Demo Faculty

Repair of DNA double-stranded breaks (DSBs) during lymphocyte development is essential for V(D)J recombination and forms the basis of immunoglobulin variable region diversity. Understanding of this process in lymphogenesis has historically been centered on the study of RAG1/2 recombinases and a set of classical non-homologous end-joining factors. Much less has been reported regarding the role of chromatin modifications on this process. Here, we show a role for the non-redundant histone H3 lysine methyltransferase, Setd2, and its modification of lysine-36 trimethylation (H3K36me3), in the processing and joining of DNA ends during V(D)J recombination. Loss leads to mis-repair of Rag-induced DNA DSBs, especially when combined with loss of Atm kinase activity. Furthermore, loss reduces immune repertoire and a severe block in lymphogenesis as well as causes post-mitotic neuronal apoptosis …

BRAF is a serine/threonine kinase that harbors activating mutations in ∼7% of human malignancies and ∼60% of melanomas. Despite initial clinical responses to BRAF inhibitors, patients frequently develop drug resistance. To identify candidate therapeutic targets for BRAF inhibitor resistant melanoma, we conduct CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAF inhibitors. To investigate the mechanisms and pathways enabling resistance to BRAF inhibitors in melanomas, we integrate expression, ATAC-seq, and CRISPR screen data. We identify the JUN family transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6, which together enable resistance to BRAF inhibitors in melanoma cells. Our findings reveal genes contributing to resistance to a selective BRAF inhibitor PLX4720, providing new insights into …

Tumor-infiltrating immune cells play critical roles in immune-mediated tumor rejection and/or progression, and are key targets of immunotherapies. Estimation of different immune subsets becomes increasingly important with the decreased cost of high-throughput molecular profiling and the rapidly growing amount of cancer genomics data. Here, we present Tumor IMmune Estimation Resource (TIMER), an in silico deconvolution method for inference of tumor-infiltrating immune components. TIMER takes bulk tissue gene expression profiles measured with RNA-seq or microarray to evaluate the abundance of six immune cell types in the tumor microenvironment: B cell, CD4+ T cell, CD8+ T cell, neutrophil, macrophage, and dendritic cell. We further introduce its associated webserver for convenient, user-friendly analysis of tumor immune infiltrates across multiple cancer types.

Over the past decade, hundreds of genome-wide association studies (GWAS) have implicated genetic variants in various diseases, including cancer. However, only few of these variants have been functionally characterized to date, mainly owing to the fact that the majority of the variants reside in noncoding regions of the human genome with unknown function. A comprehensive functional annotation of the candidate variants is thus necessary to fill the gap between the correlative findings of GWAS and the development of therapeutic strategies. By integrating large-scale multi-omics datasets such as the Cancer Genome Atlas (TCGA) and the Encyclopedia of DNA Elements (ENCODE), we performed multivariate linear regression analysis of expression quantitative trait loci, sequence permutation test of transcription factor binding perturbation, and modeling of three-dimensional chromatin interactions to analyze the potential molecular functions of 2,813 single nucleotide variants in 93 genomic loci associated with estrogen receptor-positive breast cancer. To facilitate rapid progress in functional genomics of breast cancer, we have created “Analysis of Breast Cancer GWAS” (ABC-GWAS), an interactive database of functional annotation of estrogen receptor-positive breast cancer GWAS variants. Our resource includes expression quantitative trait loci, long-range chromatin interaction predictions, and transcription factor binding motif analyses to prioritize putative target genes, causal variants and transcription factors. An embedded genome browser also facilitates convenient visualization of the GWAS loci in genomic and epigenomic context. ABC …

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V (D) J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role …

Over the past decade, hundreds of genome-wide association studies (GWAS) have implicated genetic variants in various diseases, including cancer. However, only few of these variants have been functionally characterized to date, mainly owing to the fact that the majority of the variants reside in noncoding regions of the human genome with unknown function. A comprehensive functional annotation of the candidate variants is thus necessary to fill the gap between the correlative findings of GWAS and the development of therapeutic strategies. By integrating large-scale multi-omics datasets such as the Cancer Genome Atlas (TCGA) and the Encyclopedia of DNA Elements (ENCODE), we performed multivariate linear regression analysis of expression quantitative trait loci, sequence permutation test of transcription factor binding perturbation, and modeling of three-dimensional chromatin interactions to analyze the potential molecular functions of 2,813 single nucleotide variants in 93 genomic loci associated with estrogen receptor-positive breast cancer. To facilitate rapid progress in functional genomics of breast cancer, we have created “Analysis of Breast Cancer GWAS” (ABC-GWAS), an interactive database of functional annotation of estrogen receptor-positive breast cancer GWAS variants. Our resource includes expression quantitative trait loci, long-range chromatin interaction predictions, and transcription factor binding motif analyses to prioritize putative target genes, causal variants and transcription factors. An embedded genome browser also facilitates convenient visualization of the GWAS loci in genomic and epigenomic context. ABC …

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

The human eye changes focus—accommodates—to minimize blur in the retinal image. Previous work has shown that stimulation of nonfoveal retina can produce accommodative responses when no competing stimulus is presented to the fovea. In everyday situations it is very common for the fovea and other parts of the retina to be stimulated simultaneously. We examined this situation by asking how nonfoveal retina contributes to accommodation when the fovea is also stimulated. There were three experimental conditions.(a) Real change in which stimuli of different sizes, centered on the fovea, were presented at different optical distances. Accommodation was, as expected, robust because there was no conflicting stimulation of other parts of the retina.(b) Simulated change, no conflict in which stimuli of different sizes, again centered on the fovea, were presented at different simulated distances using rendered chromatic blur. Accommodation was robust in this condition because there was no conflict between the central and peripheral stimuli.(c) Simulated change, conflict in which a central disk (of different diameters) was presented along with an abutting peripheral annulus. The disk and annulus underwent opposite changes in simulated distance. Here we observed a surprisingly consistent effect of the peripheral annulus. For example, when the diameter of the central stimulus was 8 (thereby stimulating the fovea and parafovea), the abutting peripheral annulus had a significant effect on accommodation. We discuss how these results may help us understand other situations in which nonfixated targets affect the ability to focus on a fixated target …

In computer-generated imagery and vision science, defocus blur is often rendered to simulate objects closer or farther than the focal plane. But depth-dependent optical effects, like longitudinal chromatic aberration (LCA), are not implemented in a physically correct manner. Recent evidence has shown that incorporating LCA into rendered images produces a powerful cue for driving accommodation and depth perception. But implementing correct LCA effects is computationally expensive. Applied implementations of defocus blur with LCA are possible, but require approximations in order to run in real-time. We investigated whether real-time implementation of blur with LCA using approximate blur kernels and simplified treatment of occlusions can still drive accommodation and improve perceived depth compared to conventional methods that do not incorporate LCA. We measured accommodative responses with an …

Genome-wide association studies (GWAS) have hitherto identified several germline variants associated with cancer susceptibility, but the molecular functions of these risk modulators remain largely uncharacterized. Recent studies have begun to uncover the regulatory potential of non-coding GWAS SNPs by using epigenetic information in corresponding cancer cell types and matched normal tissues. However, this approach does not explore the potential effect of risk germline variants on other important cell types that constitute the microenvironment of tumor or its precursor. This paper presents evidence that the breast cancer-associated variant rs3903072 may regulate the expression of CTSW in tumor infiltrating lymphocytes. CTSW is a candidate tumor-suppressor gene, with expression highly specific to immune cells and also positively correlated with breast cancer patient survival. Integrative analyses suggest a putative causative variant in a GWAS-linked enhancer in lymphocytes that loops to the 3’ end of CTSW through three-dimensional chromatin interaction. Our work thus poses the possibility that a cancer-associated genetic variant could regulate a gene not only in the cell of cancer origin, but also in immune cells in the microenvironment, thereby modulating the immune surveillance by T lymphocytes and natural killer cells and affecting the clearing of early cancer initiating cells.

Purpose: Experiments suggest that hyperopic focus in the peripheral retina increases the risk of developing myopia. This implies that signed defocus can trigger eye growth. But does this mean blur in the periphery affects other oculomotor mechanisms such as accommodation? Stimulation of the human peripheral retina when no foveal stimulus is present elicits accommodation. But in natural viewing, the fovea and periphery are nearly always both stimulated. We investigated accommodative responses when the fovea and periphery are both stimulated, but with different signs of defocus.Methods: 10 participants (18-25 yrs) viewed black-white textures monocularly. The stimuli varied in depth (±1.5 D) sinusoidally (0.1, 0.2, 0.5, or 1.0 Hz). Three conditions were tested: Real Blur, Defocus+ LCA, and Defocus+ LCA Conflict. In Real Blur, the optical distance of the textures (disks subtending 1, 2, 4, 6, 8, or 14) was varied …

Characterizing the ontologies of genes directly regulated by a transcription factor (TF), can help to elucidate the TF’s biological role. Previously, we developed a widely used method, BETA, to integrate TF ChIP-seq peaks with differential gene expression (DGE) data to infer direct target genes. Here, we provide Cistrome-GO, a website implementation of this method with enhanced features to conduct ontology analyses of gene regulation by TFs in human and mouse. Cistrome-GO has two working modes: solo mode for ChIP-seq peak analysis; and ensemble mode, which integrates ChIP-seq peaks with DGE data. Cistrome-GO is freely available at http://go.cistrome.org/.

The degree and type of T cell infiltration influence rectal cancer prognosis regardless of classical tumor staging. We asked whether clonal expansion and tumor infiltration are restricted to selected-phenotype T cells; which clones are accessible in peripheral blood; and what the spatial distribution of their target antigens is.From five rectal cancer patients, we isolated paired tumor-infiltrating T cells (TILs) and T cells from unaffected rectum mucosa (TUM) using 13-parameter FACS single cell index sorting. TCRαβ sequences, cytokine, and transcription factor expression were determined with single cell sequencing. TILs and TUM occupied distinct phenotype compartments and clonal expansion predominantly occurred within CD8+ T cells. Expanded TIL clones identified by paired TCRαβ sequencing and exclusively detectable in the tumor showed characteristic PD-1 and TIM-3 expression. TCRβ repertoire sequencing …

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-wide association studies have hitherto identified several common genetic variants that may significantly modulate cancer susceptibility. However, the precise molecular mechanisms behind these associations remain largely uncharacterized, creating barriers to understanding the biological processes behind oncogenesis. This thesis presents an integrated computational method for identifying functional regulatory variants associated with cancer and for revealing their precise gene-regulation role by combining analyses of heterogeneous high-throughput sequencing data. Application of the method to breast cancer susceptibility regions reveals functional variants and their perturbation on cis-regulatory elements that act on cancer-associated genes. It is also shown that a cancer-associated variant may interact with the tumor microenvironment. Overall, these computational methods built on multi-omics data have helped move toward the goal of understanding the genotype-phenotype association for personalized medicine.

Androgen deprivation therapy for prostate cancer (PCa) benefits patients with early disease, but becomes ineffective as PCa progresses to a castration-resistant state (CRPC). Initially CRPC remains dependent on androgen receptor (AR) signaling, often through increased expression of full-length AR (ARfl) or expression of dominantly active splice variants such as ARv7. We show in ARv7-dependent CRPC models that ARv7 binds together with ARfl to repress transcription of a set of growth-suppressive genes. Expression of the ARv7-repressed targets and ARv7 protein expression are negatively correlated and predicts for outcome in PCa patients. Our results provide insights into the role of ARv7 in CRPC and define a set of potential biomarkers for tumors dependent on ARv7.

Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR–Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control (QC), normalization, batch effect removal, copy-number bias correction, gene hit identification and downstream functional enrichment analysis for CRISPR …

CD8 T-cell-mediated antitumor immunity is required for the control and elimination of tumors. However, tumors are able to overcome immune response resulting in immunosuppression, tumor growth, and metastatic spread. CD8 T-cells are controlled through a homeostatic network of positive and negative feedback loops. These negative signals are exploited by the tumor and associated suppressive cell populations in the tumor microenvironment. Immunotherapies targeted to receptors that control these negative signals, termed immune checkpoint inhibitors, have provided robust and durable responses to a number of cancers. Unfortunately, only a subset of patients will respond to current immunotherapies. This is due to the accruement of suppressive and inhibitory mechanisms employed by the tumor and its microenvironment. These include expression of inhibitory ligands, release of immune-suppressive factors …