Differential Requirement for the mRNP Biogenesis Factor Thoc1 in the Adult Mouse
Pitzonka, Laura B.
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Thoc1 encodes a protein (Thoc1) that is an essential component of the TREX/THO complex involved in ribonucleoprotein biogenesis (mRNP) and the regulation of transcripts for coordinated gene expression. The goal of this dissertation is to perform a conditional deletion of Thoc1 in adult mice using the Tamoxifen (TAM)/CreERT2 recombination system and assessing the consequences of Thoc1 deletion on various tissues. To investigate the Thoc1 deletion phenotypes noted, two specific aims were established for this dissertation: 1) to determine the role for Thoc1 in intestinal tissue homeostasis in the adult mouse and 2) to investigate how Thoc1 deletion affects primary and secondary hematopoietic tissues in the adult mouse. In our first aim Thoc1 F/F :Rosa26Cre ERT2/+ (test) and Thoc1 +/+ :Rosa26Cre ERT2/+ (control) adult mice were treated with 2 mg/day TAM for 1, 2, 3, 4, 5, or 6 days to establish a time course of Thoc1 deletion in the adult mouse and allow a detailed examination of the aforementioned Thoc1 deletion phenotypes. We find Thoc1 deletion specifically disrupts S.I. crypt cell function and viability, which leads to a complete loss of S.I. epithelial cell turnover. We also confirm that the colon is not affected by Thoc1 deletion. In our second aim Thoc1 F/F :Rosa26Cre ERT2/+ (test) and Thoc1 +/+ :Rosa26Cre ERT2/+ (control) adult mice were treated with 2 mg/day TAM for 3, 4, 5, or 6 days and a complete blood count (CBC) was performed to preliminarily assess if Thoc1 deletion affects hematopoietic cells. The bone marrow (BM) contains the hematopoietic stem cells (HSCs)/progenitor cells that give rise to all hematopoietic lineages and was of interest given the previously observed peripheral blood phenotype. We find that Thoc1 deletion significantly increases the number of neutrophils and decreases the number of lymphocytes in the peripheral blood. However, we conclude that this peripheral blood phenotype was the result of a stress response induced by S.I. deterioration and malnutrition and was not reflective of the effect of Thoc1 deletion in the hematopoietic cells. To explore the effect of Thoc1 deletion on the bone marrow independently of S.I. effects, we performed reconstitution experiments that allowed us to examine the consequence of Thoc1 deletion specifically in hematopoietic cells. Fully reconstituted Thoc1 F/F :Rosa26Cre ERT2/+ BM (test) and Thoc1 +/+ :Rosa26Cre ERT2+ BM (control) adult mice were treated with 2 mg/day TAM for 5 days. Initial examination of the BM revealed that less live cells are isolated from BM lacking Thoc1 versus BM that retained the gene and that BM cells lacking Thoc1 cannot form colonies ex vivo. Given these findings, and since PreGM/GMPs make up the majority of the BM, we hypothesized Thoc1 deletion affects PreGM/GMP cell viability in the BM. Indeed, we find the number of PreGM/GMPs and PreMegEs (red blood cell precursors) significantly decrease in the BM, whereas CD150+ hematopoietic stem cells (HSCs) and multi-potent progenitors (MPPs) do not change, following Thoc1 deletion. Furthermore, ex vivo experiments demonstrate PreGM/GMPs undergo apoptosis when Thoc1 is deleted leading to the conclusion that Thoc1 is required for primary hematopoietic tissue (bone marrow) homeostasis. In both of our experimental designs, histological examination of the spleen and thymus suggests secondary hematopoietic tissues are not affected by Thoc1 deletion within the time frame of our experiments. (Abstract shortened by UMI.)