INVESTIGATOR: Steven Weicksel, Bryant University
THEME: Cancer

ABSTRACT: The 3-D organization of chromatin (chromatin topology) has been shown to play an important role in gene regulation. Some disease states (such as cancer) display changes in chromatin topology and subsequently gene regulation. Using the model organism Danio rerio (zebrafish) I will be defining the chromatin topology during early embryogenesis of the HOX clusters that control hindbrain development. The HOX genes are evolutionarily conserved in metazoans andaberrant chromatin topology and HOX expression have been linked to several forms of cancer. Understanding how HOX gene topology is controlled will give insights into the mechanism that regulate chromatin topology and have a potential impact on human health and treating cancer.
The Aims of this project are to (Aim-1) identify important cis- and trans-factors that have the potential to regulate hox chromatin topology. Using circular chromosome confirmation capture followed by high-throughput paired-end sequencing (4C) topological maps of hox cluster interactions at four time points of early zebrafish embryogenesis will be generated. The time points represent different periods of embryonic gene expression, 2 hours post fertilization (hpf) where no zygotic genes are expressed, 4hpf where the zygotic genome is first activated but no hox genes are active, 6hpf shortly after gastrulation and the first hox genes are transcribe, and 9hpf where the first hindbrain cells are created. These stage specific maps will then be compared using computational analysis to identify over-represented sequence motifs at focal points of chromatin loop formation which would correspond to DNA-binding factors that are important for gene regulation. (Aim-2) The function of known hox regulators will be disrupted using technologies such as CRISPR to generate novel mutants or morpholinos to alter gene expression. Changes within the hox topology will be monitored in the mutants using 3C/4C and changes in embryonic development will be observed using in situ hybridization and transcriptome analysis through quantitative PCR. In addition, cis- and trans-factors identified from Aim-1 will also be assayed.

RELEVANCE: The organization of the genome within the nucleus of a cell determines if a gene is turned off or turned on. In diseases such as cancer, this organization is disrupted from the norm and genes that were off are turned on (or vice versa). This project aims to identify factors that regulate genome organization for the HOX genes, a group of genes important for disease and vertebrate development.