Simultaneous sequencing of genetic and epigenetic bases in DNA.

DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine.

Impact of constitutional TET2 haploinsufficiency on molecular and clinical phenotype in humans.

Clonal hematopoiesis driven by somatic heterozygous TET2 loss is linked to malignant degeneration via consequent aberrant DNA methylation, and possibly to cardiovascular disease via increased cytokine and chemokine expression as reported in mice. Here, we discover a germline TET2 mutation in a lymphoma family. We observe neither unusual predisposition to atherosclerosis nor abnormal pro-inflammatory cytokine or chemokine expression. The latter finding is confirmed in cells from three additional unrelated TET2 germline mutation carriers. The TET2 defect elevates blood DNA methylation levels, especially at active enhancers and cell-type specific regulatory regions with binding sequences of master transcription factors involved in hematopoiesis. The regions display reduced methylation relative to all open chromatin regions in four DNMT3A germline mutation carriers, potentially due to TET2-mediated oxidation. Our findings provide insight into the interplay between epigenetic modulators and transcription factor activity in hematological neoplasia, but do not confirm the putative role of TET2 in atherosclerosis.

Exome and immune cell score analyses reveal great variation within synchronous primary colorectal cancers.

BACKGROUND: Approximately 4% of colorectal cancer (CRC) patients have at least two simultaneous cancers in the colon. Due to the shared environment, these synchronous CRCs (SCRCs) provide a unique setting to study colorectal carcinogenesis. Understanding whether these tumours are genetically similar or distinct is essential when designing therapeutic approaches. METHODS: We performed exome sequencing of 47 primary cancers and corresponding normal samples from 23 patients. Additionally, we carried out a comprehensive mutational signature analysis to assess whether tumours had undergone similar mutational processes and the first immune cell score analysis (IS) of SCRC to analyse the interplay between immune cell invasion and mutation profile in both lesions of an individual. RESULTS: The tumour pairs shared only few mutations, favouring different mutations in known CRC genes and signalling pathways and displayed variation in their signature content. Two tumour pairs had discordant mismatch repair statuses. In majority of the pairs, IS varied between primaries. Differences were not explained by any clinicopathological variable or mutation burden. CONCLUSIONS: The study shows major diversity within SCRCs. Rather than rely on data from one tumour, our study highlights the need to evaluate both tumours of a synchronous pair for optimised targeted therapy.

The interaction landscape between transcription factors and the nucleosome.

Nucleosomes cover most of the genome and are thought to be displaced by transcription factors in regions that direct gene expression. However, the modes of interaction between transcription factors and nucleosomal DNA remain largely unknown. Here we systematically explore interactions between the nucleosome and 220 transcription factors representing diverse structural families. Consistent with earlier observations, we find that the majority of the studied transcription factors have less access to nucleosomal DNA than to free DNA. The motifs recovered from transcription factors bound to nucleosomal and free DNA are generally similar. However, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many transcription factors preferentially bind close to the end of nucleosomal DNA, or to periodic positions on the solvent-exposed side of the DNA. In addition, several transcription factors usually bind to nucleosomal DNA in a particular orientation. Some transcription factors specifically interact with DNA located at the dyad position at which only one DNA gyre is wound, whereas other transcription factors prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals notable differences in the binding of transcription factors to free and nucleosomal DNA, and uncovers a diverse interaction landscape between transcription factors and the nucleosome.

Comprehensive evaluation of coding region point mutations in microsatellite-unstable colorectal cancer.

Microsatellite instability (MSI) leads to accumulation of an excessive number of mutations in the genome, mostly small insertions and deletions. MSI colorectal cancers (CRCs), however, also contain more point mutations than microsatellite-stable (MSS) tumors, yet they have not been as comprehensively studied. To identify candidate driver genes affected by point mutations in MSI CRC, we ranked genes based on mutation significance while correcting for replication timing and gene expression utilizing an algorithm, MutSigCV Somatic point mutation data from the exome kit-targeted area from 24 exome-sequenced sporadic MSI CRCs and respective normals, and 12 whole-genome-sequenced sporadic MSI CRCs and respective normals were utilized. The top 73 genes were validated in 93 additional MSI CRCs. The MutSigCV ranking identified several well-established MSI CRC driver genes and provided additional evidence for previously proposed CRC candidate genes as well as shortlisted genes that have to our knowledge not been linked to CRC before. Two genes, SMARCB1 and STK38L, were also functionally scrutinized, providing evidence of a tumorigenic role, for SMARCB1 mutations in particular.

A protein activity assay to measure global transcription factor activity reveals determinants of chromatin accessibility.

No existing method to characterize transcription factor (TF) binding to DNA allows genome-wide measurement of all TF-binding activity in cells. Here we present a massively parallel protein activity assay, active TF identification (ATI), that measures the DNA-binding activity of all TFs in cell or tissue extracts. ATI is based on electrophoretic separation of protein-bound DNA sequences from a highly complex DNA library and subsequent mass-spectrometric identification of the DNA-bound proteins. We applied ATI to four mouse tissues and mouse embryonic stem cells and found that, in a given tissue or cell type, a small set of TFs, which bound to only ∼10 distinct motifs, displayed strong DNA-binding activity. Some of these TFs were found in all cell types, whereas others were specific TFs known to determine cell fate in the analyzed tissue or cell type. We also show that a small number of TFs determined the accessible chromatin landscape of a cell, suggesting that gene regulatory logic may be simpler than previously appreciated.

Exome-wide somatic mutation characterization of small bowel adenocarcinoma.

Small bowel adenocarcinoma (SBA) is an aggressive disease with limited treatment options. Despite previous studies, its molecular genetic background has remained somewhat elusive. To comprehensively characterize the mutational landscape of this tumor type, and to identify possible targets of treatment, we conducted the first large exome sequencing study on a population-based set of SBA samples from all three small bowel segments. Archival tissue from 106 primary tumors with appropriate clinical information were available for exome sequencing from a patient series consisting of a majority of confirmed SBA cases diagnosed in Finland between the years 2003-2011. Paired-end exome sequencing was performed using Illumina HiSeq 4000, and OncodriveFML was used to identify driver genes from the exome data. We also defined frequently affected cancer signalling pathways and performed the first extensive allelic imbalance (AI) analysis in SBA. Exome data analysis revealed significantly mutated genes previously linked to SBA (TP53, KRAS, APC, SMAD4, and BRAF), recently reported potential driver genes (SOX9, ATM, and ARID2), as well as novel candidate driver genes, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We also identified clear mutation hotspot patterns in ERBB2 and BRAF. No BRAF V600E mutations were observed. Additionally, we present a comprehensive mutation signature analysis of SBA, highlighting established signatures 1A, 6, and 17, as well as U2 which is a previously unvalidated signature. Finally, comparison of the three small bowel segments revealed differences in tumor characteristics. This comprehensive work unveils the mutational landscape and most frequently affected genes and pathways in SBA, providing potential therapeutic targets, and novel and more thorough insights into the genetic background of this tumor type.

Comprehensive Evaluation of Protein Coding Mononucleotide Microsatellites in Microsatellite-Unstable Colorectal Cancer.

Approximately 15% of colorectal cancers exhibit microsatellite instability (MSI), which leads to accumulation of large numbers of small insertions and deletions (indels). Genes that provide growth advantage to cells via loss-of-function mutations in microsatellites are called MSI target genes. Several criteria to define these genes have been suggested, one of them being simple mutation frequency. Microsatellite mutation rate, however, depends on the length and nucleotide context of the microsatellite. Therefore, assessing the general impact of mismatch repair deficiency on the likelihood of mutation events is paramount when following this approach. To identify MSI target genes, we developed a statistical model for the somatic background indel mutation rate of microsatellites to assess mutation significance. Exome sequencing data of 24 MSI colorectal cancers revealed indels at 54 million mononucleotide microsatellites of three or more nucleotides in length. The top 105 microsatellites from 71 genes were further analyzed in 93 additional MSI colorectal cancers. Mutation significance and estimated clonality of mutations determined the most likely MSI target genes to be the aminoadipate-semialdehyde dehydrogenase AASDH and the solute transporter SLC9A8 Our findings offer a systematic profiling of the somatic background mutation rate in protein-coding mononucleotide microsatellites, allowing a full cataloging of the true targets of MSI in colorectal cancer. Cancer Res; 77(15); 4078-88. ©2017 AACR.

Impact of cytosine methylation on DNA binding specificities of human transcription factors.

The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion.

To identify cell cycle regulators that enable cancer cells to replicate DNA and divide in an unrestricted manner, we performed a parallel genome-wide RNAi screen in normal and cancer cell lines. In addition to many shared regulators, we found that tumor and normal cells are differentially sensitive to loss of the histone genes transcriptional regulator CASP8AP2. In cancer cells, loss of CASP8AP2 leads to a failure to synthesize sufficient amount of histones in the S-phase of the cell cycle, resulting in slowing of individual replication forks. Despite this, DNA replication fails to arrest, and tumor cells progress in an elongated S-phase that lasts several days, finally resulting in death of most of the affected cells. In contrast, depletion of CASP8AP2 in normal cells triggers a response that arrests viable cells in S-phase. The arrest is dependent on p53, and preceded by accumulation of markers of DNA damage, indicating that nucleosome depletion is sensed in normal cells via a DNA-damage -like response that is defective in tumor cells.

Exome Sequencing of Uterine Leiomyosarcomas Identifies Frequent Mutations in TP53, ATRX, and MED12.

Uterine leiomyosarcomas (ULMSs) are aggressive smooth muscle tumors associated with poor clinical outcome. Despite previous cytogenetic and molecular studies, their molecular background has remained elusive. To examine somatic variation in ULMS, we performed exome sequencing on 19 tumors. Altogether, 43 genes were mutated in at least two ULMSs. Most frequently mutated genes included tumor protein P53 (TP53; 6/19; 33%), alpha thalassemia/mental retardation syndrome X-linked (ATRX; 5/19; 26%), and mediator complex subunit 12 (MED12; 4/19; 21%). Unlike ATRX mutations, both TP53 and MED12 alterations have repeatedly been associated with ULMSs. All the observed ATRX alterations were either nonsense or frameshift mutations. ATRX protein levels were reliably analyzed by immunohistochemistry in altogether 44 ULMSs, and the majority of tumors (23/44; 52%) showed clearly reduced expression. Loss of ATRX expression has been associated with alternative lengthening of telomeres (ALT), and thus the telomere length was analyzed with telomere-specific fluorescence in situ hybridization. The ALT phenotype was confirmed in all ULMSs showing diminished ATRX expression. Exome data also revealed one nonsense mutation in death-domain associated protein (DAXX), another gene previously associated with ALT, and the tumor showed ALT positivity. In conclusion, exome sequencing revealed that TP53, ATRX, and MED12 are frequently mutated in ULMSs. ALT phenotype was commonly seen in tumors, indicating that ATR inhibitors, which were recently suggested as possible new drugs for ATRX-deficient tumors, could provide a potential novel therapeutic option for ULMS.

DNA-dependent formation of transcription factor pairs alters their binding specificity.

Gene expression is regulated by transcription factors (TFs), proteins that recognize short DNA sequence motifs. Such sequences are very common in the human genome, and an important determinant of the specificity of gene expression is the cooperative binding of multiple TFs to closely located motifs. However, interactions between DNA-bound TFs have not been systematically characterized. To identify TF pairs that bind cooperatively to DNA, and to characterize their spacing and orientation preferences, we have performed consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX) analysis of 9,400 TF-TF-DNA interactions. This analysis revealed 315 TF-TF interactions recognizing 618 heterodimeric motifs, most of which have not been previously described. The observed cooperativity occurred promiscuously between TFs from diverse structural families. Structural analysis of the TF pairs, including a novel crystal structure of MEIS1 and DLX3 bound to their identified recognition site, revealed that the interactions between the TFs were predominantly mediated by DNA. Most TF pair sites identified involved a large overlap between individual TF recognition motifs, and resulted in recognition of composite sites that were markedly different from the individual TF's motifs. Together, our results indicate that the DNA molecule commonly plays an active role in cooperative interactions that define the gene regulatory lexicon.

Flight-induced changes in gene expression in the Glanville fritillary butterfly.

Insect flight is one of the most energetically demanding activities in the animal kingdom, yet for many insects flight is necessary for reproduction and foraging. Moreover, dispersal by flight is essential for the viability of species living in fragmented landscapes. Here, working on the Glanville fritillary butterfly (Melitaea cinxia), we use transcriptome sequencing to investigate gene expression changes caused by 15 min of flight in two contrasting populations and the two sexes. Male butterflies and individuals from a large metapopulation had significantly higher peak flight metabolic rate (FMR) than female butterflies and those from a small inbred population. In the pooled data, FMR was significantly positively correlated with genome-wide heterozygosity, a surrogate of individual inbreeding. The flight experiment changed the expression level of 1513 genes, including genes related to major energy metabolism pathways, ribosome biogenesis and RNA processing, and stress and immune responses. Males and butterflies from the population with high FMR had higher basal expression of genes related to energy metabolism, whereas females and butterflies from the small population with low FMR had higher expression of genes related to ribosome/RNA processing and immune response. Following the flight treatment, genes related to energy metabolism were generally down-regulated, while genes related to ribosome/RNA processing and immune response were up-regulated. These results suggest that common molecular mechanisms respond to flight and can influence differences in flight metabolic capacity between populations and sexes.

Systematic search for rare variants in Finnish early-onset colorectal cancer patients.

The heritability of colorectal cancer (CRC) is incompletely understood, and the contribution of undiscovered rare variants may be important. In search of rare disease-causing variants, we exome sequenced 22 CRC patients who were diagnosed before the age of 40 years. Exome sequencing data from 95 familial CRC patients were available as a validation set. Cases with known CRC syndromes were excluded. All patients were from Finland, a country known for its genetically homogenous population. We searched for rare nonsynonymous variants with allele frequencies below 0.1% in 3,374 Finnish and 58,112 non-Finnish controls. In addition, homozygous and compound heterozygous variants were studied. No genes with rare loss-of-function variants were present in more than one early-onset CRC patient. Three genes (ADAMTS4, CYTL1, and SYNE1) harbored rare loss-of-function variants in both early-onset and familial CRC cases. Five genes with homozygous variants in early-onset CRC cases were found (MCTP2, ARHGAP12, ATM, DONSON, and ROS1), including one gene (MCTP2) with a homozygous splice site variant. All discovered homozygous variants were exclusive to one early-onset CRC case. Independent replication is required to associate the discovered variants with CRC. These findings, together with a lack of family history in 19 of 22 (86%) early-onset patients, suggest genetic heterogeneity in unexplained early-onset CRC patients, thus emphasizing the requirement for large sample sizes and careful study designs to elucidate the role of rare variants in CRC susceptibility.

Whole-Genome Sequencing Identifies STAT4 as a Putative Susceptibility Gene in Classic Kaposi Sarcoma.

BACKGROUND: Classic Kaposi sarcoma (cKS) is an inflammatory tumor caused by human herpesvirus 8 (HHV-8) commonly observed in elderly men of Mediterranean origin. We studied a Finnish family of 5 affected individuals in 2 generations. Except for atypical mycobacterial infection of the index case, the affected individuals did not have notable histories of infection. METHODS: We performed genome and exome sequencing and mapped shared chromosomal regions to identify genetic predisposition in the family. RESULTS: We identified 12 protein-coding candidate variants that segregated in the 3 affected cousins from whom we had samples. The affected mother of the index case was an obligatory carrier. Among the 12 candidates was a rare heterozygous substitution rs141331848 (c.1337C>T, p.Thr446Ile) in the DNA-binding domain of STAT4. The variant was not present in 242 Finnish control genomes or 180 additional regional controls. Activated T-helper cells from the HHV-8-negative variant carriers showed reduced interferon γ production, compared with age and sex matched wild-type individuals. We screened STAT4 in additional 18 familial KS cases and the variant site from 56 sporadic KS cases but detected no pathogenic mutations. CONCLUSIONS: Our data suggest that STAT4 is a potential cKS-predisposition gene, but further functional and genetic validation is needed.

The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera.

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.

Transcriptome analysis reveals signature of adaptation to landscape fragmentation.

We characterize allelic and gene expression variation between populations of the Glanville fritillary butterfly (Melitaea cinxia) from two fragmented and two continuous landscapes in northern Europe. The populations exhibit significant differences in their life history traits, e.g. butterflies from fragmented landscapes have higher flight metabolic rate and dispersal rate in the field, and higher larval growth rate, than butterflies from continuous landscapes. In fragmented landscapes, local populations are small and have a high risk of local extinction, and hence the long-term persistence at the landscape level is based on frequent re-colonization of vacant habitat patches, which is predicted to select for increased dispersal rate. Using RNA-seq data and a common garden experiment, we found that a large number of genes (1,841) were differentially expressed between the landscape types. Hexamerin genes, the expression of which has previously been shown to have high heritability and which correlate strongly with larval development time in the Glanville fritillary, had higher expression in fragmented than continuous landscapes. Genes that were more highly expressed in butterflies from newly-established than old local populations within a fragmented landscape were also more highly expressed, at the landscape level, in fragmented than continuous landscapes. This result suggests that recurrent extinctions and re-colonizations in fragmented landscapes select a for specific expression profile. Genes that were significantly up-regulated following an experimental flight treatment had higher basal expression in fragmented landscapes, indicating that these butterflies are genetically primed for frequent flight. Active flight causes oxidative stress, but butterflies from fragmented landscapes were more tolerant of hypoxia. We conclude that differences in gene expression between the landscape types reflect genomic adaptations to landscape fragmentation.

Exome sequencing reveals frequent inactivating mutations in ARID1A, ARID1B, ARID2 and ARID4A in microsatellite unstable colorectal cancer.

ARID1A has been identified as a novel tumor suppressor gene in ovarian cancer and subsequently in various other tumor types. ARID1A belongs to the ARID domain containing gene family, which comprises of 15 genes involved, for example, in transcriptional regulation, proliferation and chromatin remodeling. In this study, we used exome sequencing data to analyze the mutation frequency of all the ARID domain containing genes in 25 microsatellite unstable (MSI) colorectal cancers (CRCs) as a first systematic effort to characterize the mutation pattern of the whole ARID gene family. Genes which fulfilled the selection criteria in this discovery set (mutations in at least 4/25 [16%] samples, including at least one nonsense or splice site mutation) were chosen for further analysis in an independent validation set of 21 MSI CRCs. We found that in addition to ARID1A, which was mutated in 39% of the tumors (18/46), also ARID1B (13%, 6/46), ARID2 (13%, 6/46) and ARID4A (20%, 9/46) were frequently mutated. In all these genes, the mutations were distributed along the entire length of the gene, thus distinguishing them from typical MSI target genes previously described. Our results indicate that in addition to ARID1A, other members of the ARID gene family may play a role in MSI CRC.

Eleven candidate susceptibility genes for common familial colorectal cancer.

Hereditary factors are presumed to play a role in one third of colorectal cancer (CRC) cases. However, in the majority of familial CRC cases the genetic basis of predisposition remains unexplained. This is particularly true for families with few affected individuals. To identify susceptibility genes for this common phenotype, we examined familial cases derived from a consecutive series of 1514 Finnish CRC patients. Ninety-six familial CRC patients with no previous diagnosis of a hereditary CRC syndrome were included in the analysis. Eighty-six patients had one affected first-degree relative, and ten patients had two or more. Exome sequencing was utilized to search for genes harboring putative loss-of-function variants, because such alterations are likely candidates for disease-causing mutations. Eleven genes with rare truncating variants in two or three familial CRC cases were identified: UACA, SFXN4, TWSG1, PSPH, NUDT7, ZNF490, PRSS37, CCDC18, PRADC1, MRPL3, and AKR1C4. Loss of heterozygosity was examined in all respective cancer samples, and was detected in seven occasions involving four of the candidate genes. In all seven occasions the wild-type allele was lost (P = 0.0078) providing additional evidence that these eleven genes are likely to include true culprits. The study provides a set of candidate predisposition genes which may explain a subset of common familial CRC. Additional genetic validation in other populations is required to provide firm evidence for causality, as well as to characterize the natural history of the respective phenotypes.

Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites.

During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M phase. These results suggest that cohesin-binding functions as a cellular memory that promotes re-establishment of TF clusters after DNA replication and chromatin condensation.