Thymidine phosphorylase facilitates retinoic acid inducible gene-I induced endothelial dysfunction.

Activation of nucleic acid sensors in endothelial cells (ECs) has been shown to drive inflammation across pathologies including cancer, atherosclerosis and obesity. We previously showed that enhancing cytosolic DNA sensing by inhibiting three prime exonuclease 1 (TREX1) in ECs led to EC dysfunction and impaired angiogenesis. Here we show that activation of a cytosolic RNA sensor, Retinoic acid Induced Gene 1 (RIG-I) diminishes EC survival, angiogenesis and triggers tissue specific gene expression programs. We discovered a RIG-I dependent 7 gene signature that affects angiogenesis, inflammation and coagulation. Among these, we identified the thymidine phosphorylase TYMP as a key mediator of RIG-I induced EC dysfunction via its regulation of a subset of interferon stimulated genes. Our RIG-I induced gene signature was also conserved in the context of human diseases - in lung cancer vasculature and herpesvirus infection of lung endothelial cells. Pharmacological or genetic inhibition of TYMP rescues RIG-I induced EC death, migration arrest and restores sprouting angiogenesis. Interestingly, using RNAseq we identified a gene expression program that was RIG-I induced but TYMP dependent. Analysis of this dataset indicated that IRF1 and IRF8 dependent transcription is diminished in RIG-I activated cells when TYMP is inhibited. Functional RNAi screen of our TYMP dependent EC genes, we found that a group of 5 genes - Flot1, Ccl5, Vars2, Samd9l and Ube2l6 are critical for endothelial cell death mediated by RIG-I activation. Our observations identify mechanisms by which RIG-I drives EC dysfunction and define pathways that can be pharmacologically targeted to ameliorate RIG-I induced vascular inflammation.

Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy.

During routine genome duplication, many potential replication origins remain inactive or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that are distinguished from baseline (i.e. ordinarily active) origins by their preferential association with two phospho-isoforms of the helicase component MCM2. During normal unperturbed cell growth, baseline origins, but not dormant origins, associate with a form of MCM2 that is phosphorylated by DBF4-dependent kinase (DDK) on serine 139 (pS139-MCM2). This association facilitates the initiation of DNA replication from baseline origins. Concomitantly, SIRT1 inhibits Ataxia Telangiectasia and Rad3-related (ATR)-kinase-mediated phosphorylation of MCM2 on serine 108 (pS108-MCM2) by deacetylating the ATR-interacting protein DNA topoisomerase II binding protein 1 (TOPBP1), thereby preventing ATR recruitment to chromatin. In cells devoid of SIRT1 activity, or challenged by replication stress, this inhibition is circumvented, enabling ATR-mediated S108-MCM2 phosphorylation. In turn, pS108-MCM2 enables DDK-mediated phosphorylation on S139-MCM2 and facilitates replication initiation at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational MCM modifications that reflect a balance between SIRT1 activity and ATR signaling.

Mutated SETBP1 activates transcription of Myc programs to accelerate CSF3R-driven myeloproliferative neoplasms.

Colony stimulating factor 3 receptor (CSF3R) mutations lead to JAK pathway activation and are the molecular hallmark of chronic neutrophilic leukemia (CNL). Approximately half of patients with CNL also have mutations in SET binding protein 1 (SETBP1). In this study, we developed models of SETBP1-mutated leukemia to understand the role that SETBP1 plays in CNL. SETBP1 mutations promote self-renewal of CSF3R-mutated hematopoietic progenitors in vitro and prevent cells from undergoing terminal differentiation. In vivo, SETBP1 mutations accelerate leukemia progression, leading to the rapid development of hepatosplenomegaly and granulocytosis. Through transcriptomic and epigenomic profiling, we found that SETBP1 enhances progenitor-associated programs, most strongly upregulating Myc and Myc target genes. This upregulation of Myc can be reversed by LSD1 inhibitors. In summary, we found that SETBP1 mutations promote aggressive hematopoietic cell expansion when expressed with mutated CSF3R through the upregulation of Myc-associated gene expression programs.

Nucleic Acid Sensing in the Tumor Vasculature.

Endothelial cells form a powerful interface between tissues and immune cells. In fact, one of the underappreciated roles of endothelial cells is to orchestrate immune attention to specific sites. Tumor endothelial cells have a unique ability to dampen immune responses and thereby maintain an immunosuppressive microenvironment. Recent approaches to trigger immune responses in cancers have focused on activating nucleic acid sensors, such as cGAS-STING, in combination with immunotherapies. In this review, we present a case for targeting nucleic acid-sensing pathways within the tumor vasculature to invigorate tumor-immune responses. We introduce two specific nucleic acid sensors-the DNA sensor TREX1 and the RNA sensor RIG-I-and discuss their functional roles in the vasculature. Finally, we present perspectives on how these nucleic acid sensors in the tumor endothelium can be targeted in an antiangiogenic and immune activation context. We believe understanding the role of nucleic acid-sensing in the tumor vasculature can enhance our ability to design more effective therapies targeting the tumor microenvironment by co-opting both vascular and immune cell types.

MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells.

Defects in stress responses are important contributors in many chronic conditions including cancer, cardiovascular disease, diabetes, and obesity-driven pathologies like non-alcoholic steatohepatitis (NASH). Specifically, endoplasmic reticulum (ER) stress is linked with these pathologies and control of ER stress can ameliorate tissue damage. MicroRNAs have a critical role in regulating diverse stress responses including ER stress. Here, we show that miR-494 plays a functional role during ER stress. Pharmacological ER stress inducers (tunicamycin (TCN) and thapsigargin) and hyperglycemia robustly increase the expression of miR-494 in vitro. ATF6 impacts the primary miR-494 levels whereas all three ER stress pathways are necessary for the increase in mature miR-494. Surprisingly, miR-494 pretreatment dampens the induction and magnitude of ER stress in response to TCN in endothelial cells and increases cell viability. Conversely, inhibition of miR-494 increases ER stress de novo and amplifies the effects of ER stress inducers. Using Mass Spectrometry (TMT-MS) we identified 23 proteins that are downregulated by both TCN and miR-494 in cultured human umbilical vein endothelial cells. Among these, we found 6 transcripts which harbor a putative miR-494 binding site. We validated the anti-apoptotic gene BIRC5 (survivin) and GINS4 as targets of miR-494 during ER stress. In summary, our data indicates that ER stress driven miR-494 may act in a feedback inhibitory loop to dampen downstream ER stress signaling.

Dynamics of replication origin over-activation.

Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.

BAMscale: quantification of next-generation sequencing peaks and generation of scaled coverage tracks.

BACKGROUND: Next-generation sequencing allows genome-wide analysis of changes in chromatin states and gene expression. Data analysis of these increasingly used methods either requires multiple analysis steps, or extensive computational time. We sought to develop a tool for rapid quantification of sequencing peaks from diverse experimental sources and an efficient method to produce coverage tracks for accurate visualization that can be intuitively displayed and interpreted by experimentalists with minimal bioinformatics background. We demonstrate its strength and usability by integrating data from several types of sequencing approaches. RESULTS: We have developed BAMscale, a one-step tool that processes a wide set of sequencing datasets. To demonstrate the usefulness of BAMscale, we analyzed multiple sequencing datasets from chromatin immunoprecipitation sequencing data (ChIP-seq), chromatin state change data (assay for transposase-accessible chromatin using sequencing: ATAC-seq, DNA double-strand break mapping sequencing: END-seq), DNA replication data (Okazaki fragments sequencing: OK-seq, nascent-strand sequencing: NS-seq, single-cell replication timing sequencing: scRepli-seq) and RNA-seq data. The outputs consist of raw and normalized peak scores (multiple normalizations) in text format and scaled bigWig coverage tracks that are directly accessible to data visualization programs. BAMScale also includes a visualization module facilitating direct, on-demand quantitative peak comparisons that can be used by experimentalists. Our tool can effectively analyze large sequencing datasets (~ 100 Gb size) in minutes, outperforming currently available tools. CONCLUSIONS: BAMscale accurately quantifies and normalizes identified peaks directly from BAM files, and creates coverage tracks for visualization in genome browsers. BAMScale can be implemented for a wide set of methods for calculating coverage tracks, including ChIP-seq and ATAC-seq, as well as methods that currently require specialized, separate tools for analyses, such as splice-aware RNA-seq, END-seq and OK-seq for which no dedicated software is available. BAMscale is freely available on github (https://github.com/ncbi/BAMscale).

The RepID-CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation.

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin-RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.

The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin.

Cell cycle progression in mammals is modulated by two ubiquitin ligase complexes, CRL4 and SCF, which facilitate degradation of chromatin substrates involved in the regulation of DNA replication. One member of the CRL4 complex, the WD-40 containing protein RepID (DCAF14/PHIP), selectively binds and activates a group of replication origins. Here we show that RepID recruits the CRL4 complex to chromatin prior to DNA synthesis, thus playing a crucial architectural role in the proper licensing of chromosomes for replication. In the absence of RepID, cells rely on the alternative ubiquitin ligase, SKP2-containing SCF, to progress through the cell cycle. RepID depletion markedly increases cellular sensitivity to SKP2 inhibitors, which triggered massive genome re-replication. Both RepID and SKP2 interact with distinct, non-overlapping groups of replication origins, suggesting that selective interactions of replication origins with specific CRL components execute the DNA replication program and maintain genomic stability by preventing re-initiation of DNA replication.

Replication timing and nuclear structure.

DNA replication proceeds along spatially and temporally coordinated patterns within the nucleus, thus protecting the genome during the synthesis of new genetic material. While we have been able to visualize replication patterns on DNA fibers for 50 years, recent developments and discoveries have provided a greater insight into how DNA replication is controlled. In this review, we highlight many of these discoveries. Of great interest are the physiological role of the replication timing program, cis and trans-acting factors that modulate replication timing and the effects of chromatin structure on the replication timing program. We also discuss future directions in the study of replication timing.