Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors.

Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.

KMT2C-deficient tumors have elevated APOBEC mutagenesis and genomic instability in multiple cancers.

The histone methyltransferase KMT2C is among the most frequently mutated epigenetic modifier genes in cancer and plays an essential role in MRE11-dependent DNA replication fork restart. However, the effects of KMT2C deficiency on genomic instability during tumorigenesis are unclear. Analyzing 9,663 tumors from 30 cancer cohorts, we report that KMT2C mutant tumors have a significant excess of APOBEC mutational signatures in several cancer types. We show that KMT2C deficiency promotes APOBEC expression and deaminase activity, and compromises DNA replication speed and delays fork restart, facilitating APOBEC mutagenesis targeting single stranded DNA near stalled forks. APOBEC-mediated mutations primarily accumulate during early replication and tend to cluster along the genome and also in 3D nuclear domains. Excessive APOBEC mutational signatures in KMT2C mutant tumors correlate with elevated genome maintenance defects and signatures of homologous recombination deficiency. We propose that KMT2C deficiency is a likely promoter of APOBEC mutagenesis, which fosters further genomic instability during tumor progression in multiple cancer types.

Clustered 8-Oxo-Guanine Mutations and Oncogenic Gene Fusions in Microsatellite-Unstable Colorectal Cancer.

PURPOSE: Colorectal carcinomas (CRCs) with microsatellite-instability (MSI) are enriched for oncogenic kinase fusions (KFs), including NTRK1, RET, and BRAF, but the mechanism underlying this finding is unclear. METHODS: The genomic profiles of 32,218 advanced CRC tumor specimens were analyzed to assess the fusion breakpoints of oncogenic alterations including KFs in microsatellite-stable and microsatellite-unstable CRC. Genomic contexts of such alterations were analyzed to obtain mechanistic insights. RESULTS: Genomic analysis demonstrated that oncogenic fusion breakpoints in MSI tumors do not preferentially involve repetitive or low-complexity sequences. Instead, their junction regions showed pronounced guanine and cytosine bias and elevated mutation frequency at G:C contexts. Elevated mutation frequency at G:C bases in relevant introns predicted prevalence of associated oncogenic fusions in MSI CRCs. CRCs harboring mismatch repair signatures had enrichment of butyrate-producing microbial species, reported to be associated with induction of 8-oxoguanine lesions in the intestine. CONCLUSION: Detailed analysis of breakpoints in MSI-associated KFs support a model in which inefficient repair and/or processing of microbiome-induced clustered 8-oxoguanine damage in MSI CRC contributes to the increased incidence of specific oncogenic fusions.

Mutational signatures associated with exposure to carcinogenic microplastic compounds bisphenol A and styrene oxide.

Microplastic pollutants in oceans and food chains are concerning to public health. Common plasticizing compounds Bisphenol-A (BPA) and Styrene-7,8-Oxide (SO) are now labeled as carcinogens. We show that BPA and SO cause deoxyribonucleic acid damage and mutagenesis in human cells, and analyze the genome-wide point mutation and genomic rearrangement patterns associated with BPA and SO exposure. A subset of the single- and doublet base substitutions shows mutagenesis near or at guanine, consistent with these compounds' preferences to form guanosine adducts. Presence of other mutational signatures suggest additional mutagenesis probably due to complex effects of BPA and SO on diverse cellular processes. Analyzing data for 19 cancer cohorts, we find that tumors of digestive and urinary organs show relatively high similarity in mutational profiles, and the burden of such mutations increases with age. Even within the same cancer type, proportions of corresponding mutational patterns vary among the cohorts from different countries, as does the amount of microplastic waste in ocean waters. BPA and SO are relatively mild mutagens, and other environmental agents can also potentially generate similar, complex mutational patterns in cancer genomes. Nonetheless, our findings call for systematic evaluation of public health consequences of microplastic exposure worldwide.

Characteristics of mutational signatures of unknown etiology.

Although not all somatic mutations are cancer drivers, their mutational signatures, i.e. the patterns of genomic alterations at a genome-wide scale, provide insights into past exposure to mutagens, DNA damage and repair processes. Computational deconvolution of somatic mutation patterns and expert curation pan-cancer studies have identified a number of mutational signatures associated with point mutations, dinucleotide substitutions, insertions and deletions, and rearrangements, and have established etiologies for a subset of these signatures. However, the mechanisms underlying nearly one-third of all mutational signatures are not yet understood. The signatures with established etiology and those with hitherto unknown origin appear to have some differences in strand bias, GC content and nucleotide context diversity. It is possible that some of the hitherto 'unknown' signatures predominantly occur outside gene regions. While nucleotide contexts might be adequate to establish etiologies of some mutational signatures, in other cases additional features, such as broader (epi)genomic contexts, including chromatin, replication timing, processivity and local mutational patterns, may help fully understand the underlying DNA damage and repair processes. Nonetheless, remarkable progress in characterization of mutational signatures has provided fundamental insights into the biology of cancer, informed disease etiology and opened up new opportunities for cancer prevention, risk management, and therapeutic decision making.

Mutational signature SBS8 predominantly arises due to late replication errors in cancer.

Although a majority of somatic mutations in cancer are passengers, their mutational signatures provide mechanistic insights into mutagenesis and DNA repair processes. Mutational signature SBS8 is common in most cancers, but its etiology is debated. Incorporating genomic, epigenomic, and cellular process features for multiple cell-types we develop genome-wide composite epigenomic context-maps relevant for mutagenesis and DNA repair. Analyzing somatic mutation data from multiple cancer types in their epigenomic contexts, we show that SBS8 preferentially occurs in gene-poor, lamina-proximal, late replicating heterochromatin domains. While SBS8 is uncommon among mutations in non-malignant tissues, in tumor genomes its proportions increase with replication timing and speed, and checkpoint defects further promote this signature - suggesting that SBS8 probably arises due to uncorrected late replication errors during cancer progression. Our observations offer a potential reconciliation among different perspectives in the debate about the etiology of SBS8 and its relationship with other mutational signatures.

Non-Genetic Intra-Tumor Heterogeneity Is a Major Predictor of Phenotypic Heterogeneity and Ongoing Evolutionary Dynamics in Lung Tumors.

Impacts of genetic and non-genetic intra-tumor heterogeneity (ITH) on tumor phenotypes and evolvability remain debated. We analyze ITH in lung squamous cell carcinoma at the levels of genome, transcriptome, and tumor-immune interactions and histopathological characteristics by multi-region bulk and single-cell sequencing. Genomic heterogeneity alone is a weak indicator of intra-tumor non-genetic heterogeneity at immune and transcriptomic levels that impact multiple cancer-related pathways, including those related to proliferation and inflammation, which in turn contribute to intra-tumor regional differences in histopathology and subtype classification. Tumor subclones have substantial differences in proliferation score, suggestive of non-neutral clonal dynamics. Proliferation and other cancer-related pathways also show intra-tumor regional differences, sometimes even within the same subclones. Neo-epitope burden negatively correlates with immune infiltration, indicating immune-mediated purifying selection on somatic mutations. Taken together, our observations suggest that non-genetic heterogeneity is a major determinant of heterogeneity in histopathological characteristics and impacts evolutionary dynamics in lung cancer.

Comparative mRNA and miRNA expression in European mouflon (Ovis musimon) and sheep (Ovis aries) provides novel insights into the genetic mechanisms for female reproductive success.

Prolific breeds of domestic sheep (Ovis aries) are important genetic resources due to their reproductive performance, which is characterized by multiple lambs per birth and out-of-season breeding. However, the lack of a comprehensive understanding of the genetic mechanisms underlying the important reproductive traits, particularly from the evolutionary genomics perspective, has impeded the efficient advancement of sheep breeding. Here, for the first time, by performing RNA-sequencing we built a de novo transcriptome assembly of ovarian and endometrial tissues in European mouflon (Ovis musimon) and performed an mRNA-miRNA integrated expression profiling analysis of the wild species and a highly prolific domestic sheep breed, the Finnsheep. We identified several novel genes with differentially expressed mRNAs (e.g., EREG, INHBA, SPP1, AMH, TDRD5, and ZP2) between the wild and domestic sheep, which are functionally involved in oocyte and follicle development and fertilization, and are significantly (adjusted P-value < 0.05) enriched in the Gene Ontology (GO) terms of various reproductive process, including the regulation of fertilization, oogenesis, ovarian follicle development, and sperm-egg recognition. Additionally, we characterized 58 differentially expressed miRNAs and 210 associated target genes that are essential for the regulation of female reproduction cycles through specific regulatory networks [e.g., (miR-136, miR-374a, miR-9-5p)-(EREG, INHBA)]. Furthermore, our integrated mRNA and miRNA expression profiling analysis elucidated novel direct and indirect miRNA/mRNA causal regulatory relationships related to the reproductive traits of the Ovis species. This study provides in-depth insights into the genomic evolution underlying the reproductive traits of the Ovis species and valuable resources for ovine genomics.

The Genome Landscape of Tibetan Sheep Reveals Adaptive Introgression from Argali and the History of Early Human Settlements on the Qinghai-Tibetan Plateau.

Tibetan sheep are the most common and widespread domesticated animals on the Qinghai-Tibetan Plateau (QTP) and have played an essential role in the permanent human occupation of this high-altitude region. However, the precise timing, route, and process of sheep pastoralism in the QTP region remain poorly established, and little is known about the underlying genomic changes that occurred during the process. Here, we investigate the genomic variation in Tibetan sheep using whole-genome sequences, single nucleotide polymorphism arrays, mitochondrial DNA, and Y-chromosomal variants in 986 samples throughout their distribution range. We detect strong signatures of selection in genes involved in the hypoxia and ultraviolet signaling pathways (e.g., HIF-1 pathway and HBB and MITF genes) and in genes associated with morphological traits such as horn size and shape (e.g., RXFP2). We identify clear signals of argali (Ovis ammon) introgression into sympatric Tibetan sheep, covering 5.23-5.79% of their genomes. The introgressed genomic regions are enriched in genes related to oxygen transportation system, sensory perception, and morphological phenotypes, in particular the genes HBB and RXFP2 with strong signs of adaptive introgression. The spatial distribution of genomic diversity and demographic reconstruction of the history of Tibetan sheep show a stepwise pattern of colonization with their initial spread onto the QTP from its northeastern part ∼3,100 years ago, followed by further southwest expansion to the central QTP ∼1,300 years ago. Together with archeological evidence, the date and route reveal the history of human expansions on the QTP by the Tang-Bo Ancient Road during the late Holocene. Our findings contribute to a depth understanding of early pastoralism and the local adaptation of Tibetan sheep as well as the late-Holocene human occupation of the QTP.

Global genomic diversity and conservation priorities for domestic animals are associated with the economies of their regions of origin.

Domestic animals play a key role in human survival and the development of civilization. However, the genetic resources of domestic animals are facing an alarming rate of erosion due to socioeconomic changes, economic globalization and financial constraints. In this study, through genome-wide SNP analysis, we estimated the heterozygosity, inbreeding coefficient, effective population size, and runs of homozygosity to identify the breeds facing the risk of extinction for sheep and cattle across the world. In particular, we quantified the contribution of 97 sheep breeds and 53 cattle breeds to genomic diversity (within-breed, between-breed and total) and prioritized the breeds for conservation. Additionally, we compared the average values of genomic diversity between breeds from regions (or countries) in different economic categories (underdeveloped, developing and developed), and found that breeds in developed regions exhibit significantly higher levels of total genomic diversity than those in underdeveloped and developing regions. Altogether, our results suggested that conservation priority should be given to breeds in developed regions to secure the future genomic diversity hotspots of domestic animal resources.

Genomic Reconstruction of the History of Native Sheep Reveals the Peopling Patterns of Nomads and the Expansion of Early Pastoralism in East Asia.

China has a rich resource of native sheep (Ovis aries) breeds associated with historical movements of several nomadic societies. However, the history of sheep and the associated nomadic societies in ancient China remains poorly understood. Here, we studied the genomic diversity of Chinese sheep using genome-wide SNPs, mitochondrial and Y-chromosomal variations in > 1,000 modern samples. Population genomic analyses combined with archeological records and historical ethnic demographics data revealed genetic signatures of the origins, secondary expansions and admixtures, of Chinese sheep thereby revealing the peopling patterns of nomads and the expansion of early pastoralism in East Asia. Originating from the Mongolian Plateau ∼5,000‒5,700 years ago, Chinese sheep were inferred to spread in the upper and middle reaches of the Yellow River ∼3,000‒5,000 years ago following the expansions of the Di-Qiang people. Afterwards, sheep were then inferred to reach the Qinghai-Tibetan and Yunnan-Kweichow plateaus ∼2,000‒2,600 years ago by following the north-to-southwest routes of the Di-Qiang migration. We also unveiled two subsequent waves of migrations of fat-tailed sheep into northern China, which were largely commensurate with the migrations of ancestors of Hui Muslims eastward and Mongols southward during the 12th‒13th centuries. Furthermore, we revealed signs of argali introgression into domestic sheep, extensive historical mixtures among domestic populations and strong artificial selection for tail type and other traits, reflecting various breeding strategies by nomadic societies in ancient China.

Identification and characterization of miRNAs in the ovaries of a highly prolific sheep breed.

Until recently, there have been few studies concerning miRNAs or miRNA-mediated biological processes in sheep (Ovis aries). In the present study, we used a deep-sequencing approach to examine ovarian miRNAs and the mRNA transcriptomes in two ewes of a highly prolific breed, Finnsheep. We identified 113 known sheep miRNAs, 131 miRNAs conserved in other mammals and 60 novel miRNAs, the expression levels of which accounted for 78.22%, 21.73% and 0.05% of the total respectively. Furthermore, the 10 most abundantly expressed miRNAs in the two libraries were characterized in detail, and the putative target genes of these miRNAs were annotated using GO annotation and KEGG pathway enrichment analyses. Among the target genes, intracellular transducers (SMAD1, SMAD4, SMAD5 and SMAD9) and bone morphogenetic protein (BMP) receptors (BMPR1B and BMPR2) were involved in the transforming growth factor ß (TGFß) signaling pathway in the reproductive axis, and the most significant GO terms were intracellular part (GO:0044424), binding (GO:0005488) and biological_process (GO:0008150) for cellular component, molecular function and biological process respectively. Thus, these results expanded the sheep miRNA database and provided additional information on the prolificacy trait regulated through specific miRNAs in sheep and other mammals.

Phylogeography of Nanorana parkeri (Anura: Ranidae) and multiple refugia on the Tibetan Plateau revealed by mitochondrial and nuclear DNA.

Quaternary climatic changes have been recognized to influence the distribution patterns and evolutionary histories of extant organisms, but their effects on alpine species are not well understood. To investigate the Pleistocene climatic oscillations on the genetic structure of amphibians, we sequenced one mitochondrial and three nuclear DNA fragments in Nanorana parkeri, a frog endemic to the Tibetan Plateau, across its distribution range in the southern plateau. Mitochondrial cytochrome b (Cytb) and three nuclear genes (c-Myc2, Rhod, and Tyr) revealed two distinct lineages (i.e. the lineages East and West), which were strongly geographically structured. The split of the two divergent lineages was dated back earlier than the Middle Pleistocene, probably being associated with climatic and ecological factors. Species distribution modeling, together with the phylogeographic structuring, supported the hypothesis of multiple refugia for N. parkeri on the Tibetan Plateau during the Pleistocene glaciations, and suggested the Yarlung Zangbo valley and the Kyichu catchment to be the potential refugia. Our findings indicate that Pleistocene climatic changes have had a great impact on the evolution and demographic history of N. parkeri. Our study has important implications for conservation of this and other frog species in the Tibetan Plateau.

Systematic identification and characterization of long intergenic non-coding RNAs in fetal porcine skeletal muscle development.

Long intergenic non-coding RNAs (lincRNAs) play important roles in many cellular processes. Here, we present the first systematic identification and characterization of lincRNAs in fetal porcine skeletal muscle. We obtained a total of 55.02 million 90-bp paired-end reads and assembled 54,550 transcripts using cufflinks. We developed a pipeline to identify 570 multi-exon lincRNAs by integrating a set of previous approaches. These putative porcine lincRNAs share many characteristics with mammalian lincRNAs, such as a relatively short length, small number of exons and low level of sequence conservation. We found that the porcine lincRNAs were preferentially located near genes mediating transcriptional regulation rather than those with developmental functions. We further experimentally analyzed the features of a conserved mouse lincRNA gene and found that isoforms 1 and 4 of this lincRNA were enriched in the cell nucleus and were associated with polycomb repressive complex 2 (PRC2). Our results provide a catalog of fetal porcine lincRNAs for further experimental investigation of the functions of these genes in the skeletal muscle developmental process.