Comparisons between transcriptional regulation and RNA expression in human embryonic stem cell lines.
ABSTRACT Recent studies have focused on transcriptional regulation and gene expression profiling of human embryonic stem cells (hESCs). However, little information is available regarding the relationship between RNA expression and transcriptional regulation, which is critical in the complete understanding of pluripotency and differentiation of hESCs. In the current study, we determined RNA expression of three different hESC lines compared to Human universal reference RNA expression (HuU-RNA) using a full genome expression microarray, and compared our results to target genes previously identified using ChIP-on-chip analysis. The objective was to identify genes common between the two methods, and generate a more reliable list of embryonic signature genes. Even though hESCs were obtained from different sources and maintained under different conditions, a considerable number of genes could be identified as common between RNA expression and transcriptional regulation analyses. As an example, results from ChIP-on-chip studies show that OCT4, SOX2, and NANOG co-occupy SOX2, OCT4, TDGF1, GJA1, SET, and DPPA4 genes. The results are consistent with RNA expression analyses that demonstrate these genes as differently expressed in our hESC lines, further substantiating their role across cell types and confirming their importance as embryonic signatures. In addition, we report the differential expression of growth arrest-specific (GAS) family of genes in hESC. GAS2L1 and GAS3 members of this family appear to be transcriptionally regulated by OCT4, SOX2, or NANOG, whereas GAS5 and GAS6 are not; all of the genes are differentially expressed, as determined by microarray and validated via quantitative (Q)- PCR. Collectively, these data provide insight into the relationship between gene expression and transcriptional regulation, resulting in a reliable list of genes associated with hESCs.
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ABSTRACT: Pluripotent stem cell lines with similar phenotypes can be derived from both blastocysts (embryonic stem cells, ESC) and primordial germ cells (embryonic germ cells, EGC). Here, we present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6). Even in the differentiation-promoting conditions, these pluripotent cells showed the same general trends of gene expression changes regardless of their origin and genetic background. These data indicate that ESCs and EGCs are indistinguishable based on global gene expression patterns alone. On the other hand, a detailed comparison between a group of ESC lines and a group of EGC lines identified 20 signature genes whose average expression levels were consistently higher in ESC lines, and 84 signature genes whose average expression levels were consistently higher in EGC lines, irrespective of mouse strains. Similar analysis identified 250 signature genes whose average expression levels were consistently higher in a group of 129 cell lines, and 337 signature genes whose average expression levels were consistently higher in a group of C57BL/6 cell lines. Although none of the genes was exclusively expressed in either ESCs versus EGCs or 129 versus C57BL/6, in combination these signature genes provide a reliable separation and identification of each cell type. Differentiation-promoting conditions also revealed some minor differences between the cell lines. For example, in the presence of RA, EGCs showed a lower expression of muscle- and cardiac-related genes and a higher expression of gonad-related genes than ESCs. Taken together, the results provide a rich source of information about the similarities and differences between ESCs and EGCs as well as 129 lines and C57BL/6 lines. Such information will be crucial to our understanding of pluripotent stem cells. The results also underscore the importance of studying multiple cell lines from different strains when making comparisons based on gene expression analysis.Developmental Biology 08/2007; 307(2):446-59. DOI:10.1016/j.ydbio.2007.05.004 · 3.64 Impact Factor
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ABSTRACT: ChIP-chip studies have revealed that many in vivo binding sites have a weak match to the consensus sequence for the transcription factor being analyzed. Possible explanations for these observations include (1) the in vitro-derived consensus site does not represent the in vivo binding site and/or (2) the factor is recruited to a weak binding site via interaction with another protein. To address these possibilities, we developed an approach (ChIPMotifs) that incorporates a bootstrap resampling method to statistically infer the optimal cutoff threshold for a position weight matrix (PWM) of a motif identified from ChIP-chip data by ab initio motif discovery programs. Using OCT4 ChIP-chip data and the ChIPMotifs approach, we first developed a refined OCT4 PWM. We then used the refined PWM and a ChIPModules approach to identify transcription factors colocalizing with OCT4 in Ntera2 testicular embryonal carcinoma cells. We found that the consensus binding site for SRY, a transcription factor critical for testis development, colocalizes with the OCT4 PWM. To further characterize the relationship between OCT4 and SRY, we performed ChIP-chip experiments with human promoter microarrays, and found that 49% of the top approximately 1000 OCT4 target promoters were also bound by SRY. This analysis represents the first identification of SRY target promoters. Interestingly, we determined that promoters bound by OCT4 and SRY, but not those bound by SRY alone, were also bound by the transcriptional repressor KAP1. Our studies not only validate the ChIPMotifs and ChIPModules combinatorial approach but also identify a possible new regulatory partner of OCT4.Genome Research 07/2007; 17(6):807-17. DOI:10.1101/gr.6006107 · 13.85 Impact Factor
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ABSTRACT: The most important sepiolite and palygorskite deposits and occurrences were formed in continental environments by authigenesis, through direct precipitation from solution (neoformation), or by transformation of precursor minerals, mainly detrital clays. In the sedimentary environment, transformation is mostly related to diagenesis, whereas neoformation can take place in both syngenetic (depositional) and diagenetic environments. Sepiolite and palygorskite and other Mg-clay (saponite, stevensite and kerolite) are the result of the interaction between silica- and magnesium-bearing solutions in an environment with suitable physico-chemical conditions (mostly pH and salinity), and on the activity of aluminium in solution or reactive phases in the case of palygorskite. The physico-chemical conditions of the environment control the formation of the magnesian clay mineral, also determining its fibrous or laminar nature and, consequently, its composition. These conditions occur in specific continental sedimentary environments. This contribution suggests a model for the pathways leading to sepiolite–palygorskite (and other Mg-clays) formation based on worldwide continental occurrences. Also, it establishes the sedimentological models and related lithological associations, according to the updated experimental and geological data.