Key transcription factors have also been identified that form an intrinsic core regulatory circuit that maintains mouse ES cells in the pluripotent state in vitro
. Of these, Oct4, an atypical homeodomain protein, was originally cloned on the basis of its highly restricted expression pattern; it is expressed exclusively in murine ES cells, ICM and germ cells [7
]. Oct4-deficient murine ES cells differentiate into trophectoderm and fail to form all three germ layers (mesoderm, ectoderm and endoderm) [8
]. Sox2 is an HMG-family protein that occupies many gene targets with Oct4 and is also required to form the ICM [9
]. A recent addition to the 'pluripotency factors' is Nanog, another atypical homeodomain protein related to the Nkx subfamily. Forced expression of Nanog in ES cells lifts the requirement for LIF to maintain pluripotency, suggesting that Nanog is a major regulator of the pluripotent state [5
]. Through genome-wide chromatin immunoprecipitation followed by DNA microarray analysis (ChIP-chip) [12
] or ChIP-PET [13
] experiments (based upon high-throughput sequencing to determine gene expression patterns), numerous target genes bound by Nanog, Oct4 and Sox2 have been identified. These factors appear to act combinatorially to regulate a limited repertoire of target genes, thereby forming a tight transcriptional regulatory circuit that maintains ES cells in a pluripotent state.
Nanog's distinguishing role in the maintenance of murine ES cells in vitro
is an ability to bypass the requirement for the LIF-STAT pathway [5
]. In addition, fusion experiments with mouse cells have shown the dominance of the ES cell phenotype over that of somatic cells, implying that proteins in the nucleus of ES cells are able to reprogram more differentiated cells to an embryonic-like state [14
]. The full repertoire of factors involved in establishing or maintaining pluripotency, and also competent to reprogram cells, is unknown and, until recently, there had been no comprehensive effort to delineate factors necessary for the maintenance of the mouse ES cell in vitro
phenotype. Ihor Lemischka and his colleagues [2
] have now tackled just this issue using a functional genomics approach designed to identify novel factors required for self-renewal in mouse ES cells. They began with microarray data from mouse ES cells as they progress from an undifferentiated state into cells representing all three germ layers upon retinoic-induced differentiation [2
], and hypothesized that factors required for pluripotency and self-renewal would be rapidly downregulated. Of 901 downregulated genes, 65 DNA-binding proteins or transcription factors were selected for further functional analysis.
Ivanova et al
] then assessed the effects of the loss of each of these proteins on the ES cells' capacity for self-renewal. To do this they devised an assay in which wild-type ES cells were mixed with ES cells transduced with lentiviruses containing short hairpin RNAs (shRNAs) to trigger RNA interference (RNAi), along with an expressed green fluorescent protein (GFP) marker. Compromised self-renewal would be reflected in a decreasing percentage of GFP-marked cells in the culture. Six genes were identified by this assay and were characterized further for their effects on mouse ES cell pluripotency. Among these six were the 'old friends' Nanog
, consistent with substantial previous evidence in support of their roles as core self-renewal factors. The three other genes were Esrrb
. To further characterize the possible roles of these genes, Ivanova et al
] carried out extensive marker-gene analysis following the shRNA inhibition. These experiments revealed that each factor appeared to repress distinct differentiation programs, although there was significant overlap.
Further characterization by microarray analysis revealed three distinct patterns of gene expression on knockdown. The expression of approximately 771 genes appeared independent of gene knockdown; expression of 474 genes was either up- or downregulated by knockdown of Nanog, Oct4 or Sox2, but unaffected by knockdown of Esrrb, Tbx3 or Tcl1; and the expression of 272 genes was upregulated by the loss of Esrrb, Tbx3 and Tcl1, but unperturbed by inhibition of Nanog, Oct4 and Sox2. These data provide the first evidence in favor of two independent transcriptional pathways in mouse ES cells; one is controlled by Nanog, Oct4 and Sox2, and may be predominantly responsible for pluripotency and suppressing differentiation. A second pathway involving Esrrb, Tbx3 and Tcl1 seems to be responsible for blocking differentiation along specific cell lineages (Figure ). Surprisingly, modestly raised levels of Nanog compensated for the loss of Esrrb, Tbx and Tcl1, implying cross-talk between the two pathways, with Nanog perhaps serving as a master regulator.
Figure 1 A schematic view of the transcriptional pathways involved in self-renewal and blocking differentiation of murine embryonic stem cells. Self-renewal appears to be regulated by two distinct transcriptional pathways, one involving Esrrb, Tcl1 and Tbx3 and (more ...)