Date of Award

8-2018

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Animal Science

Committee Chair

Ryan Cabot

Committee Member 1

Zoltan Machaty

Committee Member 2

Karen Plaut

Abstract

Epigenetic marks are globally remodeled during mammalian embryo development. Chromatin remodeling mediated by the SWI/SNF (Switch/Sucrose non-fermentable) complexes can participate in epigenetic remodeling during cleavage development. SWI/SNF complexes are multi-subunit complexes that contain a core catalytic subunit (SMARCA4 or SMARCA2) and a collection of additional subunits that guide the complexes to their appropriate loci. It has been reported that discrete SWI/SNF subunits adopt different intracellular localization patterns as porcine embryos proceed through cleavage development. In the work presented here, we investigated how these SWI/SNF subunits are trafficked during embryo development. We hypothesized that the localization patterns of the SWI/SNF chromatin remodeling complex subunits ARID1A, BRD7, SMARCB1, and ARID2 were transported out of nuclei in a CRM1-dependent manner and that they are transported into the nucleus by karyopherin α mediated import.

In our first experiment porcine embryos were treated with leptomycin B (LMB), an inhibitor of CRM1-mediated nuclear export to test this hypothesis. BRD7 displayed a significant redistribution upon LMB treatment (as compared to control embryos). This finding suggests that BRD7, unlike other SWI/SNF subunits, actively shuttles between the nuclear and cytoplasm during cleavage development. The shuttling of BRD7 indicates that it may serve a unique role mediating chromatin remodeling during this critical developmental window.

Our second experiment we investigated how SWI/SNF complexes are transported by karyopherins through the embryo during development. We hypothesized that the SWI/SNF chromatin remodeling subunits ARID1A, BRD7, SMARCB1 and ARID2 were transported into the nuclei using karyopherin α mediated transport. Porcine embryos were treated with ivermectin (IVER), a karpherin α mediated transport inhibitor to test this hypothesis. While we found no difference in localization patterns in ARID1A, ARID2, and BRD7 intracellular location between embryos treated with IVER and controls, SMARCB1 had a significant redistribution upon IVER treatment when compared to controls. This suggests SMARCB1 is shuttled into the nucleus utilizing karyopherin α mediated transport at the four cells stage of development. We then utilized a specific interfering RNA to inhibit KPNA7 mediated transport to understand how this effects BRD7 distribution during embryo development. We saw no significant difference between the control injected and the RNAi injected embryos.

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