Phosphatase

: Despite the identification of master regulators of stem cell homeostasis, Sox2 , Nanog , and Oct4 , many mechanisms underlying the maintenance of self-renewal and pluripotency in embryonic stem cells are not well understood (Boyer et al. 2005, Cinghu et al. 2014). The proteins of the box H/ACA snoRNP have been identified as potentially of importance for stem cell maintenance (Cinghu et al. 2014) and are connected to regulation of stem cell-related gene expression (Fong et al. 2014), maintenance of self-renewal (Zhang et al. 2016), and accurate differentiation of stem cells (Bellodi et al. 2013). Using RNAi we investigated the connection between two members of the box H/ACA snoRNP, Dkc1 and Nop10, and stem cell homeostasis. We found that loss of Dkc1 lead to a disruption in stem cell homeostasis. To investigate if this connection was due to the pseudouridylation function of Dkc1, we constructed a genetic system to rescue the stem cell phenotype in Dkc1 knockdown. While we were able to establish exogenous expression of Dkc1 in our rescue system, the expression levels were not sufficient to rescue the loss of stem cell phenotype, requiring further optimization. Although we were not able to determine if pseudouridylation by the box H/ACA snoRNP is essential for stem cell homeostasis, we were able to demonstrate that this complex is important for the maintenance of a stem cell state.


Table of Contents
Introduction -pg. 5           Table 1: Knockdown of Dkc1 by shDkc1B leads to a defect in stem cell phenotype.

Introduction
Embryonic stem cells (ESCs) are derived from the epiblast lineage of the blastocyst and have the developmental potential to differentiate into all three primary germ layers, defined as pluripotency.This ability, along with the capacity for self-renewal, are the key features defining ESCs (Czechanski et al 2014).These properties also make ESCs a particularly useful system for studying development and modelling disease, as they can be expanded in culture indefinitely and can be theoretically directed toward any lineage.The identification of regulators of stem cell homeostasis, which includes maintenance of stem cell gene expression profile, the ability to self renew, and pluripotency, is essential to fully understand these unique cells and can help researchers further understand development and disease.However, establishing a consensus on identified genes between these various screens is not always straight-forward.Recently, a bioinformatics based screen attempted to establish a unbiased method for identifying genes important for ESC identity (Cinghu et al. 2014).Of these genes, a large number are highly expressed in ESCs and downregulated during the course of differentiation.Interestingly, four genes that fall into this category are the genes encoding the four proteins of the box H/ACA snoRNP complex, which were all found to be in the top 1% of genes suspected to be of importance in stem cell maintenance (Cinghu et al 2014).
The mammalian box H/ACA snoRNP is a complex of four proteins, the pseudouridine synthase (PUS) dyskerin (Dkc1), Nop10, Nhp2, and Gar1, and a small nucleolar RNA known as the box H/ACA snoRNA (Figure 1A).Pseudouridine (Ψ) is the C5-glycoside isomer of uridine, formed from the breakage of the N1-glycosyl bond followed by a 180° rotation of the base and formation of the C5-glycosyl bond, and is particularly abundant in rRNA although Ψ can be found in tRNA and mRNA as well (  The box H/ACA snoRNP has previously been identified as a co-activator of Oct4/Sox2 gene activation, including activation of Nanog expression in a human cell system (Fong et al 2014).This activity was attributed to direct binding of the box H/ACA snoRNP along with the XPC DNA repair complex to enhancer regions of activated genes, however researchers were not able to completely rule out the impact of other Dkc1 functions, particularly rRNA processing by pseudouridylation, on stem cell phenotype (Fong et al 2014).Box H/ACA snoRNAs have been connected directly to proliferation and self-renewal of mesenchymal stem cells (Zhang et al. 2016), strengthening the connection between the box H/ACA snoRNP to stem cell phenotype.
Additionally, the catalytic activity of Dkc1, and therefore the pseudouridylation function of the box H/ACA snoRNP, has been implicated in accurate differentiation of hematopoietic stem cells (Bellodi et al. 2013).
Pseudouridylation is the key function of the box H/ACA snoRNP.We hypothesize that pseudouridylation by the box H/ACA snoRNP is important for ESC homeostasis.Therefore, we aim to investigate the impact of Dkc1 and Nop10 on mouse (m)ESC homeostasis, and determine if this impact can be attributed to the pseudouridylation activity of the box H/ACA snoRNP.

RNA Extraction:
RNA was extracted 4 days post-transfection using the RNeasy Mini Kit (Qiagen, 74104) with on-column DNA digestion (Qiagen, 79254) according to manufacturer's instructions.
Concentration of RNA was measured by NanoDrop Spectrophotometer at 260 nm (ThermoFisher Scientific).

qPCR Data Analysis:
To analyze the RT-qPCR data the delta-delta Cq method was used (Livak and Schmittgen 2001).The difference between the raw quantitation cycle (Cq) value for each gene of interest (Dkc1, Nop10, Trub1, Sox2, Nanog, and Oct4) and the housekeeping gene Actin was found for each sample to generate the delta Cq (ΔCq = Cq(gene of interest) -Cq(housekeeping gene)).The delta delta Cq was found by finding the difference between the delta Cq for a given gene for each sample and the delta Cq for that same gene for the control, cells transfected with shGFP (ΔΔCq = ΔCq(sample) -ΔCq(control)).To find the relative gene expression between the sample and the control, the fold expression was calculated by raising 2 to the power of the negative delta delta Cq (fold expression = 2 -ΔΔCq ).For each replicate these values were then averaged.Bar graphs presenting expression of genes from RT-qPCR were generated in GraphPad Prism 7. Statistical significance was determined by two-sided paired t-test with alpha = 0.05 (95% confidence).All error bars represent standard error of the mean (SEM).

Alkaline Phosphatase (AP) Staining:
Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, P6148) in phosphate-buffered saline (PBS).AP staining was performed for phenotypic assessment of mESCs in experimental conditions using the Alkaline Phosphatase Detection Kit (Millipore Sigma, SCR004) according to manufacturer's protocol.

Protein Extraction:
Protein was isolated on day 2 post-transfection from cells that were not put under antibiotic selection, with the exception of the final western blot presented (Figure 7E

Knockdown of pseudouridine synthases by RNAi
To test the impact of the two members of the box H/ACA snoRNP Dkc1 and Nop10 on regulation of stem cell (SC) homeostasis in mESCs, we designed and constructed plasmids expressing shRNAs for the knockdown of Dkc1, Nop10, Trub1, and GFP.Trub1 is an RNA independent PUS that is not suspected to be of importance in the maintenance of a SC state, as it is not differentially expressed between SCs and differentiated cells (Cinghu et al. 2014).As the predominant mRNA PUS (Safra et al. 2017), Trub1 served as a control for mRNA pseudouridylation.By controlling for mRNA pseudouridylation, the impact of the box H/ACA snoRNP could be distinguished from the impact, if any, of mRNA pseudouridylation on ESC homeostasis.The shRNA targeting GFP served as a negative control for transfection conditions and shRNA production.Multiple shRNAs were tested for each gene due to potential variability in knockdown efficiency between shRNAs and off-target effects.Each of these plasmids was tested for knockdown efficiency and impact on gene expression by transfection into mESCs followed by antibiotic selection, then RNA extraction and RT-qPCR to determine the levels of expression of the PUSs along with expression of stem cell markers (Sox2, Nanog, Oct4).A significant knockdown of the genes of interest in given condition when compared to the shGFP negative control indicates a functional shRNA for that gene.A decrease in expression of the three SC markers when compared to the shGFP negative control would indicate a loss of stem cell phenotype and disruption in stem cell homeostasis.Three shRNAs (shNop10A, B, & C) were tested for the knockdown of Nop10.shNop10A and shNop10C both showed significant knockdown of Nop10 with 0.11 fold expression (p = 0.0002) and 0.22 fold expression (p = 0.0019) of Nop10 respectively (Figure 3A), while shNop10B did not show significant knockdown of Nop10 (p = 0.0949) (data not presented).
shNop10A also showed a significant reduction in expression of Sox2, 0.52 fold expression (p = 0.0462), and Nanog, 0.31 fold expression (p = 0.0071), but no significant change in Oct4 expression (Figure 3B-D).shNop10C showed only significant changes in expression of Nanog, with a 0.77 fold expression (p = 0.0215), but no significant changes in expression of Sox2 and Oct4 (Figure 3B-D).Alkaline phosphatase (AP) is known to be expressed at high levels on the surface of pluripotent cells, and can be used as a marker for pluripotency (Pera, Reubinoff, andTrounson 2000, Thomson et al 1998).This stain uses a substrate of AP that turns dark red on contact with AP; therefore, red color is indicative of a stem cell phenotype.Loss of staining as well as the loss of the compact, colony morphology indicative of ESCs indicates differentiation.The AP staining of the shGFP sample showed that these cells maintained a stem cell phenotype.AP staining of mESCs after shRNA knockdown of Dkc1, Nop10, and Trub1 was categorized into four categories: no differentiation: no distinction between the sample and control, mild differentiation: dark red staining but a larger number of cells outside of colonies (shTrub1A), moderate differentiation: light red staining and a larger number of cells outside of colonies (shNop10A, shNop10C), and severe differentiation: very light red staining and a large portion of cells outside of colonies (shDkc1B) (Figure 5).Using the criteria of a reduction in expression in all three stem cell markers, Sox2, Nanog, and Oct4, and a visual determination of loss of stem cell phenotype by AP staining, knockdown of Dkc1 by shDkc1B was determined to cause a defect in ESC homeostasis (Table 1).While the less significant knockdown of Dkc1 by shDkc1A and knockdown of Nop10 by shNop10A and shNop10C lead to a reduction in some stem cell makers and some differentiation phenotype, they were not determined to have a true loss in stem cell homeostasis because expression of all three markers was not modulated (Table 1).Furthermore, knockdown of Trub1 by shTrub1A and shTrub1B was also not determined to lead to a defect in ESC homeostasis, despite variability in expression of Nanog and Oct4 between shTrub1A and shTrub1B (Table 1).

Rescue of loss of stem cell phenotype by overexpression of Dkc1
Once we determined that the knockdown of Dkc1 resulted in a loss of stem cell homeostasis, the question remained if this change was dependent upon the pseudouridylation activity of Dkc1.To determine if Dkc1's catalytic activity is necessary for mESC homeostasis, we developed a genetic rescue assay (Figure 6A).This assay makes use of a co-transfection system of pLKO-shDkc1B, which results in knockdown of endogenous Dkc1 expression, and pEF-DEST51 plasmids overexpressing V5-tagged Dkc1.ShDkc1B was selected due to its robust knockdown of Dkc1, significant changes in all three stem cell markers, and notable loss of stem cell phenotype by AP staining.While the shRNA would lead to knockdown of endogenous Dkc1, we generated an shRNA resistant (shBR) mutant of Dkc1, which could be simultaneously overexpressed.This was done by introducing a series of silent mutations in the region of the Dkc1 gene that is targeted by shDkc1B using mutagenesis PCR.In addition to the resistance   The co-transfection system was then adapted to model the shRNA protocol by expanding to a four day time course, with transfection at day zero, expansion of transfections at day 1, selection with puromycin and blasticidin at day 2, and harvest of RNA at day 4 post-transfection.
In addition to the combination of pLKO shDkc1B and the four pEF-DEST Dkc1 plasmids, pLKO shGFP and pLKO shDkc1B were also co-transfected with the empty pEF-DEST51 plasmid (EV) to serve as controls for transfection conditions and shDkc1B knockdown of Dkc1.
In the co-transfection system, shDkc1B had less efficient knockdown of Dkc1 (0.21 fold expression (p < 0.0001)) when compared to the pLKO only transfection system (0.Although there was clear exogenous expression of Dkc1 protein from the shRNA resistant pEF-DEST plasmids in the co-transfection system at two days after transfection, Dkc1 mRNA levels were not restored at four days after transfection.From all four pEF-DEST Dkc1 plasmids, there was only a 2.20-2.43fold expression of Dkc1 when compared to the knockdown condition (Figure 7A).Compared to the shGFP+EV control, each of the pEF-DEST plasmids had fold expression of Dkc1 in the range of 0.46-0.51,indicating overexpression of Dkc1 from the pEF-DEST vectors was not enough to restore Dkc1 expression to levels without knockdown (Figure 7A).Additionally, when compared to the shDkc1B+EV control, there were no significant changes in stem cell markers between any of the rescue conditions, except for a slight and mildly significant increase in Oct4 expression in the catalytically active, shRNA resistant (shDkc1B+Dkc1 shBR) condition (Figure 7B-D).In order to determine if exogenous Dkc1 was still being produced at the time point of RNA harvest, protein was harvested at the same time point (day 4 post-transfection) in the same conditions (expansion on day 1 and antibiotic selection on day 2) and analyzed by western blot.This blot showed no detectable exogenous expression of Dkc1 at four days post-transfection in any of the rescue conditions (Figure 7E).The knockdown of Trub1 was investigated to determine if changes in stem cell phenotype as a result of knockdown of the RNA-dependent PUS complex would be seen in an RNA-independent PUS that is not highly expressed in mESCs when compared to differentiated cells.Even though both shTrub1A (fold expression 0.11) and shTrub1B (fold expression 0.15) had similar knockdown efficiency of Trub1, there were marked differences in stem cell marker expression between the two shRNAs.One potential explanation for this finding is off-target effects of the shTrub1A or shTrub1B shRNA.By potentially targeting mRNAs that are not encoding for Trub1, these shRNAs may have had additional impacts on mESC gene expression that cannot be attributed to changes in Trub1 expression.Therefore, we cannot conclude if Trub1 itself regulates expression of the stem cell markers Sox2, Nanog, and Oct4, or if the changes in stem cell markers and phenotype after knockdown of Dkc1 and Nop10 are specific to their identity as members of the RNA-dependent PUS complex the box H/ACA snoRNP, not due to general pseudouridylation activity.

Discussion
A co-transfection system was developed to simultaneously knockdown endogenous expression of Dkc1 and exogenously express either catalytically active (WT) or inactive (D125A) Dkc1 that were resistant to knockdown by shDkc1B (shBR), in order to determine if the PUS function of Dkc1 is essential for the maintenance of stem cell homeostasis.The efficacy of the resistance mutations was confirmed by transient co-transfection of pLKO shDkc1B and the four pEF-DEST51 plasmids, which showed exogenous expression of Dkc1 from shRNA resistant plasmids in the presence of shDkc1B.This short-term screen was interpreted as a validation of the co-transfection system for exogenous expression of Dkc1 in the presence of shDkc1B.This system allowed us to explore if expression of catalytically active or inactive Dkc1 has an impact on the expression of stem cell markers when endogenous Dkc1 expression is reduced by shRNA, thus discerning if the pseudouridylation activity of Dkc1 is required for the maintenance of a stem cell state.
The co-transfection system was then used to evaluate rescue of loss of stem cell homeostasis by overexpression of Dkc1 with and without pseudouridylation activity.In the cotransfection system, knockdown of Dkc1 by shDkc1B was less efficient than in the pLKO only knockdown condition.This corresponded to a less significant disruption in stem cell state, as measured by stem cell marker expression, in the co-transfection system when compared to the pLKO only knockdown condition.At four days after transfection, Dkc1 expression in the rescue conditions was higher than that of the knockdown condition (2.2-2.4 fold expression), but did not reach levels similar to stem cells without knockdown of Dkc1.This level of exogenous Dkc1 expression was greater than the level at which a full disruption in stem cell state was seen in the knockdown conditions, but expression of all three stem cells markers were not rescued to levels of shGFP control cells.At the level of exogenous Dkc1 expression seen in all four rescue conditions, there were no changes in expression of stem cell markers between the knockdown condition and any of the rescue conditions, with the exception of a small and slightly significant increase in Oct4 expression in the shDkc1B + Dkc1 shBR condition.Therefore, we cannot draw conclusions about the impact of pseudouridylation function of Dkc1 on expression of stem cell markers in mESCs.
Although we were able to confirm the functionality of the co-transfection system for overexpression of Dkc1 and its mutants in a short term screen, we were not able to show that this

Figure 4 :
Figure 4: ShRNA knockdown of Trub1 leads to variability in stem cell marker expression.

Figure 5 :
Figure 5: Alkaline phosphatase staining of shRNA transfected cells shows phenotypic

Figure 6 :
Figure 6: Confirmed exogenous expression of catalytically active and catalytically inactive

Figure 7 :
Figure 7: Rescue of Dkc1 expression and loss of stem cell homeostasis was not accomplished in Cell identity is maintained by key regulators, often transcription factors, that maintain the gene expression profile of a specific cell type and control the induction of differentiation or transformation into new cell types.Functional studies have identified three transcription factors, Sox2, Nanog, and Oct4 as the core regulators in the maintenance of a pluripotent state in ESCs (Chen et al 2008, Young 2011, Jaenisch and Young 2008, Ng and Surani 2011).The expression of these three transcription factors are frequently used as indicators of the pluripotent potential of ESCs.Despite the identification of these key transcription factors for maintaining self-renewal and pluripotency, mechanisms regulating these master regulators remain poorly understood (Boyer et al. 2005, Cinghu et al 2014).In an attempt to address this gap in knowledge, RNAibased screens have identified more than 400 genes with a role in ESC maintenance (Ivanoza et al 2006, Fazzio, Huff, and Panning 2008, Hu et al 2009, Ding et al 2009, Chia et al 2010, Bilodeau et al 2009).
Ge and Yu 2013, Carlile et al 2014, Lovejoy et al. 2014, Schwartz et al. 2014) (Figure 1B).This modification creates an additional hydrogen bonding site, which leads to a stabilizing effect of Ψ on RNA (Figure 1B).As with many other RNA modifications, the functions of Ψ are not yet well understood, but the process of pseudouridylation is dynamic and may play a regulatory role in response to cellular stressors (Carlile et al 2014, Courtes et al 2014, Lovejoy et al 2014, Schwartz et al 2014, Wu et al 2011).Additionally, the documented impacts of Ψ on RNA structure suggest a potential influence by post-transcriptional regulation including translation efficiency of mRNAs, ribosome pausing, and alteration of the genetic code by non-canonical base pairing (Carlile et al 2015).

Figure 1 :
Figure 1: (A) Schematic representation of the eukaryotic box H/ACA snoRNP, adapted from the archaeal Box H/ACA snoRNP structure (Watkins and Bohnsack 2011).The four proteins (Dkc1, Nop10, Nhp2, and Gar1) are bound to a box H/ACA snoRNA, which is named for the box H and box ACA sequences.The box H/ACA snoRNA has a double hairpin structure with two pseudouridylation pockets that can target different RNAs for pseudouridylation.(B) Structures of uridine and pseudouridine.Pseudouridine is a structural isomer of uridine in which the uracil ring has been rotated 180° about the N-3/C-6 axis, breaking the N-1 glycosyl bond and forming a C-5 glycosyl bond.This exposes a fourth hydrogen bonding site at N-1 (arrow).Dkc1 is an RNA-dependent PUS and functions as the catalytic element of the snoRNP ), where protein was harvested from cells on day 4 post-transfection (after antibiotic selection).Cells were harvested with Trypsin-EDTA (0.25%) (Invitrogen, 1540054), neutralized with DMEM (Invitrogen, 11965092) supplemented with 10% FBS (Gibco, 16141079), pelleted by centrifugation (113 xg), and washed 2X with PBS.Cells were lysed with RIPA Lysis Buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS) supplemented with 2X Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher, 78440).The lysate was separated by centrifugation (18213 xg) and the protein harvested in the supernatant was quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher, 23225) following the manufacturer's protocol.Western Blotting: 20-40 µg of protein was diluted to a final volume of 20 µL and mixed with NuPAGE™ LDS Sample (4X) (Invitrogen, NP0007) and boiled at 100 °C for 4 min.Samples were then run on a NuPAGE™ 4-12% Bis-Tris Protein Gel (Invitrogen, NP0322BOX), transferred to a nitrocellulose membrane, and immunoblotted with primary antibodies against the V5 tag raised in mouse (Invitrogen, R962-25) at 1:5000, for detection of Dkc1 from the pEF-DEST51 constructs, and GAPDH raised in mouse (abcam, ab9484) at 1:10000, for loading control, and with the secondary antibody IRDye® 680RD Donkey anti-Mouse IgG (H + L), 0.1 mg (Li-Cor, P/N 925-68072) at 1:10000 for visualization using the Li-Cor Odyssey Imaging System.Signal intensity of bands was quantified in ImageJ.Dkc1 expression was normalized to GAPDH expression in all samples and averaged across samples of the same condition.Bar graphs presenting expression of proteins from western blots were generated in GraphPad Prism 7.

Figure 5 :
Figure 5: Alkaline phosphatase staining of PUS knockdowns.Red colonies indicate a stem cell phenotype.shGFP control show little to no differentiation, as evidenced by dark red colonies and few cells outside of colonies.Knockdown conditions were determined to have no, mild (shTrub1A, shTrub1B), moderate (shNop10A, shNop10C, shDkc1A), or severe differentiation (shDkc1B).*shTrub1B was split 1:3 at day 1 to ensure appropriate cell density for staining.
mutation, a catalytically inactive version of Dkc1 was generated by the conversion of an aspartic acid (D) residue in the catalytic site of Dkc1 to an alanine (A) (D125A) in the pEF-DEST51 vector by mutagenesis PCR.This resulted in the generation of four independent plasmids for the exogenous expression of Dkc1, pEF-DEST Dkc1, pEF-DEST Dkc1 D125A, pEF-DEST Dkc1 shBR, pEF-DEST Dkc1 D125A shBR.Exogenous expression of Dkc1 from each of the plasmids was visualized by western blot.Dkc1 expression was determined by α-V5 western blot, and this expression was normalized to GAPDH expression (Figure 6B).Although there was variability in expression between plasmids, each plasmid expressed Dkc1 protein.The variability may be due, in part, to differences in transfection efficiency between samples as antibiotic selection was not used for these transient transfections.
Due to the high expression of proteins of the box H/ACA snoRNP in stem cells(Cinghu et al 2014) and previous findings that Dkc1 is a regulator of stem cell marker expression(Fong et al 2014), it was anticipated that the knockdown of the RNA dependent PUS Dkc1 as well as the box H/ACA snoRNP member Nop10, which is essential for the pseudouridylation function of Dkc1(Charpentier et al 2005, Li et al 2011), would lead to a loss of stem cell homeostasis, as measured by expression of stem cell markers and phenotypic staining for stem cell colonies.This finding was confirmed, particularly in the case of Dkc1, where an approximately 90% efficient knockdown of Dkc1 by shDkc1B (fold expression 0.09) led to a disruption in stem cell homeostasis.A less efficient knockdown by shDkc1A (fold expression 0.22) lead to some changes in stem cell marker expression, further supporting the claim that Dkc1 expression can impact a stem cell phenotype, although it appears that Nanog expression is most readily impacted by a reduction in Dkc1 expression.Furthermore, knockdown of Nop10 by shNop10A and shNop10C also resulted in changes in expression of some stem cell markers, but not full loss of stem cell homeostasis.This discrepancy between the phenotypic response of stem cells to the knockdown of Dkc1 and the knockdown of Nop10 was to be expected, as Dkc1 is the catalytic element of the box H/ACA snoRNP, so loss of this protein would render the complex nonfunctional.While Nop10 is an important structural component of the box H/ACA snoRNP, reduced expression by knockdown of Nop10 may still result in the assembly of some small number of functional box H/ACA snoRNP complexes.The results from RT-qPCR analysis of stem cell markers in Dkc1 and Nop10 knockdowns supports the finding that a stem cell phenotype can be modulated by the expression of proteins in the box H/ACA snoRNP, and that Nanog is particularly responsive to changes in expression of the RNA-dependent PUS complex.This finding is interesting within the traditional dogma of the master regulators, in which Nanog, Oct4, and Sox2 act in partnership through transcriptional maintenance of pluripotency.However, studies since the identification of these three factors have found that Nanog expression is highly variable in ESCs, while expression of Oct4 and Sox2 is fairly homogeneous(Silva and Smith 2008).Additionally, cells lacking Nanog can remain undifferentiated and pluripotent, but these cells have a greater tendency to differentiate(Chambers et al. 2007, Mitsui et al. 2003).This suggests that Nanog stabilizes the pluripotent state, while not being essential for the maintenance of pluripotency in ESCs(Jaenisch and Young 2008, Chambers et al. 2007).Therefore, a change in Nanog expression without a change in expression of the other two stem cell markers, may indicate a loss of stability in ESC homeostasis without a full loss of homeostasis, which could be a kind of priming prior to differentiation.This finding is also in line with previous literature that identifies Dkc1 as a coactivator along with Oct4 and Sox2 of Nanog gene expression in ESCs(Fong et al. 2014).
functionality was retained as time progressed.Potential explanations for this finding are ineffective selection of pEF-DEST51 containing cells during the antibiotic selection stage of the experimental design, improper folding of exogenous Dkc1 proteins due to the presence of a 6X-His and V5-tag on the C-terminal end of each protein leading to lack of functionality of exogenous Dkc1 and protein degradation, or silencing of transcription from the pEF-DEST51 plasmid over time by epigenetic modification such as methylation of the EF-1α promoter.In order to test the likelihood of these hypotheses further experimentation with the pEF-DEST51 plasmids, including baseline protein expression after selection and four days after transfection, need to be conducted.Furthermore, re-cloning of the Dkc1 overexpression constructs to have an N-terminal tag may aid in loss of functionality due to improper folding.These steps must be taken first in order to determine if the pseudouridylation function of Dkc1 is responsible for its impacts on stem cell phenotype and homeostasis.Previous literature had identified the proteins of the box H/ACA snoRNP as potentially important for the maintenance of a stem cell state (Cinghu et al 2014) and Dkc1 as a co-activator of Sox2/Oct4 mediated gene expression(Fong et al 2014), but its role as a PUS in the maintenance of stem cell homeostasis had not been fully explored.Through shRNA mediated knockdown of Dkc1, we were able to establish significant changes in all three stem cell markers (Sox2, Nanog, and Oct4) as well as phenotypic changes and loss of AP activity, indicative of differentiation.Furthermore, we were able to establish that knockdown of Nop10, another essential component of the box H/ACA snoRNP, lead to changes in expression of stem cell markers but not a full disruption of stem cell homeostasis.These findings confirm those in previous literature that expression of the box H/ACA snoRNP proteins is essential for stem cell homeostasis, and we have now established by RT-qPCR that expression of the three master regulators of stem cell identity are modulated by knockdown of Dkc1.However further work is still needed to establish if the relationship between Dkc1 and stem cell phenotype is due to its function as a PUS.Despite the lack of functional connection drawn from these experiments, the validation of Dkc1 as essential for the maintenance of a stem cell state furthers the goal of understanding the full network of genes responsible for stem cell self-renewal and pluripotency.and underlined bases are the mutated bases introduced by these primers through mutagenesis PCR indicated on the forward primers

Table 1 : Summary of knockdown efficiency, expression of stem cell markers, phenotypic evaluation of AP staining, and assessment of stem cell homeostasis
. (Abbreviations: Sev.severe, mod.-moderate, diff.-differentiation)