Genevieve Welch

A Genome-wide RNAi Screen for Modifiers of the Circadian Clock in Human Cells

Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.

Gene Set Enrichment in eQTL Data Identifies Novel Annotations and Pathway Regulators

Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on “trans-eQTL bands”, defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets.

Tumor suppressor protein (p)53, is a regulator of NF-κB repression by the glucocorticoid receptor

Glucocorticoids can inhibit inflammation by abrogating the activity of NF-κB, a family of transcription factors that regulates the production of proinflammatory cytokines. To understand the molecular mechanism of repression of NF-κB activity by glucocorticoids, we performed a high-throughput siRNA oligo screen to identify novel genes involved in this process. Here, we report that loss of p53, a tumor suppressor protein, impaired repression of NF-κB target gene transcription by glucocorticoids. Additionally, loss of p53 also impaired transcription of glucocorticoid receptor (GR) target genes, whereas upstream NF-κB and glucocorticoid receptor signaling cascades remained intact. We further demonstrate that p53 loss severely impaired glucocorticoid rescue of death in a mouse model of LPS shock. Our findings unveil a new role for p53 in the repression of NF-κB by glucocorticoids and suggest important implications for treatment of the proinflammatory microenvironments found in tumors with aberrant p53 activity.

Identification of small molecule and genetic modulators of AON-induced dystrophin exon skipping by high-throughput screening.

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened ∼10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.

High-content screening of functional genomic libraries.

Recent advances in functional genomics have enabled genome-wide genetic studies in mammalian cells. These include the establishment of high-throughput transfection and viral propagation methodologies, the production of large-scale cDNA and siRNA libraries, and the development of sensitive assay detection processes and instrumentation. The latter has been significantly facilitated by the implementation of automated microscopy and quantitative image analysis, collectively referred to as high-content screening (HCS), toward cell-based functional genomics application. This technology can be applied to whole genome analysis of discrete molecular and phenotypic events at the level of individual cells and promises to significantly expand the scope of functional genomic analyses in mammalian cells. This chapter provides a comprehensive guide for curating and preparing function genomics libraries and performing HCS at the level of the genome.

Cellular Ser/Thr-Kinase Assays Using Generic Peptide Substrates

High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves a) coexpression of targeted kinases with promiscuous peptide substrates and b) quantification of intracellular substrate phosphorylation by homogeneous TR-FRET. Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2δ assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC50-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.

Reporter Gene Assays Using Transfectable Functional Genomics Libraries

Transfectable functional genomics libraries are traditionally the workhorses of functional genomics screening using reporter gene assays. These libraries offer insight into fundamental cellular processes governing health and disease and can be utilized in an arrayed fashion which makes them uniquely suited to deconvolute complicated disease phenotypes and dissect biological networks that would otherwise be inaccessible. Here we give an overview of the principles for the generation, screening and data analysis of such arrayed libraries. Specifically we cover the differences between the various transfectable reagents, library selection and handling, and data analysis to offer a comprehensive understanding of these important technologies and how to apply them.

Making It All Work: Functional Genomics and Reporter Gene Assays

Functional genomics is the study of the function of genes on a genome-wide level. Reporter gene assays can be utilized in this context to dissect signaling cascades, find new drug targets, or decipher the function of gene expression. The genome-wide scale of these experiments necessitates a different approach toward science than traditional single hypothesis driven research. High-throughput experimentation requires large project teams, automation, and discrete validation of each step in the automation and assay process. The purpose of this chapter is to provide a general outline of a standard functional genomics project with a reporter gene assay as readout, give an overview of the methodologies employed and familiarize the reader with the subsequent data analysis. The advantages of such high throughput experimentation are speed, quantitative results, and insights into biology on a genome-wide scale all of which enable a more rapid progress of science.