Measuring multiple omics profiles from the same single cell opens up the opportunity to decode molecular regulation that underlies intercellular heterogeneity in development and disease. Here, we ...present co-sequencing of microRNAs and mRNAs in the same single cell using a half-cell genomics approach. This method demonstrates good robustness (~95% success rate) and reproducibility (R
= 0.93 for both microRNAs and mRNAs), yielding paired half-cell microRNA and mRNA profiles, which we can independently validate. By linking the level of microRNAs to the expression of predicted target mRNAs across 19 single cells that are phenotypically identical, we observe that the predicted targets are significantly anti-correlated with the variation of abundantly expressed microRNAs. This suggests that microRNA expression variability alone may lead to non-genetic cell-to-cell heterogeneity. Genome-scale analysis of paired microRNA-mRNA co-profiles further allows us to derive and validate regulatory relationships of cellular pathways controlling microRNA expression and intercellular variability.
Ten-Eleven-Translocation-2 (Tet2) is a DNA methylcytosine dioxygenase that functions as a tumor suppressor in hematopoietic malignancies. We examined the role of Tet2 in tumor-tissue myeloid cells ...and found that Tet2 sustains the immunosuppressive function of these cells. We found that Tet2 expression is increased in intratumoral myeloid cells both in mouse models of melanoma and in melanoma patients and that this increased expression is dependent on an IL-1R-MyD88 pathway. Ablation of Tet2 in myeloid cells suppressed melanoma growth in vivo and shifted the immunosuppressive gene expression program in tumor-associated macrophages to a proinflammatory one, with a concomitant reduction of the immunosuppressive function. This resulted in increased numbers of effector T cells in the tumor, and T cell depletion abolished the reduced tumor growth observed upon myeloid-specific deletion of Tet2. Our findings reveal a non-cell-intrinsic, tumor-promoting function for Tet2 and suggest that Tet2 may present a therapeutic target for the treatment of non-hematologic malignancies.
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•Deletion of Tet2 in myeloid cells reduces melanoma tumor burden•Tet2 expression is induced via the IL-1R-MyD88 axis in tumor-associated macrophages•Tet2 maintains myeloid immunosuppressive function and the associated genetic program•Myeloid-specific Tet2 deletion results in higher numbers of tumor-infiltrating T cells
The DNA methylcytosine dioxygenase Tet2 functions as a tumor suppressor in multiple contexts, including hematopoietic malignancies. Pan et al. now reveal a tumor-promoting role for Tet2, whereby Tet2 functions to sustain an immunosuppressive program in myeloid cells that in turn dampens the anti-tumor T cell response.
Mature microRNAs (miRNAs) are processed from hairpin-containing primary miRNAs (pri-miRNAs). However, rules that distinguish pri-miRNAs from other hairpin-containing transcripts in the genome are ...incompletely understood. By developing a computational pipeline to systematically evaluate 30 structural and sequence features of mammalian RNA hairpins, we report several new rules that are preferentially utilized in miRNA hairpins and govern efficient pri-miRNA processing. We propose that a hairpin stem length of 36 ± 3 nt is optimal for pri-miRNA processing. We identify two bulge-depleted regions on the miRNA stem, located ∼16-21 nt and ∼28-32 nt from the base of the stem, that are less tolerant of unpaired bases. We further show that the CNNC primary sequence motif selectively enhances the processing of optimal-length hairpins. We predict that a small but significant fraction of human single-nucleotide polymorphisms (SNPs) alter pri-miRNA processing, and confirm several predictions experimentally including a disease-causing mutation. Our study enhances the rules governing mammalian pri-miRNA processing and suggests a diverse impact of human genetic variation on miRNA biogenesis.
Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient and is thought to be a stochastic process. We identified a privileged somatic cell state, from ...which acquisition of pluripotency could occur in a nonstochastic manner. Subsets of murine hematopoietic progenitors are privileged whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after four to five divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ∼8 hr. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression and is increased by p53 knockdown. This ultrafast cycling population accounts for >99% of the bulk reprogramming activity in wild-type or p53 knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state and suggest that cell-cycle acceleration toward a critical threshold is an important bottleneck for reprogramming.
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•Reprogramming occurs nonstochastically from a privileged somatic cell state•Subsets of granulocyte-monocyte progenitors (GMPs) are privileged•Privileged GMPs cycle with an ultrafast speed: about 8 hr per cycle•Cells of similar cycling speed are induced prior to reprogramming of fibroblasts
Contrary to the widely accepted view that reprogramming by Yamanaka factors is stochastic, there is a somatic cell state characterized by an unusually fast cell cycle that confers the ability on specific cells to undergo reprogramming nonstochastically.
In cystic fibrosis (CF) patients, hyper-inflammation is a key factor in lung destruction and disease morbidity. We have previously demonstrated that macrophages drive the lung hyper-inflammatory ...response to LPS in CF mice, because of reduced levels of the scaffold protein CAV1 with subsequent uncontrolled TLR4 signalling. Here we show that reduced CAV1 and, consequently, increased TLR4 signalling, in human and murine CF macrophages and murine CF lungs, is caused by high microRNA-199a-5p levels, which are PI3K/AKT-dependent. Downregulation of microRNA-199a-5p or increased AKT signalling restores CAV1 expression and reduces hyper-inflammation in CF macrophages. Importantly, the FDA-approved drug celecoxib re-establishes the AKT/miR-199a-5p/CAV1 axis in CF macrophages, and ameliorates lung hyper-inflammation in Cftr-deficient mice. Thus, we identify the AKT/miR-199a-5p/CAV1 pathway as a regulator of innate immunity, which is dysfunctional in CF macrophages contributing to lung hyper-inflammation. In addition, we found that this pathway can be targeted by celecoxib.
Genetic variations within microRNA (miRNA) binding sites can affect miRNA-mediated gene regulation, which may lead to phenotypes and diseases. We perform a transcriptome-scale analysis of genetic ...variants and miRNA:target interactions identified by CLASH. This analysis reveals that rare variants tend to reside in CDSs, whereas common variants tend to reside in the 3' UTRs. miRNA binding sites are more likely to reside within those targets in the transcriptome with lower variant densities, especially target regions in which nucleotides have low mutation frequencies. Furthermore, an overwhelming majority of genetic variants within or near miRNA binding sites can alter not only the potential of miRNA:target hybridization but also the structural accessibility of the binding sites and flanking regions. These suggest an interpretation for certain associations between genetic variants and diseases, i.e. modulation of miRNA-mediated gene regulation by common or rare variants within or near miRNA binding sites, likely through target structure alterations. Our data will be valuable for discovering new associations among miRNAs, genetic variations and human diseases.
Actin cytoskeleton is well-known for providing structural/mechanical support, but whether and how it regulates chromatin and cell fate reprogramming is far less clear. Here, we report that MKL1, the ...key transcriptional co-activator of many actin cytoskeletal genes, regulates genomic accessibility and cell fate reprogramming. The MKL1-actin pathway weakens during somatic cell reprogramming by pluripotency transcription factors. Cells that reprogram efficiently display low endogenous MKL1 and inhibition of actin polymerization promotes mature pluripotency activation. Sustained MKL1 expression at a level seen in typical fibroblasts yields excessive actin cytoskeleton, decreases nuclear volume and reduces global chromatin accessibility, stalling cells on their trajectory toward mature pluripotency. In addition, the MKL1-actin imposed block of pluripotency can be bypassed, at least partially, when the Sun2-containing linker of the nucleoskeleton and cytoskeleton (LINC) complex is inhibited. Thus, we unveil a previously unappreciated aspect of control on chromatin and cell fate reprogramming exerted by the MKL1-actin pathway.
Background
Patient‐derived organoids and xenografts (PDXs) have emerged as powerful models in functional diagnostics with high predictive power for anticancer drug response. However, limitations such ...as engraftment failure and time‐consuming for establishing and expanding PDX models followed by testing drug efficacy, and inability to subject to systemic drug administration for ex vivo organoid culture hinder realistic and fast decision‐making in selecting the right therapeutics in the clinic. The present study aimed to develop an advanced PDX model, namely MiniPDX, for rapidly testing drug efficacy to strengthen its value in personalized cancer treatment.
Methods
We developed a rapid in vivo drug sensitivity assay, OncoVee® MiniPDX, for screening clinically relevant regimens for cancer. In this model, patient‐derived tumor cells were arrayed within hollow fiber capsules, implanted subcutaneously into mice and cultured for 7 days. The cellular activity morphology and pharmacokinetics were systematically evaluated. MiniPDX performance (sensitivity, specificity, positive and negative predictive values) was examined using PDX as the reference. Drug responses were examined by tumor cell growth inhibition rate and tumor growth inhibition rate in PDX models and MiniPDX assays respectively. The results from MiniPDX were also used to evaluate its predictive power for clinical outcomes.
Results
Morphological and histopathological features of tumor cells within the MiniPDX capsules matched those both in PDX models and in original tumors. Drug responses in the PDX tumor graft assays correlated well with those in the corresponding MiniPDX assays using 26 PDX models generated from patients, including 14 gastric cancer, 10 lung cancer and 2 pancreatic cancer. The positive predictive value of MiniPDX was 92%, and the negative predictive value was 81% with a sensitivity of 80% and a specificity of 93%. Through expanding to clinical tumor samples, MiniPDX assay showed potential of wide clinical application.
Conclusions
Fast in vivo MiniPDX assay based on capsule implantation was developed‐to assess drug responses of both PDX tumor grafts and clinical cancer specimens. The high correlation between drug responses of paired MiniPDX and PDX tumor graft assay, as well as translational data suggest that MiniPDX assay is an advanced tool for personalized cancer treatment.
Clustered regularly-interspaced palindromic repeats (CRISPR)-based genetic screens using single-guide-RNA (sgRNA) libraries have proven powerful to identify genetic regulators. Applying CRISPR ...screens to interrogate functional elements in noncoding regions requires generating sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. Here we present a Molecular Chipper technology for generating dense sgRNA libraries for genomic regions of interest, and a proof-of-principle screen that identifies novel cis-regulatory domains for miR-142 biogenesis. The Molecular Chipper approach utilizes a combination of random fragmentation and a type III restriction enzyme to derive a densely covering sgRNA library from input DNA. Applying this approach to 17 microRNAs and their flanking regions and with a reporter for miR-142 activity, we identify both the pre-miR-142 region and two previously unrecognized cis-domains important for miR-142 biogenesis, with the latter regulating miR-142 processing. This strategy will be useful for identifying functional noncoding elements in mammalian genomes.
Hematopoietic stem and progenitor cells are often undesired targets of chemotherapies, leading to hematopoietic suppression requiring careful clinical management. Whether microRNAs control ...hematopoietic injury response is largely unknown. We report an in vivo gain-of-function screen and the identification of miR-150 as an inhibitor of hematopoietic recovery upon 5-fluorouracil-induced injury. Utilizing a bone marrow transplant model with a barcoded microRNA library, we screened for barcode abundance in peripheral blood of recipient mice before and after 5-fluorouracil treatment. Overexpression of screen-candidate miR-150 resulted in significantly slowed recovery rates across major blood lineages, with associated impairment of bone marrow clonogenic potential. Conversely, platelets and myeloid cells from miR-150 null marrow recovered faster after 5-fluorouracil treatment. Heterozygous knockout of c-myb, a conserved target of miR-150, partially phenocopied miR-150-forced expression. Our data highlight the role of microRNAs in controlling hematopoietic injury response and demonstrate the power of in vivo functional screens for studying microRNAs in normal tissue physiology.
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► In vivo miRNA screen identified regulators of hematopoietic injury response ► miR-150 expression inhibits hematopoietic recovery across multiple lineages ► Upon injury, miR-150 null cells contribute faster to peripheral blood recovery ► Partial loss of the miR-150 target c-myb delays hematopoietic recovery
The ability to control the hematopoietic-suppressive side effects of chemotherapy is highly desired but understudied. Lu and colleagues report an in vivo gain-of-function screen to delineate microRNA-mediated mechanisms that control the rate of peripheral blood cell recovery after chemotherapy. Following up on one of several validated candidates, they find that overexpression of miR-150 inhibits recovery upon 5-fluorouracil-induced injury, whereas miR-150 knockout cells recover faster. These studies highlight the role of microRNAs in controlling hematopoietic injury response and demonstrate the power of in vivo microRNA screens for complex physiological processes.
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