Methods to Record Transcription Factor Binding and Enhancer Activity throughout Cellular Differentiation

The ability to create distinct cell types is fundamental for the development of multicellular organisms. Since all cells in an organism contain the same genes, cellular diversity is achieved through the transcriptional network where transcription factors (TFs) interacts with cis-regulatory elements, leading to the selective transcription of different sets of genes. To better understand the functions of TFs and regulatory elements underlying cell fate decisions, we developed methods that are able to record their activities throughout cellular differentiation. In Chapter 2, we developed a degradation domain based induction system for ҃alling CardsӠmethod which maps the binding sites of TFs using piggybac transposons. The induction ҃alling CardsӠmethod offers an alternative to chromatin immunoprecipitation (ChIP) methods and furthermore has the ability to record TF binding at different time periods of the development. In Chapter 3, we applied the ҃alling CardӠmethod to study the role of master regulatory Brd4-bound enhancers for sex differences in glioblastoma. We revealed a set of sex-specific regulatory genes and networks, which are indicative of sex-specific transcriptional programs regulated by Brd4-bound enhancers. Finally, to record the activity of regulatory elements or enhancers, in Chapter 4, we developed a CRE recombinase-mediated method for high-throughput functional identification of active enhancers at different time periods of the development, named as Developmental Enhancer Sequencing (DevEn-seq). We demonstrated that DevEn-seq is able to detect enhancers more efficiently than regular reporter methods and trace enhancer activities throughout cellular differentiation without being disturbed by the gene silencing effect caused by lentiviral sequences. With an in vitro neural differentiation protocol, we identified two neural progenitor-specific enhancers near HB9 and Olig2 genes respectively. In summary, this dissertation contributes to the field of developmental biology by providing useful methods for recording TF binding events and enhancer activities throughout development

Transposase mapping identifies the genomic targets of BAP1 in uveal melanoma

Table summarizing the RNA-seq results. Differential gene expression results in BAP1-knockdown compared to control OCM-1A cells are shown from the RNA-seq data. Each row gives the unique Ensembl identifier, gene name, and description for each gene, as well as the log of the fold change (logFC), average expression, adjusted p-value, and linear fold change. (XLSX 1392 kb

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

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 \u3e500 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 (

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

Cooperative p16 and p21 action protects female astrocytes from transformation

Abstract 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