Functions and regulation of nuclear TULP3 in mammalian cells
Creators: Kerek, Evan
Publisher: name: University of Alberta Library
Year: 2023

<p>Since the discovery of the tubby mouse and the characterization of its founding member TUB, a related family of Tubby-like proteins (TULPs) have been unearthed. While some of their functions have been explored in developmental and ciliary contexts, there is much that remains undiscovered in this protein family. While most TULPs have a sizable fraction of their total protein levels localized to the nuclear compartment, there is little to no information on the function of any TULP there. When combined with continuously emerging phenotypes suggesting novel roles for TULPs in various cancers, signaling pathways and liver, kidney and heart diseases, further characterization of TULP biochemistry is becoming increasingly important. <br>
Here, I describe how TULPs are regulated by post-translational modifications in the nucleus for the first time via a conserved acetylation/ubiquitin switch that regulates their stability at the protein level. Initially, I demonstrated that the acetyltransferase p300 was essential in maintaining TULP3 levels in cells as pharmacological inhibition reduced TULP3 levels to zero while overexpressing did the opposite. Conversely, this acetylation was shown to be reversible as I found HDAC1 to be the primary deacetylase that destabilizes TULP3 for proteasomal turnover. A high-throughput interactome was also used to discover the E3 ubiquitin ligase that ubiquitylates TULP3 as Cullin-3. The effects of p300 and HDAC1 on ubiquitin were inverse to their acetylation phenotypes as p300 reduced ubiquitylated-TULP3 while HDAC1 increased it. Using acetylation mass spectrometry mapping, we found that the switch was operating through conserved lysines 316 and 389, and dual mutation of these lysines effectively made TULP3 immune to turnover in cells. <br>
The conservation of these lysines prompted me to address if the acetylation switch was also conserved in the TULP family where I showed that p300 and HDAC1 also modulates the stability of TULP1, 2 and 4 in cells. Lastly, I showed that TULP3 KD in developing zebrafish resulted in several detrimental phenotypes including developmental delay and hindbrain apoptosis. The impact of our dual K316/K389 lysine was shown where microinjection of 2Q TULP3 mRNA efficiently rescued the TULP3 KD phenotypes while wild-type mRNA did not, presumably due to the greatly increased protein levels from the more stable 2Q TULP3 protein. <br>
While this first project successfully showed that TULPs localize to the nucleus, in part, to participate in the acetylation/ubiquitin switch, the mystery of nuclear TULPs was far from resolved. My initial interactome identified the strongest interactor of TULP3 to be the nuclear deacetylase SIRT1. While it was surprising that SIRT1 was not the primary deacetylase that regulated TULP3 levels, I set out to discover the purpose of the SIRT1-TULP3 complex that also resides in the nucleus. I utilized a series of high-throughput approaches where I showed definitively for the first time that TULP3 binds and forms a complex with DNA in vitro using EMSAs. Next, I utilized a fully randomized DNA library with recombinant TULP3 protein to determine the DNA binding preference of TULP3 in vitro using a high throughput technique called SELEX-seq. The most enriched sequence identified from SELEX-seq was the canonical palindromic E-box motif “CACGTG”. I then showed the relevance of this motif in cells using ATAC-seq where we showed genome wide changes in chromatin accessibility in Wild-type and TULP3 knock-out cells. Knock-out cells showed increased binding of transcription factors E-box or E-box like motifs, presumably due to vacancies on the chromatin from where TULP3 was bound. Conversely, a decrease in numerous HOX and FOX transcription factors was observed elucidating a novel role for TULP3 in nuclear signaling cascades including circadian rhythm, DNA repair, hindbrain development and Wnt. <br>
Lastly, we assessed transcriptome wide changes in mRNA expression in cancerous HEK293T and immortalized retinal pigment epithelial cells from WT and TULP3 KO samples using RNA-seq. This data showed a list of 19 genes whose differential expression consistently correlated with ATAC-seq accessibility changes. This is indicative of direct TULP3 binding as a transcription factor regulation for the first time.<br>
Collectively, the work presented herein shows the numerous functions of TULP3 in the nucleus as both being regulated by acetylation and ubiquitin PTMs as well as regulating gene expression, gene accessibility and transcription factor footprinting.</p>