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The median coverage at different genes in the MiSeq data. frequency and significance in exome and MiSeq data (per microsatellite and per gene) as well as the NAFs in the MiSeq data.

Primer sequences of the primers utilized in Sanger sequencing.

Mutation significance and sequencing information on the MiSeq sequenced microsatellites.

Clinical and sequencing related information about the 24 exome and 93 MiSeq sequenced tumors.

Figures describing the following; length distribution of somatic insertions and deletions in the exome sequencing data, coverage of mononucleotide microsatellite sites in both exome and MiSeq sequencing data, mutation frequencies at mononucleotide microsatellite sites in the exome sequencing data, number of somatic deletions and insertions in the exome sequencing data, model p-values, expected -log10(p) values, mutation frequency, significance and normalized allelic fraction (NAF) of the top genes, overlap between validation sets 1,2, and 3, and mapped base pairs in the MiSeq sequencing data as well as the supplementary methods and extended literature evaluation of the candidate genes.

Summary of all the genes included in the MiSeq validation (Sets A and B); mutation frequency and significance in exome and MiSeq data (per microsatellite and per gene) as well as the NAFs in the MiSeq data. repeats. microsatellite classes

Frequency of mutations observed at different mononucleotide repeat sites.

Additional information about the mutations in sets A, B and C.

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.

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.

DNA comprises molecular information stored via genetic bases (G, C, T, A) and also epigenetic bases, principally 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Both genetic and epigenetic information are vital to our understanding of biology and disease states. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases typically fail to capture common C-to-T mutations or distinguish 5mC from 5hmC. Here, we present a single-base-resolution sequencing methodology that will simultaneously sequence complete genetics and complete epigenetics in a single workflow. The approach is non-destructive to DNA and provides a digital readout of bases, which we exemplify by simultaneous sequencing of G, C, T, A, 5mC and 5hmC; 6-Letter sequencing. We demonstrate sequencing of human genomic DNA and also cell-free DNA taken from a blood sample of a cancer patient. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. We envisage it will be versatile across many applications in life sciences.

In this work, we map the transcriptional targets of 107 previously identified Drosophila genes whose loss caused the strongest cell-cycle phenotypes in a genome-wide RNA interference screen and mine the resulting data computationally. Besides confirming existing knowledge, the analysis revealed several regulatory systems, among which were two highly-specific and interconnected feedback circuits, one between the ribosome and the proteasome that controls overall protein homeostasis, and the other between the ribosome and Myc/Max that regulates the protein synthesis capacity of cells. We also identified a set of genes that alter the timing of mitosis without affecting gene expression, indicating that the cyclic transcriptional program that produces the components required for cell division can be partially uncoupled from the cell division process itself. These genes all have a function in a pathway that regulates the phosphorylation state of Cdk1. We provide evidence showing that this pathway is involved in regulation of cell size, indicating that a Cdk1-regulated cell size checkpoint exists in metazoans.

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.

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., Somatic heterozygous TET2 loss drives clonal hematopoiesis, which is linked to malignant cell degeneration and potentially cardiovascular disease. Here, the authors investigate the molecular impact of a germline TET2 mutation in a lymphoma family, finding elevated blood DNA methylation levels and no predisposition to atherosclerosis

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.

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., Author summary Small bowel adenocarcinoma is a rare but aggressive disease with limited treatment options. Of gastrointestinal tumors, small bowel tumors account for 3%, of which around one third are adenocarcinomas. Due to the scarcity of evidence-based treatment recommendations there is a dire need for knowledge on the biology of these tumors. Here, we performed the first large exome sequencing effort of 106 small bowel adenocarcinomas from a Finnish population-based cohort to comprehensively characterize the genetic background of this tumor type. The set included tumors from all three small bowel segments allowing us to also compare the genetic differences between these subsets. We defined significantly mutated genes and frequently affected pathways, providing potential therapeutic targets, such as BRAF, ERBB2, ERBB3, ERBB4, PIK3CA, KRAS, ATM, ACVR2A, ACVR1B, BRCA2, and SMARCA4, for this disease.

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. © 2018 The Authors. Published under the terms of the CC BY 4.0 license

It is well established that transcription factors (TFs) play crucial roles in determining cell identity, and that a large fraction of all TFs are expressed in most cell types. In order to globally characterize activities of TFs in cells, we have developed a novel massively parallel protein activity assay, Active TF Identification (ATI) that measures DNA-binding activity of all TFs from any species or tissue type. In contrast to previous studies based on mRNA expression or protein abundance, we found that a set of TFs binding to only around ten distinct motifs display strong DNA-binding activity in any given cell or tissue type. Mass spectrometric identification of TFs revealed that within these highly active TFs, there were both housekeeping TFs, which were universally found in all cell types, and specific TFs, which were highly enriched in known factors that determine the fate of the analyzed tissue or cell type. The importance of a small subset of TFs for determining the overall accessible chromatin landscape of a cell suggests that gene regulatory logic may be simpler than what has previously been appreciated.

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.

Background:Genes with recurrent codon-specific somatic mutations are likely drivers of tumorigenesis and potential therapeutic targets. Hypermutable cancers may represent a sensitive system for generation and selection of oncogenic mutations.Methods:We utilised exome-sequencing data on 25 sporadic microsatellite-instable (MSI) colorectal cancers (CRCs) and searched for base-specific somatic mutation hotspots.Results:We identified novel mutation hotspots in 33 genes. Fourteen genes displayed mutations in the validation set of 254 MSI CRCs: ANTXR1, MORC2, CEP135, CRYBB1, GALNT9, KRT82, PI15, SLC36A1, CNTF, GLDC, MBTPS1, OR9Q2, R3HDM1 and TTPAL. A database search found examples of the hotspot mutations in multiple cancer types.Conclusions:This work reveals a variety of new recurrent candidate oncogene mutations to be further scrutinised as potential therapeutic targets.British Journal of Cancer advance online publication, 12 August 2014; doi:10.1038/bjc.2014.429 www.bjcancer.com.

INTRODUCTION Nearly all cells in the human body share the same primary genome sequence consisting of four nucleotide bases. One of the bases, cytosine, is commonly modified by methylation of its 5 position in CpG dinucleotides (mCpG). Most CpG dinucleotides in the human genome are methylated, but the level of CpG methylation varies with genetic location (promoter versus gene body), whether genes are active versus silenced, and cell type. Research has shown that the maintenance of a particular cellular state after cell division is dependent on faithful transmission of methylated CpGs, as well as inheritance of the mother cells’ repertoire of transcription factors by the daughter cells. These two mechanisms of epigenetic inheritance are linked to each other; the binding of transcription factors can be affected by cytosine methylation, and cytosine methylation can, in turn, be added or removed by proteins that associate with transcription factors. RATIONALE The genetic and epigenetic language, which imparts when and where genes are expressed, is understood at a conceptual level. However, a more detailed understanding is needed of the genomic regulatory mechanism by which methylated cytosines affect transcription factor binding. Because cytosine methylation changes DNA structure, it has the potential to affect binding of all transcription factors. However, a systematic analysis of binding of a large collection of transcription factors to all possible DNA sequences has not previously been conducted. RESULTS To globally characterize the effect of cytosine methylation on transcription factor binding, we systematically analyzed binding specificities of full-length transcription factors and extended DNA binding domains to unmethylated and CpG-methylated DNA by using methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment). We evaluated binding of 542 transcription factors and identified a large number of previously uncharacterized transcription factor recognition motifs. Binding of most major classes of transcription factors, including bHLH, bZIP, and ETS, was inhibited by mCpG. In contrast, transcription factors such as homeodomain, POU, and NFAT proteins preferred to bind methylated DNA. This class of binding was enriched in factors with central roles in embryonic and organismal development. The observed binding preferences were validated using several orthogonal methods, including bisulfite-SELEX and protein-binding microarrays. In addition, the preference of the pluripotency factor OCT4 to bind to a mCpG-containing motif was confirmed by chromatin immunoprecipitation analysis in mouse embryonic stem cells with low or high levels of CpG methylation (due to deficiency in all enzymes that methylate cytosines or contribute to their removal, respectively). Crystal structure analysis of the homeodomain proteins HOXB13, CDX1, CDX2, and LHX4 revealed three key residues that contribute to the preference of this developmentally important family of transcription factors for mCpG. The preference for binding to mCpG was due to direct hydrophobic interactions with the 5-methyl group of methylcytosine. In contrast, inhibition of binding of other transcription factors to methylated sequences was found to be caused by steric hindrance. CONCLUSION Our work constitutes a global analysis of the effect of cytosine methylation on DNA binding specificities of human transcription factors. CpG methylation can influence binding of most transcription factors to DNA—in some cases negatively and in others positively. Our finding that many developmentally important transcription factors prefer to bind to mCpG sites can inform future analyses of the role of DNA methylation on cell differentiation, chromatin reprogramming, and transcriptional regulation.

Uterine leiomyomas, or fibroids, are benign tumors that affect millions of women worldwide and that can cause considerable morbidity. To study the genetic basis of this tumor type, we examined 18 uterine leiomyomas derived from 17 different patients by exome sequencing and identified tumor-specific mutations in the mediator complex subunit 12 (MED12) gene in 10. Through analysis of 207 additional tumors, we determined that MED12 is altered in 70% (159 of 225) of tumors from a total of 80 patients. The Mediator complex is a 26-subunit transcriptional regulator that bridges DNA regulatory sequences to the RNA polymerase II initiation complex. All mutations resided in exon 2, suggesting that aberrant function of this region of MED12 contributes to tumorigenesis.

Small bowel adenocarcinoma (SBA) is a rare but aggressive cancer type with limited treatment options. Known predisposing factors include Crohn's disease, celiac disease, and hereditary syndromes such as familial adenomatous polyposis (FAP), Lynch syndrome, and Peutz-Jeghers syndrome. Here, our aim was to further characterize genetic susceptibility to SBA in a large population-based cohort and simultaneously demonstrate the ability to utilize tumor-only data to cost-effectively but reliably call germline variants. Information on all SBAs diagnosed in Finland between the years 2003-2011 were collected utilizing the Finnish Cancer Registry that maintains a nation-wide database on all cancer cases diagnosed in Finland since 1953. From these we selected all SBAs 1) confirmed as small bowel primary tumor, 2) with available tumor material, and 3) tumor content of at least 50%. Additionally, all relevant medical records were available for all cases. Altogether 106 tumors representing all three parts of the small bowel were selected for exome sequencing. The variant calls were produced with GATK HaplotypeCaller. Germline calls were extracted from the data by filtering out somatic calls that were originally produced by e.g. filtering SNV and indel calls against whole-genome and exome samples of the GnomAD dataset (n=138,632). To focus on possible disease-causing variants, the remaining putative germline variants with allele frequency >0.001 in the whole GnomAD and population-specific Finnish GnomAD set (n=1,747) were excluded. The germline origin of the observed, most prominent variants are being verified by Sanger sequencing, whenever corresponding normal DNA is available. First, we considered variants that were truncating or predicted damaging in silico in the 106 known cancer predisposing genes according to COSMIC. Eight of the 106 genes harbored such variants in at least two patients. We detected the pathogenic germline variant in all patients known to have a hereditary cancer syndrome (MLH1/MSH6 in three Lynch syndrome and APC in two FAP cases). We also identified two patients with a BRCA2 germline variant, one truncating and the other one predicted damaging. BRCA2 might play a role in SBA, thus far germline BRCA2 variants have been observed at least in cancers of the ampullary region. Next, we widened the analysis for other candidate genes but preliminary results show no clear candidates that would be shared by several patients. We will also look more closely into genes belonging to the same signaling pathways as the known syndrome causing genes. Finally, no single gene mutated in all the patients with celiac disease (n=10) was observed nor were there clear differences in the germlines of patients with and without celiac disease. This population-based study on predisposing variants in SBAs provides new information on their molecular genetic background, possibly having an impact also on their treatment. Citation Format: Ulrika A. Hänninen, Riku Katainen, Tomas Tanskanen, Jiri Hamberg, Ari Ristimäki, Eero Pukkala, Minna Taipale, Jukka-Pekka Mecklin, Mervi Aavikko, Linda M. Forsström, Esa Pitkänen, Netta Mäkinen, Lauri A. Aaltonen. Identification of predisposing genes for small bowel adenocarcinoma by exome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-382.

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.

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.

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.

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.

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.

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., Marie Curie International Fellowship (PIOF-GA-2011-303312)

Transforming growth factor-beta (TGF-beta) induced growth arrest of cells involves regulation of the activities of both D- and E-type cyclin kinase complexes thought to be mediated primarily by the regulation of p15(Ink4b) and p27(Kip1) cyclin kinase inhibitors. We show here that TGF-beta downregulates Cdk6 and that transient and stable expression of Cdk6 in Mv1Lu mink epithelial cells overrides TGF-beta mediated arrest. The main effect of the ectopic Cdk6 expression was to sequester TGF-beta induced p15(Ink4b) and to maintain more p27(Kip1) in cyclin D-complexes preventing the complete shift of p27(Kip1) to Cdk2 invoked by TGF-beta. This led to the presence of an active cyclinD-Cdk6-p27(Kip1) complex and partially active cyclin E-Cdk2 complex and resulted in the failure of TGF-beta to fully arrest Mv1Lu cell growth. Though dominant negative Cdk6, expressed similarly in the cells, sequestered both p15(Ink4b) and p27(Kip1), it lacks kinase activity and was unable to override the TGF-beta arrest. The results demonstrate that downregulation of Cdk6 kinase is required for the enforcement of the G(1)-phase arrest by TGF-beta and results in changes in association of the p15(Ink4b) and p27(Kip1) inhibitors with D- and E-type cyclin kinase complexes.

The p27(Kip1) cyclin-dependent kinase inhibitor translocates in response to transforming growth factor-beta to a Cdk2-cyclin E complex inhibiting its catalytic activity, but the p27(Kip1) protein levels are unaffected [1]. We show here that transforming growth factor-beta induces the accumulation of a form of p27(Kip1) representing a subpopulation of total p27(Kip1) in growth-arrested Mv1Lu epithelial cells. The inducible p27(Kip1) is detectable only by a specific p27(Kip1) monoclonal antibody recognizing a native form of p27(Kip1). The increase in this subset of p27(Kip1) correlates with G(1) arrest and withdrawal of the cells from the cycle induced by transforming growth factor-beta, serum starvation, or contact inhibition. In contrast to the majority of p27(Kip1) in the cells, the transforming growth factor-beta-inducible p27(Kip1) is devoid of cyclin-dependent kinase/cyclin interactions. The results indicate that growth arresting treatments induce the accumulation of non-cyclin-dependent kinase-bound p27(Kip1), which may function as a reservoir for inhibition of Cdk2-cyclin E activities.

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.

Microsatellite instability can be found in approximately 15% of all colorectal cancers. To detect new oncogenes we sequenced the exomes of 25 colorectal tumors and respective healthy colon tissue. Potential mutation hot spots were confirmed in 15 genes; ADAR, DCAF12L2, GLT1D1, ITGA7, MAP1B, MRGPRX4, PSRC1, RANBP2, RPS6KL1, SNCAIP, TCEAL6, TUBB6, WBP5, VEGFB, and ZBTB2; these were validated in 86 tumors with microsatellite instability. ZBTB2, RANBP2, and PSRC1 also were found to contain hot spot mutations in the validation set. The form of ZBTB2 associated with colorectal cancer increased cell proliferation. The mutation hot spots might be used to develop personalized tumor profiling and therapy.

Early-onset colorectal cancer (CRC), defined here as age of onset less than 40 years, develops frequently in genetically predisposed individuals. Next-generation sequencing is an increasingly available option in the diagnostic workup of suspected hereditary susceptibility, but little is known about the practical feasibility and additional diagnostic yield of the technology in this patient group.We analyzed 38 young CRC patients derived from a set of 1514 CRC cases. All 38 tumors had been tested in our laboratory for microsatellite instability (MSI), and Sanger sequencing had been used to screen for MLH1 and MSH2 mutations in MSI cases. Also, gastrointestinal polyposis had been diagnosed clinically and molecularly. Family histories were acquired from national registries. If inherited syndromes had not been diagnosed in routine diagnostic efforts (n = 23), normal tissue DNA was analyzed for mutations in a comprehensive set of high-penetrance genes (MLH1, MSH2, MSH6, PMS2, APC, MUTYH, SMAD4, BMPR1A, LKB1/STK11, and PTEN) by exome sequencing.CRC predisposition syndromes were confirmed in 42% (16/38) of early-onset CRC patients. Hereditary nonpolyposis colorectal cancer was diagnosed in 12 (32%) patients, familial adenomatous polyposis in three (7.9%), and juvenile polyposis in one (2.6%) patient. Exome sequencing revealed one additional MLH1 mutation. Over half of the patients had advanced cancers (Dukes C or D, 61%, 23/38). The majority of nonsyndromic patients had unaffected first-degree relatives and microsatellite-stable tumors.Microsatellite instability positivity or gastrointestinal polyposis characterized all patients with unambiguous highly penetrant germline mutations. In our series, exome sequencing produced little added value in diagnosing the underlying predisposition conditions.

SummaryDuring 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.

Colorectal cancer (CRC) is the third most common cancer type and the second leading cause for cancer deaths in the Western world. Around 3.5% of CRC patients have more than one primary colorectal carcinoma at the time of diagnosis. These so-called synchronous colorectal cancers (SCRC) are more common in men and have been associated with predisposing conditions, such as hereditary nonpolyposis colorectal cancer (HNPCC), familial adenomatosis polyposis (FAP) and inflammatory bowel disease. Such conditions account for slightly more than 10% of SCRC cases. The pathological and clinical features of the tumors have been widely studied. The underlying molecular mechanisms in SCRC have not been well described, though. The research has mainly focused on single factors such as microsatellite instability (MSI) status or APC-, KRAS-, and p53-mutations. By exome sequencing a tumor pair and corresponding normal sample from 23 patients, we aim to study the extent of genetic overlap between the synchronous tumors to assess if they derive from a common progenitor. The tumor data will be filtered against the normal tissue data to verify the somatic origin of the observed mutations. The comparison of the mutation profiles in each tumor pair will take into account the number of shared variants that occur in the exact same positions in the coding region. If the tumors share a high number of changes in exactly the same base it would suggest they have a common origin. Different mutation patterns, with possibly a few coincidental shared mutations, may support a stochastic process in the genesis of the two tumors. We will also identify commonly mutated genes - those that are mutated in both tumors of a synchronous pair or among synchronous pairs of different patients. The patient material derives mainly from a consecutive, population-based colorectal cancer collection “Suolisyöpä Keski-Suomi 2000-2010” from Jyväskylä, Finland. In addition three SCRC-cases were included from our in-house collections. Knowledge on the molecular basis of SCRC, which haven't been studied to this extent before, provides insight on how these tumors arise and allows for development of more personalized treatments. Citation Format: Ulrika A. Hänninen, Tomas Tanskanen, Riku Katainen, Jan Böhm, Minna Taipale, Jussi Taipale, Jukka-Pekka Mecklin, Netta Mäkinen, Lauri A. Aaltonen. Comparative genomic analyses of synchronous colorectal cancers by exome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 171.

Symptomatic uterine leiomyomas (ULs) occur in as many as 20% of women. These bening lesions are a major burden to women's health by causing health complications such as abnormal bleeding, pain and infertility, and they are a common cause of hysterectomy that is at present the only curative treatment. Similar to other solid tumors, genetic and epigenetic changes are likely to have value as predictors of UL clinical outcome, as well as response to treatment. Recent findings from us and others have provided evidence for existence of at least three distinct molecular UL subgroups, each displaying a characteristic genetic driver aberration; MED12 mutation, HMGA2 activation or FH mutation. We conducted DNA methylation analysis of bisulfite sequencing data from 59 tumors and 38 normal myometrium samples on targeted genomic regions (84.5Mb) such as CpG islands, shores and shelves, GENCODE promoters and DNaseI hypersensitive sites from ENCODE data using Agilent SureSelect methyl-seq target enrichment system. The studied samples consisted of 17 MED12-mutated, 11 HMGA2-upregulated and 6 FH-mutated tumors. Whole-genome sequencing and expression array profiling had been performed previously from the same specimens (Mehine, Kaasinen, et al. 2014). We developed a data processing pipeline that makes use of: (i) Trim Galore tool for quality control of sequencing data (ii) Bismark for alignment and DNA methylation calling (Krueger & Andrews 2011). Hierarchical clustering of all detected CpG sites revealed that MED12- and FH-mutated tumors cluster according to the driver changes, and HMGA2-upregulated tumors cluster in two separate branches. Global hypermethylation profile was detected in FH-mutated tumors, which is compatible with previous reports in paragangliomas (Letouzé et al. 2013). Four tumors with COL4A5/COL4A6 aberration did not display uniform DNA methylation pattern and four tumors without known genetic driver changes clustered among normal myometrium samples; one of these tumors harbors a somatic TP53 rearrangement created through massive chromothripsis rearrangement event. As myometrium samples showed uniform global DNA methylation by clustering clearly separate from majority of the tumors, statistically powerful comparison of UL subgroups with different genetic background to myometrium samples was enabled. Furthermore, integration of differentially methylated regions with significant expression changes allowed us to characterize genes which are epigenetically activated or silenced in different UL subgroups. All ULs displayed increased methylation of genes enriched in developmentally related biological processes. Our results provide strong evidence that the known UL subgroups are distinguishable by global DNA methylation profiling. The role of the identified epigenetically activated or silenced genes should be studied further to allow better understanding of the biological processes related to UL development and clinical outcome. Citation Format: Eevi Kaasinen, Amjad Alkodsi, Miika Mehine, Hanna-Riikka Heinonen, Netta Mäkinen, Mervi Aavikko, Kati Kampjärvi, Minna Taipale, Pia Vahteristo, Rainer Lehtonen, Lauri A. Aaltonen. Genome-scale DNA methylation changes delineate uterine leiomyoma subgroups. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4435.

From Man to Mouse Genome-wide association studies of humans have identified single-nucleotide polymorphisms (SNPs) that increase an individual's risk of developing common diseases like cancer. Most of these SNPs have only a modest effect on risk, and many map to noncoding regions of the genome. Sur et al. (p. 1360 , published online 1 November; see the Perspective by Lewis and Tomlinson ) used a mouse model to study the functional impact of a particular SNP that resides 300 kilobases upstream of the MYC oncogene on human chromosome 8q24 and has been linked to cancer risk in humans. When a sequence encompassing this SNP was deleted in mice that were predisposed to develop intestinal tumors, the mice displayed fewer tumors than control mice. This SNP may thus play a causal role in human cancer, presumably through altered regulation of MYC.

Many high-throughput loss-of-function analyses of the eukaryotic cell cycle have relied on the unicellular yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. In multicellular organisms, however, additional control mechanisms regulate the cell cycle to specify the size of the organism and its constituent organs. To identify such genes, here we analysed the effect of the loss of function of 70% of Drosophila genes (including 90% of genes conserved in human) on cell-cycle progression of S2 cells using flow cytometry. To address redundancy, we also targeted genes involved in protein phosphorylation simultaneously with their homologues. We identify genes that control cell size, cytokinesis, cell death and/or apoptosis, and the G1 and G2/M phases of the cell cycle. Classification of the genes into pathways by unsupervised hierarchical clustering on the basis of these phenotypes shows that, in addition to classical regulatory mechanisms such as Myc/Max, Cyclin/Cdk and E2F, cell-cycle progression in S2 cells is controlled by vesicular and nuclear transport proteins, COP9 signalosome activity and four extracellular-signal-regulated pathways (Wnt, p38betaMAPK, FRAP/TOR and JAK/STAT). In addition, by simultaneously analysing several phenotypes, we identify a translational regulator, eIF-3p66, that specifically affects the Cyclin/Cdk pathway activity.

Colorectal cancer (CRC) is the third most common cancer in Western countries. Approximately 15% of CRCs display microsatellite instability (MSI) caused by defective cellular mismatch repair. Most of the MSI CRCs are sporadic and result from biallelic inactivation of the MLH1 gene, which is most often due to hypermethylation of its promoter. Cells displaying MSI accumulate a high number of mutations throughout the genome, especially in short repeat areas called microsatellites. These mutations typically lead to a premature stop codon resulting in a truncated protein product that may inactivate the gene, a mechanism known typical of tumor suppressor genes. The aim of this study is to identify new target genes in MSI CRC. To date, several genes have been proposed as MSI target genes based on high mutation frequency in targeted searches of microsatellites. The availability of new sequencing and bioinformatic technologies has enabled genome-wide investigation of mutations in human cancers. This has led to identification of a vast amount of mutations in cancer, most of which are passenger mutations that do not confer selective growth advantage. The challenge is therefore to distinguish true driver genes from passengers. The high number of mutations in MSI tumors augments this challenge. In this study, 25 sporadic MSI CRCs and their corresponding normal samples have been exome sequenced to identify changes of somatic origin. An analysis and visualization program developed in our group, “RikuRator” (unpublished), will be utilized along with a script to estimate the accurate somatic mutation frequency of each coding mononucleotide repeat. A statistical model based on exome sequencing data will be developed that takes into account the background mutation frequency of microsatellites depending on the repeat length and nucleotide context. The most mutated repeats will be validated by Sanger sequencing in a set of additional MSI CRCs. Functional studies will be carried out to further investigate the pathogenic effect of the new target genes identified. The systematic screening of mutations in microsatellites will improve our understanding on the mutation profile typical of these tumors. With our approach that takes into account the background mutation frequency of microsatellites we aim to identify true driver genes in MSI CRC. A detailed understanding of the molecular background of this tumor type is important for the development of more efficient screening methods and personalized treatments to improve the prognosis of patients diagnosed with MSI type CRC. Citation Format: Johanna Kondelin, Esa Pitkänen, Alexandra E. Gylfe, Kimmo Palin, Heikki Ristolainen, Riku Katainen, Eevi Kaasinen, Minna Taipale, Jussi Taipale, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Pia Vahteristo, Sari Tuupanen, Lauri A. Aaltonen. Identification of new target genes in microsatellite unstable colorectal cancer by exome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2401. doi:10.1158/1538-7445.AM2014-2401

Around 15% of colorectal cancers (CRCs) show microsatellite instability (MSI). MSI CRCs are prone to repeat mutations due to defective mismatch repair. The high background mutation frequency has discouraged systematic mutations screens in this tumor type. However, these tumors might form a sensitive system for generation and selection of oncogenic mutation hot spots. The aim of this study is to identify novel oncogenes with mutation hot spots that drive MSI CRC tumorigenesis. The exomes of 25 MSI tumors and respective healthy tissues were sequenced as the discovery set. The exome data was searched for mutation hot spots, with recurrent somatic missense mutations in at least two tumors. Potential mutation hot spots were observed in 43 genes and among these were the following known oncogenes: BRAF (V600E), CTNNB1 (T41A) and PIK3CA (H1047R). Novel potential mutation hot spots were identified in 40 genes and these were validated by Sanger sequencing. Mutation hot spots in 33 genes were confirmed and these were further screened in a validation set of 254 MSI CRCs. Fourteen genes displayed hotspot mutations also in the validation set with a total hot spot mutation frequency of 1.1-3.6 %. Many of the validated genes encode for known cancer-related proteins and for proteins with molecular and cellular functions relevant to cancer development and progression. Further work is needed to clarify the functional role of the identified hot spot mutations in MSI CRC tumorigenesis. The novel mutation hotspots may be utilized to develop personalized tumor profiling and therapy. Citation Format: Alexandra E. Gylfe, Sari Tuupanen, Ulrika Hänninen, Johanna Kondelin, Heikki Ristolainen, Riku Katainen, Esa Pitkänen, Minna Taipale, Jussi Taipale, Claus Lindbjerg Andersen, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Pia Vahteristo, Lauri A. Aaltonen. Novel candidate oncogenes with mutation hot spots in microsatellite unstable colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5193. doi:10.1158/1538-7445.AM2014-5193

Around 15% of colorectal cancers (CRCs) show microsatellite instability (MSI). A characteristic high passenger mutation load has discouraged systematic mutation screens in MSI CRCs. To systematically search for novel MSI CRC oncogenes, the exomes of 25 MSI CRC-normal pairs were sequenced as the discovery set. Observed hot spots were confirmed by Sanger sequencing and further validated in a set of 86 MSI CRCs. Mutational hot spots were confirmed in 15 genes and three genes showed additional hot spot mutations in the validation set. These three sites were highly conserved across species. The hot spot sites of these three genes were screened in 75 microsatellite stable CRCs and 12 MSI CRC cell lines with negative results. Next, we analyzed the subcellular localization of wild-type and mutant proteins. The findings of this study support the idea that cancer genomes are heterogeneous and characterized by few, frequently mutated “mountains” (e.g. BRAF and KRAS) and numerous, less frequently mutated “hills”. The identified mutational hot spots may prove important in developing personalized tumor profiling and therapy. Citation Format: Alexandra E. Gylfe, Johanna Kondelin, Mikko Turunen, Heikki Ristolainen, Riku Katainen, Esa Pitkänen, Eevi Kaasinen, Ville Rantanen, Tomas Tanskanen, Heli J. Lehtonen, Kimmo Palin, Minna Taipale, Jussi Taipale, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Ari Ristimäki, Sari Tuupanen, Auli Karhu, Outi Kilpivaara, Pia Vahteristo, Lauri A. Aaltonen. New candidate oncogenes discovered in microsatellite unstable colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3156. doi:10.1158/1538-7445.AM2013-3156

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.