Oct4 targets regulatory nodes to modulate stem cell function

Abstract

Stem cells are characterized by two defining features, the ability to self-renew and to differentiate into highly specialized cell types. The POU homeodomain transcription factor Oct4 (Pou5f1) is an essential mediator of the embryonic stem cell state and has been implicated in lineage specific differentiation, adult stem cell identity, and cancer. Recent description of the regulatory networks which maintain 'ES' have highlighted a dual role for Oct4 in the transcriptional activation of genes required to maintain self-renewal and pluripotency while concomitantly repressing genes which facilitate lineage specific differentiation. However, the molecular mechanism by which Oct4 mediates differential activation or repression at these loci to either maintain stem cell identity or facilitate the emergence of alternate transcriptional programs required for the realization of lineage remains to be elucidated. To further investigate Oct4 function, we employed gene expression profiling together with a robust statistical analysis to identify genes highly correlated to Oct4. Gene Ontology analysis to categorize overrepresented genes has led to the identification of themes which may prove essential to stem cell identity, including chromatin structure, nuclear architecture, cell cycle control, DNA repair, and apoptosis. Our experiments have identified previously unappreciated roles for Oct4 for firstly, regulating chromatin structure in a state consistent with self-renewal and pluripotency, and secondly, facilitating the expression of genes that keeps the cell poised to respond to cues that lead to differentiation. Together, these data define the mechanism by which Oct4 orchestrates cellular regulatory pathways to enforce the stem cell state and provides important insight into stem cell function and cancer.

Figure 1. Oct4 Correlation Analysis.

ESC, EC, myogenic, neuronal, retinal, and hematopoietic stem cells and their differentiated derivatives underwent Affymetrix gene expression profiling as part of the Stem Cell Genomics Project. A set of 45 samples were profiled in biological triplicate and hybridized to the MOE430 GeneChip set. Mean intensity values for each biological triplicate are plotted in log scale on the Y-axis, with an approximate cutoff of 1000 demarcating detection status of each gene represented. Transcript expression levels of genes positively (Nanog, Sox2), negatively (Tdrd7 and Mef2a), and not (Myog) correlated to Oct4 are displayed. Detection calls of ‘Present’ for Oct4 are depicted by solid black squares. ‘Absent’ calls are represented by open black squares.

Figure 2. Functional classification of Oct4 correlated genes.

Manual curation of databases reveals highly enriched Gene Ontology (GO) categories for Oct4 positively (A) and negatively (B) correlated genes. Numbers displayed represent percentage of unique transcripts attributed to each category with only the most abundant categories listed individually.

Figure 3. Validation of Oct4 targets.

Chromatin immunoprecipitation (ChIP) assays were performed with Oct4 and IgG antibody and no antibody as a negative control followed by Quantitative Real-time PCR analysis (ChIP/QRT-PCR) for putative positively regulated Oct4 target (A), negatively regulated Oct4 target (B), and non-validated (C) genes. *8L16Rik represents 1110008L16Rik. Results are from two independent ChIP assays, with duplicate QRT-PCR assessment for each. Error bars denote standard error of the mean (SEM).

Figure 4. The Oct4 transcriptional regulatory network.

Validated Oct4 targets (A) are indicated by solid red or green lines. Red and green indicate negative and positive regulation, respectively for all cases. Dashed lines emanating from Oct4 indicate putatively regulated genes. Solid black lines represent potential regulatory nodes that could facilitate the directed differentiation of ESCs. The pressures that preserve stem cell function and modulate early lineage commitment are diametrically opposed. While Oct4 acts to maintain self-renewal and pluripotency in the undifferentiated ‘ES’ state by its modulation of genes that act to maintain permissive chromatin structure, DNA repair, anti-apoptosis, and inactive pRb (B), in differentiation the balance of these forces is altered to favour repressive chromatin structure, DNA checkpoint control, apoptosis, and active pRB which facilitate cellular commitment (C).