHD1L inhibitors were determined using biochemical assays, cell models, tumor organoids, patient-derived tumor organoids, and in vivo pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent in vitro antitumor activity by reversing TCF-driven EMT. The best lead CHD1L inhibitor possesses drug-like properties in pharmacokinetic/pharmacodynamic mouse models. This work validates CHD1L as a druggable target and establishes a novel therapeutic strategy for the treatment of colorectal cancer.

Smarcad1 mediates microbiota-induced inflammation in mouse and coordinates gene expression in the intestinal epithelium.

BACKGROUND: How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue. RESULTS: Specific deletion of Smarcad1 in the mouse intestinal epithelium leads to colitis resistance and substantial changes in gene expression, including a striking increase of expression of several genes linked to innate immunity. Absence of Smarcad1 leads to changes in chromatin accessibility and significant changes in histone H3K9me3 over many sites, including genes that are differentially regulated upon Smarcad1 deletion. We identify candidate members of the gut microbiome that elicit a Smarcad1-dependent colitis response, including members of the poorly understood TM7 phylum. CONCLUSIONS: Our study sheds light onto the role of the chromatin remodeling machinery in intestinal epithelial cells in the colitis response and shows how a highly conserved chromatin remodeling factor has a distinct role in anti-microbial defense. This work highlights the importance of the intestinal epithelium in the colitis response and the potential of microbial species as pharmacological and probiotic targets in the context of inflammatory diseases.

[SMARCA4-deficient thoracic tumors: A new entity]. / Les tumeurs thoraciques SMARCA4 déficientes : une nouvelle entité.

A growing number of studies suggest a tumor suppressor role for the SWI/SNF complex involved in the remodeling of chromatin. Alterations of this complex have been found in many tumors of different origins, with topographic, morphologic and phenotypic diversity. Notably, they define 2 types of thoracic tumors: SMARCA4-deficient non-small cell lung carcinoma and SMARCA4-deficient sarcoma. Some clinical features appear to be common to both, such as intrathoracic localization, smoking exposure, male predominance and poor prognosis. However, the histological distinction between these two entities is sometimes difficult and it is not excluded that these entities belong to the same tumor spectrum with different degrees of differentiation. The therapy of these tumors is not yet codified. These tumors do not seem associated with oncogenic driver mutations allowing the prescription of targeted therapy, but immunotherapy has been shown to be effective in rare reported cases. More specific treatments using EZH2 inhibitors also seem promising in SMARCA4 deficient sarcomas.

hGFAP-Positive Stem Cells Depend on Brg1 for Proper Formation of Cerebral and Cerebellar Structures.

Brahma-related gene 1 (Brg1) is one of the two mutually exclusive catalytic subunits of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. Several roles of Brg1 have been described including acting as a tumor suppressor but also functioning in neural stem cell (NSC) maintenance, neural crest development, or differentiation of oligodendrocytes and Schwann cells. Here, we generated human glial fibrillary acidic protein (hGFAP)-cre::Brg1fl/fl mice to analyze the function of Brg1 in multipotential NSCs during late stages of neural development. hGFAP-cre::Brg1fl/fl mice died approximately 2 weeks after birth. Macroscopic examination revealed a severe hydrocephalus and a decreased brain weight caused by the loss of Brg1. The cerebellum of hGFAP-cre::Brg1fl/fl mice displayed disorganized cortical layers as well as a massive hypoplasia due to a dramatically reduced number of granule neurons. The cerebrum presented with less proliferative and more apoptotic precursor cells in the subventricular zone (SVZ). Furthermore, the cerebral cortex stood out with significantly thinned upper layers and with impressive dendrite pathology. Finally, the hippocampus was severely underdeveloped with only a sparse number of detectable neurons. We conclude that NSCs depend on Brg1 to give rise to major essential brain structures including the cerebellum, the cerebral cortex, and the hippocampus.