ABSTRACT: Biomarkers unique for distributed stem cells (DSCs) have proven elusive. Previous searches for proteins expressed specifically in DSCs were hampered by difficulty obtaining pure DSCs and challenges to successfully mining complex molecular expression data. To identify novel candidates for DSC biomarkers, we combined a sparse feature selection method with combinatorial molecular expression data focused on asymmetric self-renewal, a defining DSC property. Our analyses revealed reduced expression of the histone H2A variant H2A.Z as a superior discriminator for asymmetric self-renewal, which proved to be a novel pattern-specific biomarker of DSCs. Overview: Sixteen cDNA micro-array samples were analyzed, with four replicates for each of four biologically orthogonal comparisons of congruent asymmetrically self-renewing versus symmetrically self-renewing cells. Sparse feature selection was used to identify a uniquely robust discriminator of these two different DSC self-renewal patterns. General Approach: An orthogonal-intersection strategy for identification of asymmetric self-renewal associated genes Previously, we used non-tumorigenic, immortalized mouse mammary epithelial C127 cells and mouse embryo fibroblasts (MEFs) to derive lines with conditional self-renewal patterns. The self-renewal pattern of these cells can be reversibly switched between symmetric and asymmetric by varying either culture temperature or Zn concentration, respectively, as a consequence of controlling p53 expression from respectively responsive promoters (1-6). These and related cell lines were used to design a 2 x 4 orthogonal-intersection microarray analysis for the purpose of identifying genes whose expression consistently showed the same pattern of change between asymmetric self-renewal versus symmetric self-renewal. Four different pair-wise comparisons were developed in which a state of asymmetric self-renewal was compared to a congruent state of symmetric self-renewal. The first 3 comparisons were based on a difference in p53 expression, but each had a different biological context. For asymmetric versus symmetric, respectively, these comparisons were: 1) Zn-inducible p53 MEFs (Ind-8 cells) versus p53-null control MEFs (Con-3 cells), both cultured in Zn-supplemented medium (65 micromolar ZnCl2); 2) Zn-inducible p53 MEFs in Zn-supplemented medium versus in Zn-free medium (1,3-6); and 3) low temperature (32.5?C)-inducible p53 mouse mammary epithelial cells (MMECs; 1h-3 cells) versus vector-control MMECs (i.e., no p53 transgene; 1g-1 cells), both cultured at 32.5?C (2,5,6). The fourth comparison had a special purpose. It provided a comparison of asymmetric versus symmetric self-renewal that was not due to a difference in p53 expression (1). Two previously described derivatives of the Zn-responsive p53-inducible MEFs were used to make this comparison. One line (tI-3 cells) is stably transfected with a constitutively expressed type II inosine monophosphate dehydrogenase (IMPDH II) gene (1,3,5). IMPDH II is the rate-limiting enzyme for guanine ribonucleotide biosynthesis. Its down-regulation by p53 is required for asymmetric self-renewal (3,5). Therefore, even in Zn-supplemented medium, which induces normal p53 expression, cells derived with a stably expressed IMPDH II transgene continue to undergo symmetric self-renewal (3,5). This abrogation of p53 effects on self-renewal pattern occurs even though other p53-dependent responses remain intact (3). Under the same conditions, control vector-only transfectants (tC-2 cells) continue to exhibit asymmetric self-renewal (3,5). Thus, this fourth comparison could be used to exclude genes whose change in expression was primarily due to changes p53 expression and not specifically transitions in self-renewal symmetry as well (1). We identified genes whose expression varied consistently for all 4 orthogonal comparisons of cells in states of asymmetric self-renewal versus symmetric self-renewal (7). We developed a new quality control metric called the population division cycle ratio (PDC ratio) to insure consistent degrees of asymmetric self-renewal and symmetric self-renewal across all 4 orthogonal comparisons (6,7). Prefabricated cDNA microarrays constructed with the National Institute for Aging (NIA) mouse 15K mouse clone set were used for the analysis (8,9). As detailed in supplemental Materials and Methods (see below), for each of the 4 experimental comparisons, we isolated two independent samples of RNA; and each of the RNA samples was labeled independently with Cy5 and Cy3 fluorescent dyes. Each independent set of fluorescent RNA samples was used to develop two reciprocal co-hybridizations for each experimental comparison. Thus, data from 4 microarrays, representing 2 independent experiments, were available for each of the 4 orthogonal contexts for asymmetric versus symmetric self-renewal. Materials and Methods: cDNA Microarray Data Analysis Cells for total RNA extraction were harvested at the time estimated for PDC = 2 (36 hours for Ind-8 and Con-3, 48 hours for tC-2, tI-3, 1h-3, and 1g-1), and were selected for microarray experiments based on the actual PDC ratio curves derived from replicate cell cultures grown in parallel. The same PDC ratio values were maintained to obtain similar fractions of cycling stem-like cells and non-cycling differentiating cells for all compared asymmetric models. Total RNA was extracted with the Trizol reagent (Invitrogen, Carlsbad, CA) and impurities were removed with the Qiagen RNeasy kit (Qiagen, Valencia, CA). Fifty to seventy ?g of total RNA was used for cDNA syntheses. Arabidopsis thaliana mRNAs (Stratagene, La Jolla, CA) were introduced as internal probe standards into reverse transcription reactions to normalize data between different arrays. Cy3- or Cy5-fluorescently labeled cDNAs were hybridized onto the NIA 15K mouse cDNA prefabricated arrays (8), supplied by the Massachusetts Institute of Technology (MIT)-BioMicro Center, using the procedure provided by the MIT-BioMicro Center. Hybridized microarrays were scanned with the arrayWoRxeTM Biochip Reader (Applied Precision LLC, Northwest Issaquah, WA). The fluorescence intensity of each spot was analyzed from the scanned tiff images by using the DigitalGenomeTM software (MolecularWare, Inc. Cambridge, MA). The Cy3 and Cy5 fluorescence intensities were normalized by calculating the normalization factor from total intensity normalization (10). Analyses for each self-renewal pattern comparison were performed as duplicate independent experiments. For each comparison, we performed two chip hybridizations with reciprocally labeled Cy3 or Cy5 target cDNAs to each biological sample. The entire analysis incorporated data from 16 independent chips. A gene was selected for data analyses only if the mean value of foreground pixels of the spot was greater than the sum of the mean and two standard deviations of the background pixels. For individual gene probe spots, the expression intensities of Cy5 and Cy3 channels were estimated by subtracting mean backgrounds from mean foregrounds. The ratios of the final gene expression intensities for the asymmetrically self-renewing states to the respective symmetrically self-renewing states were calculated. These ratio values (7) were used for sparse feature selection. References: 1. M. Noh, J. L. Smith, Y. H. Huh, J. L. Sherley, J. L., A resource for discovering specific and universal biomarkers for distributed stem cells. PLoS ONE 6(7): e22077. doi:10.1371/journal.pone.0022077 (2011). 2. J. L. Sherley, P. B. Stadler, D. R. Johnson, Expression of the wild-type p53 antioncogene induces guanine nucleotide-dependent stem cell division kinetics. Proc. Natl. Acad. Sci. USA 92, 136-140 (1995). 3. Y. Liu, S. A. Bohn, J. L. Sherley, Inosine-5’-monophosphate dehydrogenase is a rate-limiting factor for p53-dependent growth regulation. Mol. Biol. Cell 9, 15-28 (1998). 4. L. Rambhatla et al., Cellular senescence: ex vivo p53-dependent asymmetric cell kinetics. J. Biomed. Biotech. 1, 28-37 (2001). 5. L. Rambhatla, S. Ram-Mohan, J. J. Cheng, J. L. Sherley, Immortal DNA strand co-segregation requires p53/IMPDH-dependent asymmetric self-renewal associated with adult stem cells. Cancer Research 65, 3155-3161 (2005). 6. Y. Liu et al., Comparison of bax, waf1, and IMP dehydrogenase regulation in response to wild-type p53 expression under normal growth conditions. J. Cell Physiol. 177, 364-376 (1998). 7. M. Noh, thesis, Massachusetts Institute of Technology (2006). 8. T. S. Tanaka et al., Genome-wide expression pr
