A Systems Biology Approach Identifies a Regulatory Network in Parotid Acinar Cell Terminal Differentiation

<div><p>Objective</p><p>The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.</p><p>Methodology</p><p>A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM) was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.</p><p>Results</p><p>Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for <i>Pparg</i> which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving <i>Klf4</i>, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving <i>Prdm1</i>, <i>Sox11</i>, <i>Pax5</i>, miR-200a, and miR-30a) progressively decreases repression of <i>Xbp1</i> gene transcription, in concert with decreased translational repression by miR-214. <i>The transcription factor Xbp1</i> mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with <i>Atf6</i>. Transfection studies show that <i>Xbp1Mist1</i> promoter. In addition, <i>Xbp1</i> and <i>Mist1</i> each activate the parotid secretory protein (<i>Psp</i>) gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.</p><p>Conclusion</p><p>This study identifies novel expression patterns of <i>Pparg</i>, <i>Klf4</i>, and <i>Sox11</i> during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a genetic switch involving transcription factors and microRNAs, and transition to an <i>Xbp1</i> driven differentiation network. This proposed network suggests key regulatory interactions in parotid gland terminal differentiation.</p></div

<i>Xbp1</i> Regulates <i>Mist1</i> Expression during Parotid Differentiation.

<p>(A) Log2 plot of microarray data for <i>Xbp1</i> and <i>Mist1</i>. (B) Expression of <i>Xbp1</i> and <i>Mist1</i> is highly correlated across parotid differentiation. Plot of Log2 <i>Xbp1</i> vs. Log2 <i>Mist1</i> shows a linear trend with R² = 0.9538. (C) Luciferase assay shows activation of <i>Mist1</i> promoter by <i>Xbp1</i> in ParC5 cells. Increasing amount of <i>Xbp1</i>-S (spliced <i>Xbp1</i>) cDNA/well (0.25 μg, 0.5 μg, and 1 μg) were co-transfected with a luciferase expression plasmid driven by a <i>Mist1</i> promoter. Significant increase in luciferase expression was observed for all concentrations of <i>Xbp1</i>-S (p = 0.017, p = 0.01, and p = 0.05 respectively) (n = 3).</p

MicroRNA Expression Changes between Time Points.

<p>MicroRNA Expression Changes between Time Points.</p

Transient Activation of <i>Pparg</i> during Parotid Acinar Cell Differentiation.

<p>(A) Network showing transcription factor <i>Pparg</i> and known downstream target genes found in DE Cluster 4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.g002" target="_blank">Fig 2</a>). DE Cluster 4 contains 106 genes (including <i>Pparg</i>) with a unique expression pattern; higher expression only in stages 2 and 3. The Metacore knowledge-base identifies 18 of these as <i>Pparg</i> target genes. A green arrow indicates activation of transcription while red arrow indicates inhibition. A grey line means the interaction is uncharacterized. Although a red arrow connects <i>Pparg</i> and <i>ACP5</i>, some publications list the interaction as activating [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.ref067" target="_blank">67</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.ref068" target="_blank">68</a>] indicating it could be context dependent. (B) Log2 expression of <i>Pparg</i> from microarray data. (C) qPCR data confirming the expression profile of <i>Pparg</i>. RNA samples from independent animals were collected at three time points (E20, P5, and P25). Expression was normalized to <i>Arbp</i>, and data showed significant change in expression by ANOVA. n = 3.</p

mRNAs with a Quadratic Expression Pattern.

<p>Quadratic regression reveals late activation of acinar cell specific genes. (A) Quadratic regression analysis identified 430 genes having a significant quadratic trend (FDR < 0.05), which were clustered into eight patterns. For visualization, the expression data for each gene was scaled to a mean of zero and standard deviation of 1 before plotting. The red line traces the average expression for the cluster. (B) Log2 plot of Quadratic Cluster 6 members with at least a 4-fold expression difference between P25 and E18. This shows late up-regulation of several genes known to produce salivary proteins (i.e. <i>DNase I</i>, <i>Chitinase</i>, <i>Prp15</i>, <i>Sgp158/Prr21</i>).</p

<i>Psp</i> is Directly Regulated by both <i>Xbp1</i> and <i>Mist1</i>.

<p>(A) <i>Xbp1</i> activates the <i>Psp</i> promoter. Increasing amounts of <i>Xbp1</i>-S cDNA was co-transfected into ParC5 cells along with a luciferase expression plasmid driven by either a 500 bp or 1 kb fragment of the <i>Psp</i> promoter region. Analysis was performed by t-test. Expression of luciferase driven by 1 kb <i>Psp</i> promoter increases significantly upon increasing transfection of <i>Xbp1</i>-S (p = 0.007, 0.02, and 0.005 respectively). The same is seen with the 500bp <i>Psp</i> promoter (p = 0.01, 0.01, and 0.003 respectively) (n = 3). (B) <i>Mist1</i> activates the <i>Psp</i> promoter through interactions with intronic sequences. Luciferase expression was driven by either a 1.5 kb fragment of the <i>Psp</i> promoter or the 1.5 kb fragment along with 1320 bp of intronic sequence flanking exon 3 that contains two E-boxes. Promoter plasmids were co-transfected with <i>Mist1</i> and <i>Tcf3</i> cDNA expression plasmids. Analysis was performed by t-test (p = 0.02) (n = 4).</p

Gene Expression Comparisons between Developmental Stages.

<p>Gene Expression Comparisons between Developmental Stages.</p

Hierarchical Clustering Divides Parotid Acinar Cell Differentiation into Stages.

<p>(A) Principal component analysis (PCA) of the 27 microarrays of RNA samples taken in triplicate at nine time-points across acinar cell differentiation. The first three principal components are plotted on a three dimensional graph. Samples are largely clustered based on the replicate time points. (B) Hierarchical clustering of differentially expressed mRNAs displayed as a heatmap suggests the presence of 4 stages. 2656 genes were identified as differentially expressed by one-way ANOVA (FDR p < 0.05). Image was generated using the heatmap.2 function in R and distance was calculated as dissimilarity = 1-r (correlation coefficient). Expression values are scaled (mean 0, std dev of 1) by rows.</p

Profiles of Differentially Expressed mRNAs during Differentiation.

<p>The time course microarray data were analyzed to identify differentially expressed mRNAs, which were clustered based on statistically significant similarities in the expression profiles. For visualization, the expression data for each gene was scaled to a mean of zero and standard deviation of 1 before plotting. The red line traces the average expression for the cluster.</p