Gene Knockdown allows regulation of Heterochromatin of Zebrafish Embryos

Undergraduate #105
Discipline: Biological Sciences
Subcategory: Genetics
Session: 1
Room: Exhibit Hall A

Chauncey Ragin - Fort Valley State University
Co-Author(s): Katherine Duval, University of Georgia, Athens, GA; Mary Goll, University of Georgia

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Eukaryotic DNA is packaged into a nucleoprotein complex called chromatin. This causes the genetic material to be condensed so that it can fit within the nucleus of the cell [1]. One type of chromatin is heterochromatin which is tightly condensed, can be identified by histone 3 lysine-9 trimethylation (H3K9meᶟ), and is formed in highly repetitive sequences. The aim of this project is to determine what genes play a part in the regulation of heterochromatin. This experiment uses a transgenic line of zebrafish with 70 copies of the gene encoding fluorophore DsRed. Due to the transgene’s highly repetitive nature, it is enriched with H3K9me3, is transcriptionally silenced, and serves as a model for heterochromatin regulation. If heterochromatin regulation is disrupted the transgene can be reactivated, producing fluorescence. To identify genes involved in heterochromatin establishment, candidates were knocked down using morpholinos. Morpholinos are synthetic oligos that are able reduce expression of a gene of interest. If the gene targeted is involved in heterochromatin regulation, morpholino knockdown will result in fluorescence. For this experiment we used 2ng of a SUV39h1a targeting morpholino, and 2ng of a DMAP1 targeting morpholino. They were then allowed to develop for two days before being viewed under a fluorescence microscope. When viewed under the scope, the more fluorescence that was seen in the tail area, the more we could tell that the genes that was targeted had a role in heterochromatin formation. These experiments provide insight for complex mechanisms driving heterochromatin regulation in vertebrates and from these we discovered that when the gene suv39h1a or dmap1 is knocked down in zebrafish embryos, transgene repression is disrupted and that the genes SUV39 and dmap1 are involved in heterochromatin regulation. Some directions we could go with this include determining what else takes part in the formation of heterochromatin, figuring out if knocking down genes related to other biological processes affect heterochromatin formation, and if enabling certain genes instead of knocking them down alter the formation of heterochromatin.

Funder Acknowledgement(s): I would like to acknowledge REU program, NIH, Department of Genetics, University of Georgia and NSF TIP award # 1818695, Prof Seema Dhir for all their support.

Faculty Advisor: Dr. Seema Dhir, dhirs@fvsu.edu

Role: I did all parts of the research with help from my lab mentor, Mary Goll, and my graduate student mentor, Katherine Duval. That included cleaning and removing the outer layer of the embryos, injecting them with the various morpholinos, and looking at them under a fluorescence microscope to see if there is any fluorescence indicating gene suppression. I did that everyday excluding weekends for that ten week period.