Genome-wide Profiling Studies Research Articles

Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation profiling

BackgroundThe Illumina family of Infinium Methylation BeadChip microarrays has been widely used over the last 15 years for genome-wide DNA methylation profiling, including large-scale and population-based studies, due to their ease of use and cost effectiveness. Succeeding the popular HumanMethylationEPIC BeadChip (EPICv1), the recently released Infinium MethylationEPIC v2.0 BeadChip (EPICv2) claims to extend genomic coverage to more than 935,000 CpG sites. Here, we comprehensively characterise the reproducibility, reliability and annotation of the EPICv2 array, based on bioinformatic analysis of both manifest data and new EPICv2 data from diverse biological samples.ResultsWe find a high degree of reproducibility with EPICv1, evidenced by comparable sensitivity and precision from empirical cross-platform comparison incorporating whole genome bisulphite sequencing (WGBS), and high correlation between technical sample replicates, including between samples with DNA input levels below the manufacturer’s recommendation. We provide a full assessment of probe content, evaluating genomic distribution and changes from previous array versions. We characterise EPICv2’s new feature of replicated probes and provide recommendations as to the superior probes. In silico analysis of probe sequences demonstrates that probe cross-hybridisation remains a significant problem in EPICv2. By mapping the off-target sites at single nucleotide resolution and comparing with WGBS we show empirical evidence for preferential off-target binding.ConclusionsOverall, we find EPICv2 a worthy successor to the previous Infinium methylation microarrays, however some technical issues remain. To support optimal EPICv2 data analysis we provide an expanded version of the EPICv2 manifest to aid researchers in understanding probe design, data processing, choosing appropriate probes for analysis and for integration with methylation datasets from previous versions of the Infinium Methylation BeadChip.

Genome-wide transcriptional responses of osteoblasts to different titanium surface topographies

This is the first genome-wide transcriptional profiling study using RNA-sequencing to investigate osteoblast responses to different titanium surface topographies, specifically between machined, smooth and acid-etched, microrough surfaces. Rat femoral osteoblasts were cultured on machine-smooth and acid-etched microrough titanium disks. The culture system was validated through a series of assays confirming reduced osteoblast attachment, slower proliferation, and faster differentiation on microrough surfaces. RNA-sequencing analysis of osteoblasts at an early stage of culture revealed that gene expression was highly correlated (r = 0.975) between the two topographies, but 1.38 % genes were upregulated and 0.37 % were downregulated on microrough surfaces. Upregulated transcripts were enriched for immune system, plasma membrane, response to external stimulus, and positive regulation to stimulus processes. Structural mapping confirmed microrough surface-promoted gene sharing and networking in signaling pathways and immune system/responses. Target-specific pathway analysis revealed that Rho family G-protein signaling pathways and actin genes, responsible for the formation of stress fibers, cytoplasmic projections, and focal adhesion, were upregulated on microrough surfaces without upregulation of core genes triggered by cell-to-cell interactions. Furthermore, disulfide-linked or -targeted extracellular matrix (ECM) or membranous glycoproteins such as laminin, fibronectin, CD36, and thrombospondin were highly expressed on microrough surfaces. Finally, proliferating cell nuclear antigen (PCNA) and cyclin D1, whose co-expression reduces cell proliferation, were upregulated on microrough surfaces. Thus, osteoblasts on microrough surfaces were characterized by upregulation of genes related to a wide range of functions associated with the immune system, stress/stimulus responses, proliferation control, skeletal and cytoplasmic signaling, ECM-integrin receptor interactions, and ECM-membranous glycoprotein interactions, furthering our knowledge of the surface-dependent expression of osteoblastic biomarkers on titanium.

THU569 Steroid Receptor Coactivator 3 Is Required To Prevent Early Pregnancy Loss In The Mouse

Abstract Disclosure: V. Maurya: None. L. Hai: None. M.M. Szwarc: None. W. San-Pin: None. F.J. DeMayo: None. J.P. Lydon: None. Members of the Steroid Receptor Coactivator (SRC) family enact distinct transcriptional coregulator functions within a broad spectrum of physiologies and pathologies. We previously demonstrated that endometrial SRC-3 is critical for in vitro human endometrial stromal cell decidualization, a critical cellular process in the establishment of the maternofetal interface. To address SRC-3’s in vivo role in endometrial periimplantation biology, we recently generated a SRC-3 conditional knockout mouse model (SRC-3d/d) in which SRC-3 is selectively ablated in cells that express the progesterone receptor. A six-month breeding trial with wild type proven stud males demonstrated that the SRC-3d/d female fails to produce litters (n>10 per genotype). Unlike the SRC-2d/d female, however, both embryo attachment and implantation into the stromal compartment of the SRC-3d/d endometrium are not impaired at gestation day (GD) 5 or 6. While endometrial stromal decidualization initiates with embryo invasion, pregnant SRC-3d/d mice exhibit early pregnancy loss (EPL) by GD-7.5 (before placentation). At GD-6, the SRC-3d/d decidua is significantly smaller than those of sibling controls. Histological analyses reveal extensive decidual cell death in the SRC-3d/d decidua by GD-7, which results in the sudden demise of the embryo. These histological findings are supported by recent genome-wide RNA profiling studies that show a pro-survival role for SRC-3 in this tissue. Based on these results, we conclude that endometrial SRC-3 is critical for maintaining the functionality and survival of the decidual stromal cell and embryo developmental progression to placentation. Therefore, endometrial SRC-3 (and its downstream signaling networks) are predicted to represent new molecular determinants of non-aneuploid EPL, a poorly understood pregnancy complication for which diagnostic and treatment options are limited. Presentation Date: Thursday, June 15, 2023

Synovial gene signatures associated with the development of rheumatoid arthritis in at risk individuals: A prospective study

ObjectiveTo identify molecular changes in synovium before arthritis development in individuals at risk of developing rheumatoid arthritis (RA). Materials and methodsWe included 67 IgM rheumatoid factor and/or anti-citrullinated protein antibody positive individuals with arthralgia but without arthritis. Synovial biopsies were collected after which individuals were prospectively followed for at least 2 years during which 17 developed arthritis. An exploratory genome-wide transcriptional profiling study was performed in 13 preselected individuals to identify transcripts associated with arthritis development (n = 6). Findings were validated using quantitative real-time PCR and immunohistochemistry in the total cohort. ResultsMicroarray-based survival analyses identified 5588 transcripts whose expression levels in synovium were significantly associated with arthritis development. Pathway analysis revealed that synovial tissue of at risk individuals who later developed arthritis display higher expression of genes involved in adaptive immune response-related pathways compared to at risk individuals who did not develop arthritis. Lower expression was observed for genes involved in extracellular matrix receptor interaction, Wnt-mediated signal transduction and lipid metabolism. Two-way hierarchical clustering analyses of a 27-gene signature separated the total at risk cohort into two groups, where pre-RA individuals preferred to cluster together. Immunohistochemistry studies revealed more podoplanin positive cells and lower lipid droplet staining in synovial tissue from pre-RA individuals. ConclusionSynovial alterations in adaptive immune response and lipid metabolism are associated with future development of arthritis. Since this data show synovial changes without overt cellular infiltration, these may be attributed to preclinical changes in resident synovial tissue cells such as fibroblasts, macrophages and tissue resident T cells.

Genome-wide identification of Arabidopsis non-AUG-initiated upstream ORFs with evolutionarily conserved regulatory sequences that control protein expression levels.

This study identified four novel regulatory non-AUG-initiated upstream ORFs (uORFs) with evolutionarily conserved sequences in Arabidopsis and elucidated the mechanism by which a non-AUG-initiated uORF promotes main ORF translation. Upstream open reading frames (uORFs) are short ORFs found in the 5'-untranslated regions (5'-UTRs) of eukaryotic transcripts and can influence the translation of protein-coding main ORFs (mORFs). Recent genome-wide ribosome profiling studies have revealed that hundreds or thousands of uORFs initiate translation at non-AUG start codons. However, the physiological significance of these non-AUG uORFs has so far been demonstrated for only a few of them. In this study, to identify physiologically important regulatory non-AUG uORFs in Arabidopsis, we took an approach that combined bioinformatics and experimental analysis. Since physiologically important non-AUG uORFs are likely to be conserved across species, we first searched the Arabidopsis genome for non-AUG-initiated uORFs with evolutionarily conserved sequences. Then, we examined the effects of the conserved non-AUG uORFs on the expression of the downstream mORFs using transient expression assays. As a result, three inhibitory and one promotive non-AUG uORFs were identified. Among the inhibitory non-AUG uORFs, two exerted repressive effects on mORF expression in an amino acid sequence-dependent manner. These two non-AUG uORFs are likely to encode regulatory peptides that cause ribosome stalling, thereby enhancing their repressive effects. In contrast, one of the identified regulatory non-AUG uORFs promoted mORF expression by alleviating the inhibitory effect of a downstream AUG-initiated uORF. These findings provide insights into the mechanisms that enable non-AUG uORFs to play regulatory roles despite their low translation initiation efficiencies.

A chemical labeling of N6-formyl adenosine (f6A) RNA

N6-methyl adenosine (m6A) is an eminent epigenetic mark in mRNAs that affects a broad range of biological functions in diverse species. However, the chemically inert methyl group prevents a direct labeling of this modification for subsequent detection and sequencing. Therefore, most current approaches for the labeling of m6A still have limitations of relying on the utilization of corresponding methyltransferases, which resulted in the lacking of efficiency. Here we present an approach which selectively alkylated the N6-formyl adenosine (f6A), the key intermediate during chemical oxidation of m6A, with an alkyne functionality that can be further labeled with click reactions. This covalent labeling approach will be able to facilitate in the affinity purification, detection and genome-wide profiling studies.

Origin of translational control by eIF2α phosphorylation: insights from genome-wide translational profiling studies in fission yeast.

During amino acid limitation, the protein kinase Gcn2 phosphorylates the α subunit of eIF2, thereby regulating mRNA translation. In yeast Saccharomyces cerevisiae and mammals, eIF2α phosphorylation regulates translation of related transcription factors Gcn4 and Atf4 through upstream open reading frames (uORFs) to activate transcription genome wide. However, mammals encode three more eIF2α kinases activated by distinct stimuli. Did the translational control system involving eIF2α phosphorylation evolve from so simple (as found in yeast S. cerevisiae) to complex (as found in humans)? Recent genome-wide translational profiling studies of amino acid starvation response in the fission yeast Schizosaccharomyces pombe provide an unexpected answer to this question.

Combinatorial ETS1-dependent control of oncogenic NOTCH1 enhancers in T-cell leukemia.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to co-bind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis, but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and points to Ets1-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition.

LPA3-mediated lysophosphatidic acid signaling promotes postnatal heart regeneration in mice.

Background: Lysophosphatidic acid (LPA) is a small glycerophospholipid that acts as a potent extracellular signal in various biological processes and diseases. Our previous work demonstrated that the expression of the LPA receptors LPA1 and LPA3 is elevated in the early postnatal heart. However, the role of this stage-specific expression of LPA1 and LPA3 in the heart is unknown.Methods and Results: By using LPA3 and LPA1 knockout mice, and neonatal SD rats treated with Ki16425 (LPA1/LPA3 inhibitor), we found that the number of proliferating cardiomyocytes, detected by coimmunostaining pH3, Ki67 or BrdU with cardiac troponin T, was significantly decreased in the LPA3 knockout mice and the Ki16425-treated rats but not in the LPA1 knockout mice during the first week of postnatal life. Using a myocardial infarction (MI) model, we found that cardiac function and the number of proliferating cardiomyocytes were decreased in the neonatal LPA3 KO mice and increased in the AAV9-mediated cardiac-specific LPA3 overexpression mice. By using lineage tracing and AAV9-LPA3, we further found that LPA3 overexpression in adult mice enhances cardiac function and heart regeneration as assessed by pH3-, Ki67-, and Aurora B-positive cardiomyocytes and clonal cardiomyocytes after MI. Genome-wide transcriptional profiling and additional mechanistic studies showed that LPA induces cardiomyocyte proliferation through the PI3K/AKT, BMP-Smad1/5, Hippo/YAP and MAPK/ERK pathways in vitro, whereas only ERK was confirmed to be activated by LPA-LPA3 signaling in vivo.Conclusion: Our study reports that LPA3-mediated LPA signaling is a crucial factor for cardiomyocyte proliferation in the early postnatal heart. Cardiac-specific LPA3 overexpression improved cardiac function and promoted cardiac regeneration after myocardial injury induced by MI. This finding suggested that activation of LPA3 potentially through AAV-mediated gene therapy might be a therapeutic strategy to improve the outcome after MI.

The Regulatory Roles of Long Noncoding RNAs in Acute Myeloid Leukemia.

In this post-genomic era, long noncoding RNAs (lncRNAs) are rapidly gaining recognition for their crucial roles across diverse biological processes and contexts. The human blood system is no exception, where dozens of lncRNAs have been established as regulators of normal and/or malignant hematopoiesis, and where ongoing works continue to uncover novel lncRNA functions. Our review focuses on lncRNAs that are involved in the pathogenesis of acute myeloid leukemia (AML) and the mechanisms through which they control gene expression in this disease context. We also comment on genome-wide sequencing or profiling studies that have implicated large sets of lncRNAs in AML pathophysiology.

Translational Regulation by Upstream Open Reading Frames and Human Diseases.

Short upstream open reading frames (uORFs) are cis-acting elements located within the 5'-leader sequence of transcripts and are defined by an initiation codon in-frame with a termination codon located upstream or downstream of its main ORF (mORF) initiation codon. Recent genome-wide ribosome profiling studies have confirmed the widespread presence of uORFs and have shown that many uORFs can initiate with non-AUG codons. uORFs can impact gene expression of the downstream mORF by triggering mRNA decay or by regulating translation. Thus, disruption or creation of uORFs can elicit the development of several genetic diseases. Here, we review the mechanisms by which AUG- and non-AUG uORFs regulate translation. We also show some examples of uORF deregulation in human genetic diseases, focusing mainly on cancer. The knowledge of how uORF deregulation drives the onset of a disease, points out the need to screen the 5'-leader sequences of the transcripts in search for potential disease-related variants. This information will be relevant for the implementation of new diagnostic and/or therapeutic tools.

Targeting Non-coding RNA in Vascular Biology and Disease.

Only recently have we begun to appreciate the importance and complexity of the non-coding genome, owing in some part to truly significant advances in genomic technology such as RNA sequencing and genome-wide profiling studies. Previously thought to be non-functional transcriptional “noise,” non-coding RNAs (ncRNAs) are now known to play important roles in many diverse biological pathways, not least in vascular disease. While microRNAs (miRNA) are known to regulate protein-coding gene expression principally through mRNA degradation, long non-coding RNAs (lncRNAs) can activate and repress genes by a variety of mechanisms at both transcriptional and translational levels. These versatile molecules, with complex secondary structures, may interact with chromatin, proteins, and other RNA to form complexes with an array of functional consequences. A body of emerging evidence indicates that both classes of ncRNAs regulate multiple physiological and pathological processes in vascular physiology and disease. While dozens of miRNAs are now implicated and described in relative mechanistic depth, relatively fewer lncRNAs are well described. However, notable examples include ANRIL, SMILR, and SENCR in vascular smooth muscle cells; MALAT1 and GATA-6S in endothelial cells; and mitochondrial lncRNA LIPCAR as a powerful biomarker. Due to such ubiquitous involvement in pathology and well-known biogenesis and functional genetics, novel miRNA-based therapies and delivery methods are now in development, including some early stage clinical trials. Although lncRNAs may hold similar potential, much more needs to be understood about their relatively complex molecular behaviours before realistic translation into novel therapies. Here, we review the current understanding of the mechanism and function of ncRNA, focusing on miRNAs and lncRNAs in vascular disease and atherosclerosis. We discuss existing therapies and current delivery methods, emphasising the importance of miRNAs and lncRNAs as effectors and biomarkers in vascular pathology.

Growth Cone Tctp Is Dynamically Regulated by Guidance Cues.

Translationally controlled tumor protein (Tctp) contributes to retinal circuitry formation by promoting axon growth and guidance, but it remains unknown to what extent axonal Tctp specifically influences axon development programs. Various genome-wide profiling studies have ranked tctp transcripts among the most enriched in the axonal compartment of distinct neuronal populations, including embryonic retinal ganglion cells (RGCs), suggesting its expression can be regulated locally and that this may be important during development. Here, we report that growth cone Tctp levels change rapidly in response to Netrin-1 and Ephrin-A1, two guidance cues encountered by navigating RGC growth cones. This regulation is opposite in effect, as we observed protein synthesis- and mTORC1-dependent increases in growth cone Tctp levels after acute treatment with Netrin-1, but a decline upon exposure to Ephrin-A1, an inhibitor of mTORC1. Live imaging with translation reporters further showed that Netrin-1-induced synthesis of Tctp in growth cones is driven by a short 3′untranslated region (3′UTR) tctp mRNA isoform. However, acute inhibition of de novo Tctp synthesis in axons did not perturb the advance of retinal projections through the optic tract in vivo, indicating that locally produced Tctp is not necessary for normal axon growth and guidance.

Epigenetic and Transcriptome Profiling Identifies a Population of Visceral Adipose-Derived Progenitor Cells with the Potential to Differentiate into an Endocrine Pancreatic Lineage

Type 1 diabetes (T1D) is characterized by the loss of insulin-producing β-cells in the pancreas. T1D can be treated using cadaveric islet transplantation, but this therapy is severely limited by a lack of pancreas donors. To develop an alternative cell source for transplantation therapy, we carried out the epigenetic characterization in nine different adult mouse tissues and identified visceral adipose-derived progenitors as a candidate cell population. Chromatin conformation, assessed using chromatin immunoprecipitation (ChIP) sequencing and validated by ChIP-polymerase chain reaction (PCR) at key endocrine pancreatic gene promoters, revealed similarities between visceral fat and endocrine pancreas. Multiple techniques involving quantitative PCR, in-situ PCR, confocal microscopy, and flow cytometry confirmed the presence of measurable (2–1000-fold over detectable limits) pancreatic gene transcripts and mesenchymal progenitor cell markers (CD73, CD90 and CD105; >98%) in visceral adipose tissue-derived mesenchymal cells (AMCs). The differentiation potential of AMCs was explored in transgenic reporter mice expressing green fluorescent protein (GFP) under the regulation of the Pdx1 (pancreatic and duodenal homeobox-1) gene promoter. GFP expression was measured as an index of Pdx1 promoter activity to optimize culture conditions for endocrine pancreatic differentiation. Differentiated AMCs demonstrated their capacity to induce pancreatic endocrine genes as evidenced by increased GFP expression and validated using TaqMan real-time PCR (at least 2–200-fold relative to undifferentiated AMCs). Human AMCs differentiated using optimized protocols continued to produce insulin following transplantation in NOD/SCID mice. Our studies provide a systematic analysis of potential islet progenitor populations using genome-wide profiling studies and characterize visceral adipose-derived cells for replacement therapy in diabetes.

Epigenetic deregulation in chronic lymphocytic leukemia: Clinical and biological impact.

Deregulated transcriptional control caused by aberrant DNA methylation and/or histone modifications is a hallmark of cancer cells. In chronic lymphocytic leukemia (CLL), the most common adult leukemia, the epigenetic 'landscape' has added a new layer of complexity to our understanding of this clinically and biologically heterogeneous disease. Early studies identified aberrant DNA methylation, often based on single gene promoter analysis with both biological and clinical impact. Subsequent genome-wide profiling studies revealed differential DNA methylation between CLLs and controls and in prognostics subgroups of the disease. From these studies, it became apparent that DNA methylation in regions outside of promoters, such as enhancers, is important for the regulation of coding genes as well as for the regulation of non-coding RNAs. Although DNA methylation profiles are reportedly stable over time and in relation to therapy, a higher epigenetic heterogeneity or 'burden' is seen in more aggressive CLL subgroups, albeit as non-recurrent 'passenger' events. More recently, DNA methylation profiles in CLL analyzed in relation to differentiating normal B-cell populations revealed that the majority of the CLL epigenome reflects the epigenomes present in the cell of origin and that only a small fraction of the epigenetic alterations represents truly CLL-specific changes. Furthermore, CLL patients can be grouped into at least three clinically relevant epigenetic subgroups, potentially originating from different cells at various stages of differentiation and associated with distinct outcomes. In this review, we summarize the current understanding of the DNA methylome in CLL, the role of histone modifying enzymes, highlight insights derived from animal models and attempts made to target epigenetic regulators in CLL along with the future directions of this rapidly advancing field.

Flow-dependent epigenetic regulation of IGFBP5 expression by H3K27me3 contributes to endothelial anti-inflammatory effects.

Rationale: Atherosclerosis is a chronic inflammatory and epigenetic disease that is influenced by different patterns of blood flow. However, the epigenetic mechanism whereby atheroprotective flow controls endothelial gene programming remains elusive. Here, we investigated the possibility that flow alters endothelial gene expression through epigenetic mechanisms.Methods: En face staining and western blot were used to detect protein expression. Real-time PCR was used to determine relative gene expression. RNA-sequencing of human umbilical vein endothelial cells treated with siRNA of enhancer of zeste homolog 2 (EZH2) or laminar flow was used for transcriptional profiling.Results: We found that trimethylation of histone 3 lysine 27 (H3K27me3), a repressive epigenetic mark that orchestrates gene repression, was reduced in laminar flow areas of mouse aorta and flow-treated human endothelial cells. The decrease of H3K27me3 paralleled a reduction in the epigenetic “writer”-EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2). Moreover, laminar flow decreased expression of EZH2 via mechanosensitive miR101. Genome-wide transcriptome profiling studies in endothelial cells treated with EZH2 siRNA and flow revealed the upregulation of novel mechanosensitive gene IGFBP5 (insulin-like growth factor-binding protein 5), which is epigenetically silenced by H3K27me3. Functionally, inhibition of H3K27me3 by EZH2 siRNA or GSK126 (a specific EZH2 inhibitor) reduced H3K27me3 levels and monocyte adhesion to endothelial cells. Adenoviral overexpression of IGFBP5 also recapitulated the anti-inflammatory effects of H3K27me3 inhibition. More importantly, we observed EZH2 upregulation, and IGFBP5 downregulation, in advanced atherosclerotic plaques from human patients.Conclusion: Taken together, our findings reveal that atheroprotective flow reduces H3K27me3 as a chromatin-based mechanism to augment the expression of genes that confer an anti-inflammatory response in the endothelium. Our study exemplifies flow-dependent epigenetic regulation of endothelial gene expression, and also suggests that targeting the EZH2/H3K27me3/IGFBP5 pathway may offer novel therapeutics for inflammatory disorders such as atherosclerosis.

Chapter 19 - Cerebellar tumors

The cerebellum is the most common site of presentation of central nervous system tumors in children but exceedingly rare in adults. Children often present with acute symptoms related to increased intracranial pressure, requiring urgent surgical intervention. The differential diagnosis is broad and includes a variety of benign and malignant entities. Cerebellar low-grade gliomas are the most common and benign, slow-growing tumors, for which surgical resection alone is curative. Embryonal tumors, on the other hand - most commonly medulloblastomas - are highly aggressive and treatment includes intensive postsurgical radiotherapy and chemotherapy. Driven by multiple genomewide profiling studies, the field of neuro-oncology is making great strides towards understanding how different tumors develop and embarking on a new generation of molecularly informed clinical trials.

A field guide for cancer diagnostics using cell-free DNA: From principles to practice and clinical applications.

Recently, many genome-wide profiling studies provided insights into the molecular make-up of major cancer types. The deeper understanding of these genetic alterations and their functional consequences led to the discovery of novel therapeutic opportunities improving clinical management of cancer patients. While tissue-based molecular patient stratification is the gold standard for precision medicine, it has certain limitations: Tissue biopsies are invasive sampling procedures carrying the risk of complications and may not represent the entire tumor due to underlying genetic heterogeneity. In this context, complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Fragments of circulating cell-free DNA (cfDNA) are released from tissues of healthy individuals as well as cancer patients. The fraction of cfDNA that is released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers. Recent studies have demonstrated technical feasibility and clinical applications including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. To this end, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are being employed and several analytical methods and technologies ranging from PCR-based single locus assays to genome-wide approaches are available, which considerably differ in sensitivity, specificity, and throughput. However, broad implementation of ctDNA analysis in daily clinical practice requires a thorough understanding of theoretical, technical, and biological concepts and necessitates standardization and validation of pre-analytical and analytical procedures across different technologies. Here, we review the pertinent literature and discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics.

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data.

Upstream open reading frames (uORFs), located in transcript leaders (5′ UTRs), are potent cis-acting regulators of translation and mRNA turnover. Recent genome-wide ribosome profiling studies suggest that thousands of uORFs initiate with non-AUG start codons. Although intriguing, these non-AUG uORF predictions have been made without statistical control or validation; thus, the importance of these elements remains to be demonstrated. To address this, we took a comparative genomics approach to study AUG and non-AUG uORFs. We mapped transcription leaders in multiple Saccharomyces yeast species and applied a novel machine learning algorithm (uORF-seqr) to ribosome profiling data to identify statistically significant uORFs. We found that AUG and non-AUG uORFs are both frequently found in Saccharomyces yeasts. Although most non-AUG uORFs are found in only one species, hundreds have either conserved sequence or position within Saccharomyces. uORFs initiating with UUG are particularly common and are shared between species at rates similar to that of AUG uORFs. However, non-AUG uORFs are translated less efficiently than AUG-uORFs and are less subject to removal via alternative transcription initiation under normal growth conditions. These results suggest that a subset of non-AUG uORFs may play important roles in regulating gene expression.

Editor's Highlight: High-Throughput Functional Genomics Identifies Modulators of TCE Metabolite Genotoxicity and Candidate Susceptibility Genes.

Trichloroethylene (TCE), an industrial chemical and environmental contaminant, is a human carcinogen. Reactive metabolites are implicated in renal carcinogenesis associated with TCE exposure, yet the toxicity mechanisms of these metabolites and their contribution to cancer and other adverse effects remain unclear. We employed an integrated functional genomics approach that combined functional profiling studies in yeast and avian DT40 cell models to provide new insights into the specific mechanisms contributing to toxicity associated with TCE metabolites. Genome-wide profiling studies in yeast identified the error-prone translesion synthesis (TLS) pathway as an import mechanism in response to TCE metabolites. The role of TLS DNA repair was further confirmed by functional profiling in DT40 avian cell lines, but also revealed that TLS and homologous recombination DNA repair likely play competing roles in cellular susceptibility to TCE metabolites in higher eukaryotes. These DNA repair pathways are highly conserved between yeast, DT40, and humans. We propose that in humans, mutagenic TLS is favored over homologous recombination repair in response to TCE metabolites. The results of these studies contribute to the body of evidence supporting a mutagenic mode of action for TCE-induced renal carcinogenesis mediated by reactive metabolites in humans. Our approach illustrates the potential for high-throughput in vitro functional profiling in yeast to elucidate toxicity pathways (molecular initiating events, key events) and candidate susceptibility genes for focused study.