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5-Hydroxymethylcytosine Primes Neuronal Genes for Activation During Zebrafish Retinal Progenitor Cell Differentiation

Spengler, Jennifer (2021) 5-Hydroxymethylcytosine Primes Neuronal Genes for Activation During Zebrafish Retinal Progenitor Cell Differentiation. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

The epigenetic mark 5-hydroxymethylcytosine (5hmC) is generated on DNA by the oxidation of 5-methylcytosine (5mC) by members of the ten-eleven-translocase (tet) enzyme family. 5hmC is detected at high levels within differentiated neurons and previous results from our lab have shown that activity of tet2 and tet3 enzymes is necessary for proper retinal differentiation in zebrafish (Seritrakul et al., 2017). However, the mechanism(s) by which tet activity and 5hmC regulate gene expression and influence differentiation in retinal progenitor cells (RPCs) is unknown. This study aimed to determine how 5mC and 5hmC modulate gene expression in early and late RPCs, and if 5mC and 5hmC relate to gene expression after RPCs differentiate to retinal ganglion cells (RGCs) in the developing zebrafish. 5hmC and 5mC levels in zebrafish RPCs were determined by oxidative-bisulfite and bisulfite sequencing. Gene expression levels were determined with RNA sequencing on RPCs and RGCs. Analyses were performed on RPCs at two developmental time points: 22 hours post-fertilization (HPF), when retinal progenitor cells are proliferative and not yet committed to retinal neuron fates, and 27 HPF, when retinal ganglion cell differentiation is underway. RGCs were also collected as they differentiate at 27 HPF to inform gene expression as a result of the epigenetic changes in progenitors. The resulting epigenetic and gene expression data from RPCs were integrated to identify how 5mC and 5hmC were distributed in the progenitor. The analysis performed here revealed that both 5mC and 5hmC were distributed to repress gene expression in retinal progenitor cells. Regions that gained 5hmC between early and late RPCs were enriched for neurogenic genes but did not correlate to gene expression in the RPCs. However, the increasing 5hmC was localized to neurogenic genes that were upregulated in RGCs. The data presented here indicate that the 5hmC generated between early and late RPCs primes genes for activation after the onset of differentiation.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Spengler, Jenniferjas606@pitt.edujas6060000-0002-2271-9628
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairGhazi, Arjumandghazia@pitt.edughazia0000-0002-5859-4206
Kostka, Denniskostka@pitt.edukoskta0000-0002-1460-5487
Thesis AdvisorGross, Jeffreygrossjm@pitt.edugrossjm0000-0002-9422-6312
Date: 5 January 2021
Date Type: Publication
Defense Date: 16 December 2020
Approval Date: 5 January 2021
Submission Date: 21 December 2020
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 98
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Genetics and Developmental Biology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: retina, epigenetics, development, differentiation, DNA methylation, 5mC, 5hmC, DNA hydroxymethylation
Date Deposited: 05 Jan 2021 14:32
Last Modified: 05 Jan 2021 14:32
URI: http://d-scholarship.pitt.edu/id/eprint/40119

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