Jason A. Reuter

DNMT1 maintains progenitor function in self-renewing somatic tissue

Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

High-Throughput Sequencing Technologies

The human genome sequence has profoundly altered our understanding of biology, human diversity, and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past 10 years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them, as well as the challenges facing current sequencing platforms and their clinical application.

Recurrent somatic mutations in regulatory regions of human cancer genomes

Aberrant regulation of gene expression in cancer can promote survival and proliferation of cancer cells. Here we integrate whole-genome sequencing data from The Cancer Genome Atlas (TCGA) for 436 patients from 8 cancer subtypes with ENCODE and other regulatory annotations to identify point mutations in regulatory regions. We find evidence for positive selection of mutations in transcription factor binding sites, consistent with these sites regulating important cancer cell functions. Using a new method that adjusts for sample- and genomic locus–specific mutation rates, we identify recurrently mutated sites across individuals with cancer. Mutated regulatory sites include known sites in the TERT promoter and many new sites, including a subset in proximity to cancer-related genes. In reporter assays, two new sites display decreased enhancer activity upon mutation. These data demonstrate that many regulatory regions contain mutations under selective pressure and suggest a greater role for regulatory mutations in cancer than previously appreciated.

Genomic analysis of mycosis fungoides and Sézary syndrome identifies recurrent alterations in TNFR2

Mycosis fungoides and Sézary syndrome comprise the majority of cutaneous T cell lymphomas (CTCLs), disorders notable for their clinical heterogeneity that can present in skin or peripheral blood. Effective treatment options for CTCL are limited, and the genetic basis of these T cell lymphomas remains incompletely characterized. Here we report recurrent point mutations and genomic gains of TNFRSF1B, encoding the tumor necrosis factor receptor TNFR2, in 18% of patients with mycosis fungoides and Sézary syndrome. Expression of the recurrent TNFR2 Thr377Ile mutant in T cells leads to enhanced non-canonical NF-κB signaling that is sensitive to the proteasome inhibitor bortezomib. Using an integrative genomic approach, we additionally discovered a recurrent CTLA4-CD28 fusion, as well as mutations in downstream signaling mediators of these receptors.

Modeling Inducible Human Tissue Neoplasia Identifies an Extracellular Matrix Interaction Network Involved in Cancer Progression

To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in three-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends on specific extracellular matrix-interacting network hubs. Blockade of one such hub, the beta1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression.

Identification of significantly mutated regions across cancer types highlights a rich landscape of functional molecular alterations

Cancer sequencing studies have primarily identified cancer driver genes by the accumulation of protein-altering mutations. An improved method would be annotation independent, sensitive to unknown distributions of functions within proteins and inclusive of noncoding drivers. We employed density-based clustering methods in 21 tumor types to detect variably sized significantly mutated regions (SMRs). SMRs reveal recurrent alterations across a spectrum of coding and noncoding elements, including transcription factor binding sites and untranslated regions mutated in up to ∼15% of specific tumor types. SMRs demonstrate spatial clustering of alterations in molecular domains and at interfaces, often with associated changes in signaling. Mutation frequencies in SMRs demonstrate that distinct protein regions are differentially mutated across tumor types, as exemplified by a linker region of PIK3CA in which biophysical simulations suggest that mutations affect regulatory interactions. The functional diversity of SMRs underscores both the varied mechanisms of oncogenic misregulation and the advantage of functionally agnostic driver identification.

Simul-seq: combined DNA and RNA sequencing for whole-genome and transcriptome profiling

Paired DNA and RNA profiling is increasingly employed in genomics research to uncover molecular mechanisms of disease and to explore personal genotype and phenotype correlations. Here, we introduce Simul-seq, a technique for the production of high-quality whole-genome and transcriptome sequencing libraries from small quantities of cells or tissues. We apply the method to laser-capture-microdissected esophageal adenocarcinoma tissue, revealing a highly aneuploid tumor genome with extensive blocks of increased homozygosity and corresponding increases in allele-specific expression. Among this widespread allele-specific expression, we identify germline polymorphisms that are associated with response to cancer therapies. We further leverage this integrative data to uncover expressed mutations in several known cancer genes as well as a recurrent mutation in the motor domain of KIF3B that significantly affects kinesin–microtubule interactions. Simul-seq provides a new streamlined approach for generating comprehensive genome and transcriptome profiles from limited quantities of clinically relevant samples.

Use of Conditionally Active Ras Fusion Proteins to Study Epidermal Growth, Differentiation, and Neoplasia

Ras proteins are membrane-bound GTPases that play a central role in transmitting signals from the cell surface to the nucleus and affect a wide array of biological processes. The overall cellular response to Ras activation varies with cell type, experimental conditions, signal strength, and signal duration. Most current studies, however, rely on expression of constitutively active protein to study Ras function and thus ignore temporal variables, as well as signal strength. These experiments may provide contradictory results, as seen in the case of epidermal keratinocytes. In this setting, Ras has been shown to both promote and oppose proliferation and differentiation. By providing control over timing, duration, and signal magnitude, conditional systems allow for more precise investigation of the role of Ras in carcinogenesis, as well as normal cellular physiology. This chapter focuses on use of a ligand-responsive steroid hormone receptor fusion of Ras, ER-Ras, to study aspects of cellular transformation in epidermal keratinocytes.

A genome-wide association study identifies only two ancestry specific variants associated with spontaneous preterm birth

Preterm birth (PTB), or the delivery prior to 37 weeks of gestation, is a significant cause of infant morbidity and mortality. Although twin studies estimate that maternal genetic contributions account for approximately 30% of the incidence of PTB, and other studies reported fetal gene polymorphism association, to date no consistent associations have been identified. In this study, we performed the largest reported genome-wide association study analysis on 1,349 cases of PTB and 12,595 ancestry-matched controls from the focusing on genomic fetal signals. We tested over 2 million single nucleotide polymorphisms (SNPs) for associations with PTB across five subpopulations: African (AFR), the Americas (AMR), European, South Asian, and East Asian. We identified only two intergenic loci associated with PTB at a genome-wide level of significance: rs17591250 (P = 4.55E-09) on chromosome 1 in the AFR population and rs1979081 (P = 3.72E-08) on chromosome 8 in the AMR group. We have queried several existing replication cohorts and found no support of these associations. We conclude that the fetal genetic contribution to PTB is unlikely due to single common genetic variant, but could be explained by interactions of multiple common variants, or of rare variants affected by environmental influences, all not detectable using a GWAS alone.

Systematic identification of significantly mutated regions reveals a rich landscape of functional molecular alterations across cancer genomes

One Sentence Summary Identification of multi-scale mutational hotspots in cancer exomes facilitates understanding of mutations both within coding and non-coding elements. Cancer genome sequencing studies have identified cancer-driver genes from the increased accumulation of protein-altering mutations. However, the positional distributions of coding mutations, and the 79% of somatic variants in exome data that do not alter protein sequence or RNA splicing, remain largely unstudied. We employed density-based clustering methods on ∼4,700 exomes from 21 tumor types to detect variably-sized significantly mutated regions (SMRs). SMRs reveal recurrent alterations across a diverse spectrum of coding and non-coding elements, including microRNAs, transcription factor binding sites, and untranslated regions that are individually mutated in up to ∼15% of samples in specific cancer types. SMRs often associated with changes in gene expression and signalling. Mapping SMRs to protein structures revealed spatial clustering of somatic mutations at known and novel cancer-driver domains and molecular interfaces. Mutation frequencies in SMRs demonstrate that distinct protein regions are differentially mutated among tumor types. The functional diversity of SMRs underscores both the varied mechanisms of oncogenic misregulation and the advantage of unbiased driver identification.