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Prostate cancer is the second most prevalent malignancy in men. Biomarkers for outcome prediction are urgently needed, so that high‐risk patients could be monitored more closely postoperatively. To identify prognostic markers and to determine causal players in prostate cancer progression, we assessed changes in chromatin state during tumor development and progression. Based on this, we assessed genomewide androgen receptor/chromatin binding and identified a distinct androgen receptor/chromatin binding profile between primary prostate cancers and tumors with an acquired resistance to therapy. These differential androgen receptor/chromatin interactions dictated expression of a distinct gene signature with strong prognostic potential. Further refinement of the signature provided us with a concise list of nine genes that hallmark prostate cancer outcome in multiple independent validation series. In this report, we identified a novel gene expression signature for prostate cancer outcome through generation of multilevel genomic data on chromatin accessibility and transcriptional regulation and integration with publically available transcriptomic and clinical datastreams. By combining existing technologies, we propose a novel pipeline for biomarker discovery that is easily implementable in other fields of oncology. Synopsis Investigation of chromatin accessibility in prostate specimens identified potential driving transcription factors in prostate tumorigenesis, including androgen receptor (AR). AR ChIP‐seq unveiled distinct profiles that bear prognostic potential and can stratify patients on outcome. Chromatin is more condensed in healthy prostate as opposed to prostate tumors. Motif sequences for AR and its interactors are enriched at accessible chromatin sites. AR chromatin binding sites differ between primary and progressive prostate cancer. Genes with a proximal differentially bound AR chromatin site can stratify patients with prostate cancer on outcome. The gene classifier can be used alone or in combination with the D'Amico classification to improve risk stratification for biochemical recurrence. Graphical Abstract Investigation of chromatin accessibility in prostate specimens identified potential driving transcription factors in prostate tumorigenesis, including androgen receptor (AR). AR ChIP‐seq unveiled distinct profiles that bear prognostic potential and can stratify patients on outcome.

Evolution of cis-regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised cis-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using nubbin in the wing and hunchback in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in Tribolium, and in a cross-species context. Together, these advances will facilitate investigation of cis-evolution and morphological diversity in Tribolium and other insects.

Ecology of insects is as wide as their diversity, which reflects their high capacity of adaptation in most of the environments of our planet. Aphids, with over 4,000 species, have developed a series of adaptations including a high phenotypic plasticity and the ability to feed on the phloem sap of plants, which is enriched in sugars derived from photosynthesis. Recent analyses of aphid genomes have indicated a high level of shared ancestral gene duplications that might represent a basis for genetic innovation and broad adaptations. In addition, there are a large number of recent, species-specific gene duplications whose role in adaptation remains poorly understood. Here, we tested whether duplicates specific to the pea aphid Acyrthosiphon pisum are related to genomic innovation by combining comparative genomics, transcriptomics, and chromatin accessibility analyses. Consistent with large levels of neofunctionalization, we found that most of the recent pairs of gene duplicates evolved asymmetrically, showing divergent patterns of positive selection and gene expression. Genes under selection involved a plethora of biological functions, suggesting that neofunctionalization and tissue specificity, among other evolutionary mechanisms, have orchestrated the evolution of recent paralogs in the pea aphid and may have facilitated host–symbiont cooperation. Our comprehensive phylogenomics analysis allowed us to tackle the history of duplicated genes to pave the road toward understanding the role of gene duplication in ecological adaptation.

ABSTRACT Many yeasts differentiate into multicellular phenotypes in adverse environmental conditions. Here, we investigate pseudohyphal growth in Komagataella phaffii and the involvement of the flocculin (FLO) gene family in its regulation. The K. phaffii FLO family consists of 13 members, and the conditions inducing pseudohyphal growth are different from Saccharomyces cerevisiae. So far, this phenotype was only observed when K. phaffii was cultivated at slow growth rates in glucose-limited chemostats, but not upon nitrogen starvation or the presence of fusel alcohols. Transcriptional analysis identified that FLO11, FLO400 and FLO5-1 are involved in the phenotype, all being controlled by the transcriptional regulator Flo8. The three genes exhibit a complex mechanism of expression and repression during transition from yeast to pseudohyphal form. Unlike in S. cerevisiae, deletion of FLO11 does not completely prevent the phenotype. In contrast, deletion of FLO400 or FLO5-1 prevents pseudohyphae formation, and hampers FLO11 expression. FAIRE-Seq data shows that the expression and repression of FLO400 and FLO5-1 are correlated to open or closed chromatin regions upstream of these genes, respectively. Our findings indicate that K. phaffii Flo400 and/or Flo5-1 act as upstream signals that lead to the induction of FLO11 upon glucose limitation in chemostats at slow growth and chromatin modulation is involved in the regulation of their expression. Two novel members of the FLO gene family, FLO400 and FLO5-1, are involved in pseudohyphal growth in Komagataella phaffii.

Background Genomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. Results Here, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using FAIRE-seq approach supported by transmission electron microscopy. We identified differences in T. cruzi genome compartments, putative transcriptional start regions, and virulence factors. In addition, we also detected a developmental chromatin regulation at tRNA loci (tDNA), which could be linked to the intense chromatin remodeling and/or the translation regulatory mechanism required for parasite differentiation. We further integrated the open chromatin profile with public transcriptomic and MNase-seq datasets. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. Conclusion Taken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

Background High-throughput sequence (HTS) data exhibit position-specific nucleotide biases that obscure the intended signal and reduce the effectiveness of these data for downstream analyses. These biases are particularly evident in HTS assays for identifying regulatory regions in DNA (DNase-seq, ChIP-seq, FAIRE-seq, ATAC-seq). Biases may result from many experiment-specific factors, including selectivity of DNA restriction enzymes and fragmentation method, as well as sequencing technology-specific factors, such as choice of adapters/primers and sample amplification methods. Results We present a novel method to detect and correct position-specific nucleotide biases in HTS short read data. Our method calculates read-specific weights based on aligned reads to correct the over- or underrepresentation of position-specific nucleotide subsequences, both within and adjacent to the aligned read, relative to a baseline calculated in assay-specific enriched regions. Using HTS data from a variety of ChIP-seq, DNase-seq, FAIRE-seq, and ATAC-seq experiments, we show that our weight-adjusted reads reduce the position-specific nucleotide imbalance across reads and improve the utility of these data for downstream analyses, including identification and characterization of open chromatin peaks and transcription-factor binding sites. Conclusions A general-purpose method to characterize and correct position-specific nucleotide sequence biases fills the need to recognize and deal with, in a systematic manner, binding-site preference for the growing number of HTS-based epigenetic assays. As the breadth and impact of these biases are better understood, the availability of a standard toolkit to correct them will be important.

Background Despite substantial progress in mosquito genomic and genetic research, few cis-regulatory elements (CREs), DNA sequences that control gene expression, have been identified in mosquitoes or other non-model insects. Formaldehyde-assisted isolation of regulatory elements paired with DNA sequencing, FAIRE-seq, is emerging as a powerful new high-throughput tool for global CRE discovery. FAIRE results in the preferential recovery of open chromatin DNA fragments that are not bound by nucleosomes, an evolutionarily conserved indicator of regulatory activity, which are then sequenced. Despite the power of the approach, FAIRE-seq has not yet been applied to the study of non-model insects. In this investigation, we utilized FAIRE-seq to profile open chromatin and identify likely regulatory elements throughout the genome of the human disease vector mosquito Aedes aegypti . We then assessed genetic variation in the regulatory elements of dengue virus susceptible (Moyo-S) and refractory (Moyo-R) mosquito strains. Results Analysis of sequence data obtained through next generation sequencing of FAIRE DNA isolated from A. aegypti embryos revealed >121,000 FAIRE peaks (FPs), many of which clustered in the 1 kb 5’ upstream flanking regions of genes known to be expressed at this stage. As expected, known transcription factor consensus binding sites were enriched in the FPs, and of these FoxA1, Hunchback, Gfi, Klf4, MYB/ph3 and Sox9 are most predominant. All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays. Of the >13,000 single nucleotide polymorphisms (SNPs) recently identified in dengue virus-susceptible and refractory mosquito strains, 3365 were found to map to FPs. Conclusion FAIRE-seq analysis of open chromatin in A. aegypti permitted genome-wide discovery of CREs. The results of this investigation indicate that FAIRE-seq is a powerful tool for identification of regulatory DNA in the genomes of non-model organisms, including human disease vector mosquitoes.

Poor outcomes in diabetic patients are observed across a range of human tumors, suggesting that cancer cells develop unique characteristics under diabetic conditions. Cancer cells exposed to hyperglycemic insults acquire permanent aggressive traits of tumor growth, even after a return to euglycemic conditions. Comparative genome-wide mapping of hyperglycemia-specific open chromatin regions and concomitant mRNA expression profiling revealed that the neuregulin-1 gene, encoding an established endogenous ligand for the HER3 receptor, is activated through a putative distal enhancer. Our findings highlight the targeted inhibition of NRG1-HER3 pathways as a potential target for the treatment breast cancer patients with associated diabetes.

Background The Aedes aegypti mosquito is a threat to human health across the globe. The A. aegypti genome was recently re-sequenced and re-assembled. Due to a combination of long-read PacBio and Hi-C sequencing, the AaegL5 assembly is chromosome complete and significantly improves the assembly in key areas such as the M/m sex-determining locus. Release of the updated genome assembly has precipitated the need to reprocess historical functional genomic data sets, including cis -regulatory element (CRE) maps that had previously been generated for A. aegypti. Results We re-processed and re-analyzed the A. aegypti whole embryo FAIRE seq data to create an updated embryonic CRE map for the AaegL5 genome. We validated that the new CRE map recapitulates key features of the original AaegL3 CRE map. Further, we built on the improved assembly in the M/m locus to analyze overlaps of open chromatin regions with genes. To support the validation, we created a new method (PeakMatcher) for matching peaks from the same experimental data set across genome assemblies. Conclusion Use of PeakMatcher software, which is available publicly under an open-source license, facilitated the release of an updated and validated CRE map, which is available through the NIH GEO. These findings demonstrate that PeakMatcher software will be a useful resource for validation and transferring of previous annotations to updated genome assemblies.

Background Functional regulatory regions in eukaryotic genomes are characterized by the disruption of nucleosomes leading to accessible chromatin. The modulation of chromatin accessibility is one of the key mediators of transcriptional regulation, and variation in chromatin accessibility across individuals has been linked to complex traits and disease susceptibility. While mechanisms responsible for chromatin variation across individuals have been investigated, the overwhelming majority of chromatin variation remains unexplained. Furthermore, the processes through which the variation of chromatin accessibility contributes to phenotypic diversity remain poorly understood. Results We profiled chromatin accessibility in liver from seven strains of mice with phenotypic diversity in response to a high-fat/high-sucrose (HF/HS) diet and identified reproducible chromatin variation across the individuals. We found that sites of variable chromatin accessibility were more likely to coincide with particular classes of transposable elements (TEs) than sites with common chromatin signatures. Evolutionarily younger long interspersed nuclear elements (LINEs) are particularly likely to harbor variable chromatin sites. These younger LINEs are enriched for binding sites of immune-associated transcription factors, whereas older LINEs are enriched for liver-specific transcription factors. Genomic region enrichment analysis indicates that variable chromatin sites at TEs may function to regulate liver metabolic pathways. CRISPR-Cas9 deletion of a number of variable chromatin sites at TEs altered expression of nearby metabolic genes. Finally, we show that polymorphism of TEs and differential DNA methylation at TEs can both influence chromatin variation. Conclusions Our results demonstrate that specific classes of TEs show variable chromatin accessibility across strains of mice that display phenotypic diversity in response to a HF/HS diet. These results indicate that chromatin variation at TEs is an important contributor to phenotypic variation among populations.