Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

Brd4-bound enhancers drive cell-intrinsic sex differences in glioblastoma.

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited sex-specific responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET (bromodomain and extraterminal) inhibitors than are female cells. Thus, Brd4 activity is revealed to drive sex differences in stem cell and tumorigenic phenotypes, which can be abrogated by sex-specific responses to BET inhibition. This has important implications for the clinical evaluation and use of BET inhibitors.

Human L1 Transposition Dynamics Unraveled with Functional Data Analysis.

Long INterspersed Elements-1 (L1s) constitute >17% of the human genome and still actively transpose in it. Characterizing L1 transposition across the genome is critical for understanding genome evolution and somatic mutations. However, to date, L1 insertion and fixation patterns have not been studied comprehensively. To fill this gap, we investigated three genome-wide data sets of L1s that integrated at different evolutionary times: 17,037 de novo L1s (from an L1 insertion cell-line experiment conducted in-house), and 1,212 polymorphic and 1,205 human-specific L1s (from public databases). We characterized 49 genomic features-proxying chromatin accessibility, transcriptional activity, replication, recombination, etc.-in the ±50 kb flanks of these elements. These features were contrasted between the three L1 data sets and L1-free regions using state-of-the-art Functional Data Analysis statistical methods, which treat high-resolution data as mathematical functions. Our results indicate that de novo, polymorphic, and human-specific L1s are surrounded by different genomic features acting at specific locations and scales. This led to an integrative model of L1 transposition, according to which L1s preferentially integrate into open-chromatin regions enriched in non-B DNA motifs, whereas they are fixed in regions largely free of purifying selection-depleted of genes and noncoding most conserved elements. Intriguingly, our results suggest that L1 insertions modify local genomic landscape by extending CpG methylation and increasing mononucleotide microsatellite density. Altogether, our findings substantially facilitate understanding of L1 integration and fixation preferences, pave the way for uncovering their role in aging and cancer, and inform their use as mutagenesis tools in genetic studies.

An optimized, broadly applicable piggyBac transposon induction system.

The piggyBac (PB) transposon has been used in a number of biological applications. The insertion of PB transposons into the genome can disrupt genes or regulatory regions, impacting cellular function, so for many experiments it is important that PB transposition is tightly controlled. Here, we systematically characterize three methods for the post-translational control of the PB transposon in four cell lines. We investigated fusions of the PB transposase with ERT2 and two degradation domains (FKBP-DD, DHFR-DD), in multiple orientations, and determined (i) the fold-induction achieved, (ii) the absolute transposition efficiency of the activated construct and (iii) the effects of two inducer molecules on cellular transcription and function. We found that the FKBP-DD confers the PB transposase with a higher transposition activity and better dynamic range than can be achieved with the other systems. In addition, we found that the FKBP-DD regulates transposon activity in a reversible and dose-dependent manner. Finally, we showed that Shld1, the chemical inducer of FKBP-DD, does not interfere with stem cell differentiation, whereas tamoxifen has significant effects. We believe the FKBP-based PB transposon induction will be useful for transposon-mediated genome engineering, insertional mutagenesis and the genome-wide mapping of transcription factor binding.

Redesign of the monomer-monomer interface of Cre recombinase yields an obligate heterotetrameric complex.

Cre recombinase catalyzes the cleavage and religation of DNA at loxP sites. The enzyme is a homotetramer in its functional state, and the symmetry of the protein complex enforces a pseudo-palindromic symmetry upon the loxP sequence. The Cre-lox system is a powerful tool for many researchers. However, broader application of the system is limited by the fixed sequence preferences of Cre, which are determined by both the direct DNA contacts and the homotetrameric arrangement of the Cre monomers. As a first step toward achieving recombination at arbitrary asymmetric target sites, we have broken the symmetry of the Cre tetramer assembly. Using a combination of computational and rational protein design, we have engineered an alternative interface between Cre monomers that is functional yet incompatible with the wild-type interface. Wild-type and engineered interface halves can be mixed to create two distinct Cre mutants, neither of which are functional in isolation, but which can form an active heterotetramer when combined. When these distinct mutants possess different DNA specificities, control over complex assembly directly discourages recombination at unwanted half-site combinations, enhancing the specificity of asymmetric site recombination. The engineered Cre mutants exhibit this assembly pattern in a variety of contexts, including mammalian cells.

Immune Cell Deconvolution Reveals Possible Association of γδ T Cells with Poor Survival in Head and Neck Squamous Cell Carcinoma.

(1) Background: The role of rare immune cell subtypes in many solid tumors, chief among them head and neck squamous cell carcinoma (HNSCC), has not been well defined. The objective of this study was to assess the association between proportions of common and rare immune cell subtypes and survival outcomes in HNSCC. (2) Methods: In this cohort study, we utilized a deconvolution approach based on the CIBERSORT algorithm and the LM22 signature matrix to infer proportions of immune cell subtypes from 517 patients with untreated HPV-negative HNSCC from The Cancer Genome Atlas. We performed univariate and multivariable survival analysis, integrating immune cell proportions with clinical, pathologic, and genomic data. (3) Results: We reliably deconvolved 22 immune cell subtypes in most patients and found that the most common immune cell types were M0 macrophages, M2 macrophages, and memory resting CD4 T cells. In the multivariable analysis, we identified advanced N stage and the presence of γδ T cells as independently predictive of poorer survival. (4) Conclusions: We uncovered that γδ T cells in the tumor microenvironment were a negative predictor of survival among patients with untreated HNSCC. Our findings underscore the need to better understand the role of γδ T cells in HNSCC, including potential pro-tumorigenic mechanisms, and whether their presence may predict the need for alternative therapy approaches.

Cellular states are coupled to genomic and viral heterogeneity in HPV-related oropharyngeal carcinoma.

Head and neck squamous cell carcinoma (HNSCC) includes a subset of cancers driven by human papillomavirus (HPV). Here we use single-cell RNA-seq to profile both HPV-positive and HPV-negative oropharyngeal tumors, uncovering a high level of cellular diversity within and between tumors. First, we detect diverse chromosomal aberrations within individual tumors, suggesting genomic instability and enabling the identification of malignant cells even at pathologically negative margins. Second, we uncover diversity with respect to HNSCC subtypes and other cellular states such as the cell cycle, senescence and epithelial-mesenchymal transitions. Third, we find heterogeneity in viral gene expression within HPV-positive tumors. HPV expression is lost or repressed in a subset of cells, which are associated with a decrease in HPV-associated cell cycle phenotypes, decreased response to treatment, increased invasion and poor prognosis. These findings suggest that HPV expression diversity must be considered during diagnosis and treatment of HPV-positive tumors, with important prognostic ramifications.

Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis.

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

Digital analysis of the expression levels of multiple colorectal cancer-related genes by multiplexed digital-PCR coupled with hydrogel bead-array.

To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction. The specificity of probe hybridization was improved by using electrophoresis to remove mismatched probes on the bead's surface. The number of positive beads reflects the abundance of expressed genes; the expression levels of target genes were normalized to a housekeeping gene and expressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1% (i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is below 100 cells when using the ß-actin gene as the detection target. We have successfully employed MDHB to detect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2, MMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detection rates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for early non-invasive diagnosis of CRC.

Single-Cell Deconvolution of Head and Neck Squamous Cell Carcinoma.

Complexities in cell-type composition have rightfully led to skepticism and caution in the interpretation of bulk transcriptomic analyses. Recent studies have shown that deconvolution algorithms can be utilized to computationally estimate cell-type proportions from the gene expression data of bulk blood samples, but their performance when applied to tumor tissues, including those from head and neck, remains poorly characterized. Here, we use single-cell data (~6000 single cells) collected from 21 head and neck squamous cell carcinoma (HNSCC) samples to generate cell-type-specific gene expression signatures. We leverage bulk RNA-seq data from >500 HNSCC samples profiled by The Cancer Genome Atlas (TCGA), and using single-cell data as a reference, apply two newly developed deconvolution algorithms (CIBERSORTx and MuSiC) to the bulk transcriptome data to quantitatively estimate cell-type proportions for each tumor in TCGA. We show that these two algorithms produce similar estimates of constituent/major cell-type proportions and that a high T-cell fraction correlates with improved survival. By further characterizing T-cell subpopulations, we identify that regulatory T-cells (Tregs) were the major contributor to this improved survival. Lastly, we assessed gene expression, specifically in the Treg population, and found that TNFRSF4 (Tumor Necrosis Factor Receptor Superfamily Member 4) was differentially expressed in the core Treg subpopulation. Moreover, higher TNFRSF4 expression was associated with greater survival, suggesting that TNFRSF4 could play a key role in mechanisms underlying the contribution of Treg in HNSCC outcomes.

[Advances in early diagnosis of colorectal cancer based on detection of RNAs in stool.].

Colorectal cancer (CRC) is one of the most malignant cancers in gastrointestinal tract. In China, there are increasing rates of morbidity and mortality for CRC. As the mortality is closely related to the stage of disease at time of diagnosis, early diagnosis of CRC is important. However, current techniques used for clinical diagnosis have limitations which made them difficult to achieve the early diagnosis. The detection of RNAs in stool is a newly developed noninvasive technique for early diagnosis of CRC at molecular levels. Compared with the techniques including colonoscopy, fecal occult-blood test and stool DNA-based mutation detection, diagnosis based on the detection of RNAs in stool has the advantages of low-cost and high sensitivity. Moreover, stool RNA-based techniques are able to analyze multiplexed gene expression simultaneously and monitor cancer progression dynamically. This paper introduced the feasibility of stool RNA analysis, and systematically reviewed the genes associated with stool RNA analysis, methods of RNA isolation from stool sample, and techniques for gene expression analysis in stool RNA. Finally, further applications of stool RNA-based techniques for early diagnosis of CRC were briefly discussed.

Highly sensitive mutation detection based on digital amplification coupled with hydrogel bead-array.

In order to detect small amounts of mutants for early cancer diagnosis, we have developed the novel method of using amplicon-coated microbeads and single-molecule-PCR in water-in-oil emulsions, which we coupled with a new detection platform, the hydrogel bead-array, a 3-D polyacrylamide gel network used as a carrier to immobilize the beads.

A gel-based solid-phase amplification and its application for SNP typing and sequencing on-chip.

As conventional solid-phase amplification (SPA) on a two-dimensional slide has a low amplification capacity due to a limited amount of immobilized primers, we propose a three-dimensional SPA by immobilizing primers in hydrogel attached to a slide. One of the PCR primers, modified with an acrylamide group at the 5'-terminal, was copolymerized with both polyacrylamide gel and an acryl-modified glass slide, resulting in a high amplification capacity. The immobilization process was carried out by adding the catalysis reagent N,N,N',N'-tetramethylethylenediamine (TEMED) volatilized in vacuum, with uniform sample-concentration and gel-viscosity in the course of one-step nucleic acid immobilization. The porous structure of polyacrylamide gel, which allows PCR reagents such as Taq DNA polymerase, primers, dNTPs and DNA templates to freely enter the gel matrix, provides a homogeneous solution-mimicking environment for SPA on the interface or the inside of gel pads. Based on gel-based SPA, genotypes of different samples were accurately discriminated by either dual-color fluorescence hybridization or BAMPER (Bioluminometric Assay coupled with Modified Primer Extension Reactions). Pyrosequencing was also successfully carried out on SPA products. As the linkage between DNA molecules and gel is very strong, SPA products immobilized on gel pads could be reused several times if extended strands were removed by electrophoresis. Thus, the gel-based SPA provides a powerful tool for directly using on-chip amplicons for parallel detection.

Single-cell sequencing and its applications in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC), like many tumors, is characterized by significant intra-tumoral heterogeneity, namely transcriptional, genetic, and epigenetic differences that define distinct cellular subpopulations. While it has been established that intra-tumoral heterogeneity may have prognostic significance in HNSCC, we are only beginning to describe and define such heterogeneity at a cellular resolution. Recent advances in single-cell sequencing technologies have been critical in this regard, opening new avenues in our understanding of more nuanced tumor biology by identifying distinct cellular subpopulations, dissecting signaling within the tumor microenvironment, and characterizing cellular genomic mutations and copy number aberrations. The combined effect of these insights is likely to be robust and meaningful changes in existing diagnostic and treatment algorithms through the application of novel biomarkers as well as targeted therapeutics. Here, we review single-cell technological and computational advances at the genomic, transcriptomic, and epigenomic levels, and discuss their applications in cancer research and clinical practice, with a specific focus on HNSCC.

Transposase mapping identifies the genomic targets of BAP1 in uveal melanoma.

BACKGROUND: BAP1 is a histone deubiquitinase that acts as a tumor and metastasis suppressor associated with disease progression in human cancer. We have used the "Calling Card System" of transposase-directed transposon insertion mapping to identify the genomic targets of BAP1 in uveal melanoma (UM). This system was developed to identify the genomic loci visited by transcription factors that bind directly to DNA; our study is the first use of the system with a chromatin-remodeling factor that binds to histones but does not interact directly with DNA. METHODS: The transposase piggyBac (PBase) was fused to BAP1 and expressed in OCM-1A UM cells. The insertion of transposons near BAP1 binding sites in UM cells were identified by genomic sequencing. We also examined RNA expression in the same OCM-1A UM cells after BAP1 depletion to identify BAP1 binding sites associated with BAP1-responsive genes. Sets of significant genes were analyzed for common pathways, transcription factor binding sites, and ability to identify molecular tumor classes. RESULTS: We found a strong correlation between multiple calling-card transposon insertions targeted by BAP1-PBase and BAP1-responsive expression of adjacent genes. BAP1-bound genomic loci showed narrow distributions of insertions and were near transcription start sites, consistent with recruitment of BAP1 to these sites by specific DNA-binding proteins. Sequence consensus analysis of BAP1-bound sites showed enrichment of motifs specific for YY1, NRF1 and Ets transcription factors, which have been shown to interact with BAP1 in other cell types. Further, a subset of the BAP1 genomic target genes was able to discriminate aggressive tumors in published gene expression data from primary UM tumors. CONCLUSIONS: The calling card methodology works equally well for chromatin regulatory factors that do not interact directly with DNA as for transcription factors. This technique has generated a new and expanded list of BAP1 targets in UM that provides important insight into metastasis pathways and identifies novel potential therapeutic targets.

Cooperative p16 and p21 action protects female astrocytes from transformation.

Mechanisms underlying sex differences in cancer incidence are not defined but likely involve dimorphism (s) in tumor suppressor function at the cellular and organismal levels. As an example, sexual dimorphism in retinoblastoma protein (Rb) activity was shown to block transformation of female, but not male, murine astrocytes in which neurofibromin and p53 function was abrogated (GBM astrocytes). Correlated sex differences in gene expression in the murine GBM astrocytes were found to be highly concordant with sex differences in gene expression in male and female GBM patients, including in the expression of components of the Rb and p53 pathways. To define the basis of this phenomenon, we examined the functions of the cyclin dependent kinase (CDK) inhibitors, p16, p21 and p27 in murine GBM astrocytes under conditions that promote Rb-dependent growth arrest. We found that upon serum deprivation or etoposide-induced DNA damage, female, but not male GBM astrocytes, respond with increased p16 and p21 activity, and cell cycle arrest. In contrast, male GBM astrocytes continue to proliferate, accumulate chromosomal aberrations, exhibit enhanced clonogenic cell activity and in vivo tumorigenesis; all manifestations of broad sex differences in cell cycle regulation and DNA repair. Differences in tumorigenesis disappeared when female GBM astrocytes are also rendered null for p16 and p21. These data elucidate mechanisms underlying sex differences in cancer incidence and demonstrate sex-specific effects of cytotoxic and targeted therapeutics. This has critical implications for lab and clinical research.

Role of WNT7B-induced noncanonical pathway in advanced prostate cancer.

Advanced prostate cancer is characterized by incurable castration-resistant progression and osteoblastic bone metastasis. While androgen deprivation therapy remains the primary treatment for advanced prostate cancer, resistance inevitably develops. Importantly, mounting evidence indicates that androgen receptor (AR) signaling continues to play a critical role in the growth of advanced prostate cancer despite androgen deprivation. While the mechanisms of aberrant AR activation in advanced prostate cancer have been extensively studied, the downstream AR target genes involved in the progression of castration resistance are largely unknown. Here, we identify WNT7B as a direct AR target gene highly expressed in castration-resistant prostate cancer (CRPC) cells. Our results show that expression of WNT7B is necessary for the growth of prostate cancer cells and that this effect is enhanced under androgen-deprived conditions. Further analyses reveal that WNT7B promotes androgen-independent growth of CRPC cells likely through the activation of protein kinase C isozymes. Our results also show that prostate cancer-produced WNT7B induces osteoblast differentiation in vitro through a direct cell-cell interaction, and that WNT7B is upregulated in human prostate cancer xenografts that cause an osteoblastic reaction when grown in bone. Taken together, these results suggest that AR-regulated WNT7B signaling is critical for the growth of CRPC and development of the osteoblastic bone response characteristic of advanced prostate cancer.