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.
The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the ...processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation.
Dicer, which is required for the processing of both microRNAs (miRNAs) and small interfering RNAs (siRNAs), is essential for oocyte maturation
1, 2. Oocytes express both miRNAs and endogenous siRNAs ...(endo-siRNAs)
3, 4. To determine whether the abnormalities in
Dicer knockout oocytes during meiotic maturation are secondary to the loss of endo-siRNAs and/or miRNAs, we deleted
Dgcr8, which encodes an RNA-binding protein specifically required for miRNA processing. In striking contrast to
Dicer,
Dgcr8-deficient oocytes matured normally and, when fertilized with wild-type sperm, produced healthy-appearing offspring, even though miRNA levels were reduced to similar levels as
Dicer-deficient oocytes. Furthermore, the deletion of both maternal and zygotic
Dgcr8 alleles did not impair preimplantation development, including the determination of the inner cell mass and trophectoderm. Most surprisingly, the mRNA profiles of wild-type and
Dgcr8 null oocytes were essentially identical, whereas
Dicer null oocytes showed hundreds of misregulated transcripts. These findings show that miRNA function is globally suppressed during oocyte maturation and preimplantation development and that endo-siRNAs, rather than miRNAs, underlie the
Dicer knockout phenotype in oocytes.
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► MicroRNA function is globally suppressed in mature oocytes and early embryos ► Endo-siRNAs, not miRNAs, are essential for oocyte maturation ► Endo-siRNA and microRNA function are separable downstream of Dicer maturation
Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events ...that are driving these transitions. Here we used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA-binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation. Using this strategy, we identified highly specific patterns of mRNA recruitment to the polysomes that are synchronized with the cell cycle. A large number of the mRNAs recovered with translating ribosomes contain motifs for the RNA-binding proteins DAZL (deleted in azoospermia-like) and CPEB (cytoplasmic polyadenylation element-binding protein). Although a Dazl role in early germ cell development is well established, no function has been described during oocyte-to-embryo transition. We demonstrate that CPEB1 regulates Dazl post-transcriptionally, and that DAZL is essential for meiotic maturation and embryonic cleavage. In the absence of DAZL synthesis, the meiotic spindle fails to form due to disorganization of meiotic microtubules. Therefore, Cpeb1 and Dazl function in a progressive, self-reinforcing pathway to promote oocyte maturation and early embryonic development.
Although cancer genomes are replete with noncoding mutations, the effects of these mutations remain poorly characterized. Here we perform an integrative analysis of 930 tumor whole genomes and ...matched transcriptomes, identifying a network of 193 noncoding loci in which mutations disrupt target gene expression. These 'somatic eQTLs' (expression quantitative trait loci) are frequently mutated in specific cancer tissues, and the majority can be validated in an independent cohort of 3,382 tumors. Among these, we find that the effects of noncoding mutations on DAAM1, MTG2 and HYI transcription are recapitulated in multiple cancer cell lines and that increasing DAAM1 expression leads to invasive cell migration. Collectively, the noncoding loci converge on a set of core pathways, permitting a classification of tumors into pathway-based subtypes. The somatic eQTL network is disrupted in 88% of tumors, suggesting widespread impact of noncoding mutations in cancer.
Estimating the abundance of cell-free DNA (cfDNA) fragments shed from a tumor (i.e., circulating tumor DNA (ctDNA)) can approximate tumor burden, which has numerous clinical applications. We derived ...a novel, broadly applicable statistical method to quantify cancer-indicative methylation patterns within cfDNA to estimate ctDNA abundance, even at low levels. Our algorithm identified differentially methylated regions (DMRs) between a reference database of cancer tissue biopsy samples and cfDNA from individuals without cancer. Then, without utilizing matched tissue biopsy, counts of fragments matching the cancer-indicative hyper/hypo-methylated patterns within DMRs were used to determine a tumor methylated fraction (TMeF; a methylation-based quantification of the circulating tumor allele fraction and estimate of ctDNA abundance) for plasma samples. TMeF and small variant allele fraction (SVAF) estimates of the same cancer plasma samples were correlated (Spearman's correlation coefficient: 0.73), and synthetic dilutions to expected TMeF of 10
and 10
had estimated TMeF within two-fold for 95% and 77% of samples, respectively. TMeF increased with cancer stage and tumor size and inversely correlated with survival probability. Therefore, tumor-derived fragments in the cfDNA of patients with cancer can be leveraged to estimate ctDNA abundance without the need for a tumor biopsy, which may provide non-invasive clinical approximations of tumor burden.
The miR-294 and miR-302 microRNAs promote the abbreviated G1 phase of the embryonic stem cell (ESC) cell cycle and suppress differentiation induced by let-7. Here, we evaluated the role of the ...retinoblastoma (Rb) family proteins in these settings. Under normal growth conditions, miR-294 promoted the rapid G1-S transition independent of the Rb family. In contrast, miR-294 suppressed the further accumulation of cells in G1 in response to nutrient deprivation and cell-cell contact in an Rb-dependent fashion. We uncovered five additional miRNAs (miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218) that silenced ESC self-renewal in the absence of other miRNAs, all of which were antagonized by miR-294 and miR-302. Four of the six differentiation-inducing miRNAs induced an Rb-dependent G1 accumulation. However, all six still silenced self-renewal in the absence of the Rb proteins. These results show that the miR-294/miR-302 family acts through Rb-dependent and -independent pathways to regulate the G1 restriction point and the silencing of self-renewal, respectively.
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•miR-294/miR-302 promote G1-S in ESCs through Rb-dependent and -independent mechanisms•Multiple miRNAs silence the pluripotency program of Dgcr8-null but not WT ESCs•A subset of the diff-inducing miRNAs activate the Rb-dependent restriction point•miR-294/miR-302 antagonize the diff-inducing miRNAs through an Rb-independent mechanism
The miR-294/miR-302 family promotes embryonic stem cell proliferation with an abbreviated G1 phase while also suppressing differentiation. Whether these distinct phenotypes reflect a common underlying molecular mechanism is not known. Here, Blelloch, Wang, and colleagues tackle this problem by evaluating the epistatic relationship of the miR-294 and miR-302 miRNAs to the retinoblastoma family of proteins. They find that the cellular phenotypes reflect multiple, rather than a single, common target pathway, emphasizing the broad impact of miRNAs on the biology of stem cells.
Stem cell differentiation requires a complex coordination of events to transition from a self-renewing to a differentiated cell fate. Stem cells can be pluripotent (capable of giving rise to all ...embryonic lineages), multipotent (possessing the potential to give rise to multiple lineages) and unipotent (capable of given rise to a single cell lineage). Regardless of their potency all stem cells must silence their self-renewal program during differentiation. The self-renewal program can be defined as the integration of external and internal stimuli that enables a cell to proliferate while maintaining its potency. Two hallmarks of the self-renewal program are a self-reinforcing transcriptional network and a specialized cell-cycle profile. In this chapter we discuss the impact of various microRNAs (miRNAs) to either reinforce or inhibit the self-renewal program of stem cells and how this added regulatory layer provides robustness to cell-fate decisions. We will focus on embryonic stem cells (ESCs) describing miRNA function in self-renewal, differentiation and de-differentiation. We will compare and contrast miRNA functions in ESCs with miRNA function in lineage specific somatic stem cells and in cancer.
When embryonic stem cells (ESCs) differentiate, they must both silence the ESC self-renewal program and activate new tissue-specific programs. In the absence of DGCR8 (Dgcr8(-/-)), a protein required ...for microRNA (miRNA) biogenesis, mouse ESCs are unable to silence self-renewal. Here we show that the introduction of let-7 miRNAs-a family of miRNAs highly expressed in somatic cells-can suppress self-renewal in Dgcr8(-/-) but not wild-type ESCs. Introduction of ESC cell cycle regulating (ESCC) miRNAs into the Dgcr8(-/-) ESCs blocks the capacity of let-7 to suppress self-renewal. Profiling and bioinformatic analyses show that let-7 inhibits whereas ESCC miRNAs indirectly activate numerous self-renewal genes. Furthermore, inhibition of the let-7 family promotes de-differentiation of somatic cells to induced pluripotent stem cells. Together, these findings show how the ESCC and let-7 miRNAs act through common pathways to alternatively stabilize the self-renewing versus differentiated cell fates.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Early cancer detection could identify tumors at a time when outcomes are superior and treatment is less morbid. This prospective case-control sub-study (from NCT02889978 and NCT03085888) assessed the ...performance of targeted methylation analysis of circulating cell-free DNA (cfDNA) to detect and localize multiple cancer types across all stages at high specificity.
The 6689 participants 2482 cancer (>50 cancer types), 4207 non-cancer were divided into training and validation sets. Plasma cfDNA underwent bisulfite sequencing targeting a panel of >100 000 informative methylation regions. A classifier was developed and validated for cancer detection and tissue of origin (TOO) localization.
Performance was consistent in training and validation sets. In validation, specificity was 99.3% 95% confidence interval (CI): 98.3% to 99.8%; 0.7% false-positive rate (FPR). Stage I–III sensitivity was 67.3% (CI: 60.7% to 73.3%) in a pre-specified set of 12 cancer types (anus, bladder, colon/rectum, esophagus, head and neck, liver/bile-duct, lung, lymphoma, ovary, pancreas, plasma cell neoplasm, stomach), which account for ∼63% of US cancer deaths annually, and was 43.9% (CI: 39.4% to 48.5%) in all cancer types. Detection increased with increasing stage: in the pre-specified cancer types sensitivity was 39% (CI: 27% to 52%) in stage I, 69% (CI: 56% to 80%) in stage II, 83% (CI: 75% to 90%) in stage III, and 92% (CI: 86% to 96%) in stage IV. In all cancer types sensitivity was 18% (CI: 13% to 25%) in stage I, 43% (CI: 35% to 51%) in stage II, 81% (CI: 73% to 87%) in stage III, and 93% (CI: 87% to 96%) in stage IV. TOO was predicted in 96% of samples with cancer-like signal; of those, the TOO localization was accurate in 93%.
cfDNA sequencing leveraging informative methylation patterns detected more than 50 cancer types across stages. Considering the potential value of early detection in deadly malignancies, further evaluation of this test is justified in prospective population-level studies.
•Targeted methylation analysis of cfDNA simultaneously detected and localized >50 cancer types, including high-mortality cancers that lack screening paradigms.•Cancers were detected across all stages (stage I–III sensitivity: 43.9%; stage I–IV sensitivity: 54.9%) at a specificity of >99% and a single false positive rate of <1%.•This targeted methylation approach localized the tissue of origin with >90% accuracy, which will be critical for directing follow-up care.•This supports the continued investigation of this test with the goal of population-scale early multi-cancer detection.
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