ES cell gene expression is carefully regulated and cells either maintain the pluripotent state by self-renewal or undergo differentiation. This is the first study to investigate the coexpression of genes along the chromosome in human ES cells and their earliest stage of differentiation in culture, EB's. Significant coexpression patterns were revealed and confirmed by random tests and Monte-Carlo simulation. The coexpression is suggestive of transcriptional regulation operating at the chromosome domain level in ES and EB cells. The coexpression domains do not appear to represent amplicons or regions of chromosome imbalance that were previously described in cancer cells [23
]. The chromosome region with adjacent localization of the genes NANOG, STELLAR, and GDF3 has been considered as a hotspot for teratocarcinoma [24
]. Our study however indicated that the genes in the region were not coexpressed, suggestive of no transcriptional regulation operating at this domain in ES or EB. Nevertheless, the identified coexpression chromosome domains are biologically and physiologically significant, some of which are associated with functions important to ES development. New coexpression chromosome domains would possibly be observed when each cell line had been analyzed separately. Recent studies have shown that some ES cell lines exhibit unique morphological and genetic features [25
]. The cell line BG01V, for example, shows abnormal chromosome and karyotype, different from other ES cells [26
].It is thus important to examine cell line specific patterns of local coexpression, which will be the future direction of our studies.
The genes LIFR, GP130, STAT3, OCT3/4, SOX2, UTF-1, FOXD3, ERAS, TEL1, FGF4, NANOG, NODAL, TDFG1, CER1, and ABCG2 have shown to be critical for ESC self-renewal and self-renewal and regarded as the "signature" [15
]. Some of these genes were not coexpressed on the chromosome (Table ), suggesting that global regions still tend to be involved in determining the overall state of the ES cell and provide context for cell-type specific signaling. Nonetheless, the other ES-signature genes did show coexpression along the chromosome. The genes that were adjacent and coexpressed with the signature genes were often related to development and transcriptional regulation. STAT3, for example, is a transcription factor which plays a central role within ES self-renewal pathways and feed-back loops [29
]. The STAT3 gene, located at 17q21, was coexpressed in ES (coexpression index 0.37) but not in EB (-0.17) (Table ). The coexpression chromosome domain where STAT3 resides also contained the duplicated genes STAT5B and STAT5A, as well as TCF1, a transcription factor important in proliferation and differentiation. Other ES-signature genes, UTF1, TLE1, and OCT3/4, showed higher coexpression index in EB (0.299, 0.285, and 0.23, respectively, although slightly lower than the threshold value) than in ES (0.125, 0.08, and 0.10, respectively). UTF1 is a transcription factor, and the domain where the UTF1 gene is located (at 10q26) contained two other transcriptional factors, VENTX2 [a homeodomain protein implicated in mesodermal patterning and hemopoietic stem cell maintenance [31
]], and NKX6-2. TLE1 is an ES cell-specific gene coding a RNA-binding protein which functions downstream of the LIF and Oct3/4 pathways [32
]. The TLE1 gene domain is located at 9q21.32 and the coexpressed genes included the duplicated gene TLE4 and signal transduction genes GNAQ, GKAP42, and GNA14. OCT3/4 is also a transcriptional factor critical for ES cell self-renewal [34
]. The OCT3/4 domain, located at 6p21.31, contained NFKBIL1 and MHC class I genes. In addition to the ES signature genes, other genes important for ES cell development were also found to be coexpressed on the chromosome in ES or EB. SOX15, for example, is a transcription factor involved in the regulation of embryonic development and transcriptional control in ES cells [35
]. The gene was significantly up-regulated in ES cells (P
value 0.029, fold-change 3.25). The SOX15 domain (Fig. ) showed coexpression in ES cells but not in EB cells, as described above. Among the genes on this domain, EFNB3 belongs to the ephrin gene family and is implicated in development, TNFSF1 is a cytokine belonging to the tumor necrosis factor (TNF) ligand family, and POLR2A, ZBTB4, TP53, and FXR2 are all involved in transcription. Apparently, the differentiation or self-renewal of ES cells was evidenced not only by the differential expression of individual genes at the global level, but also by the differential coexpression of genes at the chromosomal domain level.
Chromosomal clustering of functionally related genes has been demonstrated in various eukaryotes, including the yeast, fruit fly, nematode, and human [1
]. Natural selection might have organized genes to clusters on the chromosome according to the molecular function or biological process so that their expression can be coordinately regulated. The coexpression of physically adjacent genes may be caused by the long range effect of transcription factors, chromatin structure modifications, or increased concentration of components of the transcriptional machinery (such as transcription factors) in a particular subnuclear location of chromosomal segments [18
]. The coexpression could also be due to duplicated genes, which often remain adjacent and have similarexpression patterns [18
]. Our study revealed that gene duplication had a minimal impact and was not a major contributing factor for the observed coexpression pattern in ES and EB. Our study also revealed differential local coexpression between ES and EB. Differentially coexpressed genes may not be differentially expressed, while similarly coexpressed genes may be differentially expressed. The transcriptome map thus provides a basis for examining how transcriptional regulation interacts with genomic structure and how genes clustered on the chromosome are coexpressed during the ES self-renewal and differentiation.