Cancer is the second leading cause of death worldwide and disease burden is expected to increase globally throughout the next several decades, with the majority of cancer-related deaths occurring in metastatic disease. Cancers exhibit known hallmarks that endow them with increased survival and proliferative capacities, frequently as a result of de-stabilizing mutations. However, the genomic features that resolve metastatic clones from primary tumors are not yet well-characterized, as no mutational landscape has been identified as predictive of metastasis. Further, many cancers exhibit no known mutation signature. This suggests a larger role for non-mutational genome re-organization in promoting cancer evolution and dissemination. In this review, we highlight current critical needs for understanding cell state transitions and clonal selection advantages for metastatic cancer cells. We examine links between epigenetic states, genome structure, and misregulation of tumor suppressors and oncogenes, and discuss how recent technologies for understanding domain-scale regulation have been leveraged for a more complete picture of oncogenic and metastatic potential.

Algae in the dinoflagellate family Symbiodiniaceae are endocellular photosymbionts of corals and other cnidarians. This close relationship is disrupted when seawater temperature increases, causing coral bleaching eventually affecting entire coral reefs. Although the relationship between animal host and photosymbiont has been well-studied, little is known about the bacterial community associated with Symbiodiniaceae in culture. We compared the microbial communities of three isolates from different species of the genus Symbiodinium (formerly known as Symbiodinium clade A) with different ecophysiology, levels of interaction with the animal host, and thermal adaptations. Two species, Symbiodinium microadriaticum and Symbiodinium necroappettens, exhibit intermediate thermotolerance, with a decrease of both growth rate and photochemical efficiency with increased temperature. The third species, Symbiodinium pilosum, has high thermotolerance with no difference in growth rate or photochemical efficiency at 32°C. Microbial communities were characterized after 27 days of growth under control (26°C) and high temperature (32°C). Data shows stronger grouping of bacterial assemblages based on Symbiodinium species than temperature. Microbial communities did not group phylogenetically. We found a shared set of fifteen ASVs belonging to four genera and three families that remained in all three Symbiodiniaceae species. These included Labrenzia, Phycisphaeraceae (SM1A02), Roseovarius, and Muricauda, which are all commonly associated with corals and Symbiodiniaceae cultures. Few ASVs differed significantly by temperature within species. S. pilosum displayed significantly lower levels of microbial diversity and greater individual variability in community composition at 32°C compared to 26°C. These results suggest that bacteria associated or co-cultured with thermotolerant Symbiodinium might play an important role in thermotolerance. Further research on the functional metabolic pathways of these bacteria might hold the key to understanding Symbiodinium’s ability to tolerate thermal stress.

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

Aging is affected by genetic and environmental factors, and cigarette smoking is strongly associated with accumulation of senescent cells. In this study, we wanted to identify genes that may potentially be beneficial for cell survival in response to cigarette smoke and thereby may contribute to development of cellular senescence. Primary human bronchial epithelial cells from five healthy donors were cultured, treated with or without 1.5% cigarette smoke extract (CSE) for 24 h or were passaged into replicative senescence. Transcriptome changes were monitored using RNA-seq in CSE and non-CSE exposed cells and those passaged into replicative senescence. We found that, among 1534 genes differentially regulated during senescence and 599 after CSE exposure, 243 were altered in both conditions, representing strong enrichment. Pathways and gene sets overrepresented in both conditions belonged to cellular processes that regulate reactive oxygen species, proteasome degradation, and NF-κB signaling. Our results offer insights into gene expression responses during cellular aging and cigarette smoke exposure, and identify potential molecular pathways that are altered by cigarette smoke and may also promote airway epithelial cell senescence.

Abstract MicroRNAs (miRs) regulate many fundamental biological processes primarily through inhibiting the transcription and/or translation of their target genes. MiR-486 is an intragenic miR located within ANK1, a gene that encodes for the adapter protein ankyrin-1. Although the tumor suppressor role of miR-486-5p and its down-regulation in non-small cell lung cancer (NSCLC) is well established, the mechanism(s) leading to its repression is unclear. In this study, we investigated epigenetic regulation of ANK1 and its impact on miR-486-5p expression in lung cancer. DNA methylation and expression levels of ANK1 and miR-486-5p in lung tumor-normal pairs, NSCLC cell lines, as well as various normal blood and lung epithelial cell controls were evaluated using qualitative and quantitative assays. The genome-wide impact of epigenetic repression of ANK1 was evaluated using siRNA. The results show that miR-486-5p expression in lung tumors and NSCLC cell lines was significantly reduced compared to distant normal lung tissue pairs (p = 0.0006) and strongly correlated with ANK1 expression. In NSCLC cell lines with strongly reduced expression of miR-486-5p and ANK1, treatment with the DNA methylation inhibitor 5-aza-2-deoxycytidine significantly increased the expression of both the miR and its host gene. Human ANK1 is a large (~230kb) gene with three promoter regions regulating tissue specific expression of three (ANK1B, ANK1E, and ANK1A) transcript variants. ANK1B (the longest of the three variants) is the primary transcript expressed in lung tissue, while ANK1E and ANK1A are weakly and rarely expressed in lung epithelial cells. ANK1B promoter contains a large CpG island that is methylated in 45% (118/262) of lung tumors but not in normal lung or blood samples. The prevalence for ANK1B methylation was significantly higher (p < 0.001) in lung adenocarcinoma (101/200, 51%) compared to lung squamous cell carcinomas (17/62, 27%) after adjustment for smoking. Comparison of methylation by patients’ smoking history also revealed that ANK1B methylation in lung adenocarcinoma was significantly more prevalent in smokers (57%, 73/128) than in never smokers (39%, 28/72), p = 0.014; HR = 2.086 (CI: 1.157 - 3.759). These findings were confirmed by quantitative methylation assays and independently validated using publicly available methylation data for large (n = 809) NSCLC cases from the Cancer Genome Atlas database. Finally, siRNA mediated knockdown of ANK1 in lung cancer cell lines followed by transcriptome-wide expression and pathway analysis revealed that ANK1 repression primarily alters cancer development and progression pathways. Taken together, our data indicates that aberrant methylation of ANK1B promoter is highly prevalent in lung cancer, discriminates lung tumors by histology and smoking history, and represses the expression of ANK1 along with the tumor-suppressor miR-486-5p it hosts within its last intron. Citation Format: Mathewos Tessema, Christin M. Yingling, Maria A. Picchi, Guodong Wu, Tyron Ryba, Young Lin, Steven A. Belinsky. Aberrant methylation of ANK1, the host gene for miR-486, distinguishes lung tumors by histology and smoking status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5384. doi:10.1158/1538-7445.AM2017-5384

The intragenic tumor-suppressor microRNA miR-486-5p is often down-regulated in non-small cell lung cancer (NSCLC) but the mechanism is unclear. This study investigated epigenetic co-regulation of miR-486-5p and its host gene ANK1. MiR-486-5p expression in lung tumors and cell lines was significantly reduced compared to normal lung (p < 0.001) and is strongly correlated with ANK1expression. In vitro, siRNA-mediated ANK1 knockdown in NSCLC cells also reduced miR-486-5p while the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced expression of both. ANK1 promoter CpG island was unmethylated in normal lung but methylated in 45% (118/262) lung tumors and 55% (17/31) NSCLC cell lines. After adjustment for tumor histology and smoking, methylation was significantly more prevalent in adenocarcinoma (101/200, 51%) compared to squamous cell carcinoma (17/62, 27%), p < 0.001; HR = 3.513 (CI: 1.818-6.788); and in smokers (73/128, 57%) than never-smokers (28/72, 39%), p = 0.014; HR = 2.086 (CI: 1.157-3.759). These results were independently validated using quantitative methylation data for 809 NSCLC cases from The Cancer Genome Atlas project. Together, our data indicate that aberrant ANK1 methylation is highly prevalent in lung cancer, discriminate tumors by histology and patients' smoking history, and contributes to miR-486-5p repression. (C) 2017 Elsevier B.V. All rights reserved.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

Background: Cellular differentiation and reprogramming are accompanied by changes in replication timing and 3D organization of large-scale (400 to 800 Kb) chromosomal domains (‘replication domains’), but few gene products have been identified whose disruption affects these properties. Results: Here we show that deletion of esBAF chromatin-remodeling complex components BAF250a and Brg1, but not BAF53a, disrupts replication timing at specific replication domains. Also, BAF250a-deficient fibroblasts reprogrammed to a pluripotency-like state failed to reprogram replication timing in many of these same domains. About half of the replication domains affected by Brg1 loss were also affected by BAF250a loss, but a much larger set of domains was affected by BAF250a loss. esBAF binding in the affected replication domains was dependent upon BAF250a but, most affected domains did not contain genes whose transcription was affected by loss of esBAF. Conclusions: Loss of specific esBAF complex subunits alters replication timing of select replication domains in pluripotent cells.

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400-800 kilobases ('replication domains'), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.

Background: Cellular differentiation and reprogramming are accompanied by changes in replication timing and 3D organization of large-scale (400 to 800 Kb) chromosomal domains ('replication domains'), but few gene products have been identified whose disruption affects these properties.Results: Here we show that deletion of esBAF chromatin-remodeling complex components BAF250a and Brg1, but not BAF53a, disrupts replication timing at specific replication domains. Also, BAF250a-deficient fibroblasts reprogrammed to a pluripotency-like state failed to reprogram replication timing in many of these same domains. About half of the replication domains affected by Brg1 loss were also affected by BAF250a loss, but a much larger set of domains was affected by BAF250a loss. esBAF binding in the affected replication domains was dependent upon BAF250a but, most affected domains did not contain genes whose transcription was affected by loss of esBAF.Conclusions: Loss of specific esBAF complex subunits alters replication timing of select replication domains in pluripotent cells.

Several 400- to 800-kb murine chromosome domains switch from early to late replication during loss of pluripotency, accompanied by a stable form of gene silencing that is resistant to reprogramming. We found that, whereas enhanced nuclease accessibility correlated with early replication genome-wide, domains that switch replication timing during differentiation were exceptionally inaccessible even when early-replicating. Nonetheless, two domains studied in detail exhibited substantial changes in transcriptional activity and higher-order chromatin unfolding confined to the region of replication timing change. Chromosome conformation capture (4C) data revealed that in the unfolded state in embryonic stem cells, these domains interacted preferentially with the early-replicating chromatin compartment, rarely interacting even with flanking late-replicating domains, whereas after differentiation, these same domains preferentially associated with late-replicating chromatin, including flanking domains. In both configurations they retained local boundaries of self-interaction, supporting the replication domain model of replication-timing regulation. Our results reveal a principle of developmentally regulated, large-scale chromosome folding involving a subnuclear compartment switch of inaccessible chromatin. This unusual level of regulation may underlie resistance to reprogramming in replication-timing switch regions.

Abnormal replication timing has been observed in cancer but no study has comprehensively evaluated this misregulation. We generated genome-wide replication-timing profiles for pediatric leukemias from 17 patients and three cell lines, as well as normal B and T cells. Nonleukemic EBV-transformed lymphoblastoid cell lines displayed highly stable replication-timing profiles that were more similar to normal T cells than to leukemias. Leukemias were more similar to each other than to B and T cells but were considerably more heterogeneous than nonleukemic controls. Some differences were patient specific, while others were found in all leukemic samples, potentially representing early epigenetic events. Differences encompassed large segments of chromosomes and included genes implicated in other types of cancer. Remarkably, differences that distinguished leukemias aligned in register to the boundaries of developmentally regulated replication-timing domains that distinguish normal cell types. Most changes did not coincide with copy-number variation or translocations. However, many of the changes that were associated with translocations in some leukemias were also shared between all leukemic samples independent of the genetic lesion, suggesting that they precede and possibly predispose chromosomes to the translocation. Altogether, our results identify sites of abnormal developmental control of DNA replication in cancer that reveal the significance of replication-timing boundaries to chromosome structure and function and support the replication domain model of replication-timing regulation. They also open new avenues of investigation into the chromosomal basis of cancer and provide a potential novel source of epigenetic cancer biomarkers.

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

We recently identified a set of chromosome domains that are early replicating uniquely in pluripotent cells. Their switch from early to late replication occurs just prior to germ layer commitment, associated with a stable form of gene silencing that is difficult to reverse. Here, we discuss results demonstrating that these domains are among the least sensitive regions in the genome to global digestion by either MNase or restriction enzymes. This inaccessible chromatin state persists whether these regions are in their physically distended early replicating or compact late replicating configuration, despite dramatic changes in 3D chromatin folding and long-range chromatin interactions, and despite large changes in transcriptional activity. This contrasts with the strong correlation between early replication, accessibility, transcriptional activity and open chromatin configuration that is observed genome-wide. We put these results in context with findings from other studies indicating that many structural (DNA sequence) and functional (density and activity of replication origins) properties of developmentally regulated replication timing ("switching") domains resemble properties of constitutively late replicating domains. This suggests that switching domains are a type of late replicating domain within which both replication timing and transcription are subject to unique or additional layers of control not experienced by the bulk of the genome. We predict that understanding the unusual structure of these domains will reveal a novel principle of chromosome folding.