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Title: Systematic Identification of cis-Regulatory Sequences Active in Mouse and Human Embryonic Stem Cells}
Article:
Abstract:
Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem( ES) cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.
Introduction:
Pluripotent stem cells can give rise to all fetal and adult cell lineages, including the germline. The prototypical pluripotent stem cells are embryonic stem( ES) cells[1, 2]. ES cells are a remarkable model for the study of early development and hold promise as a source for cell replacement therapies[3]. To successfully manipulate ES cells in culture, it is important to understand the mechanisms by which ES cells maintain their self renewal and pluripotency. ES cells are derived from the inner cell mass of the blastocyst, a group of cells that gives rise to all cells of the fetus. After the blastocyst implants in the uterus and gastrulation ensues, most cells of the embryo lose the ability to give rise to pluripotent stem cells, except for primordial germ cells( PGCs)[4, 5]. PGCs are the germline precursors that give rise to sperm or eggs. When cultured in vitro, PGCs give rise to embryonic germ( EG) cells, pluripotent stem cells very similar to ES cells[6, 7]. Several regulatory pathways that control ES cell pluripotency and self renewal have recently been identified( reviewed in[8]). Factors involved include the leukemia inhibitory factor( LIF) and BMP signaling pathways[9–12], and transcription factors Nanog[13, 14] and Oct4[15, 16]. Interestingly, the signaling pathways do not appear to be conserved between mouse and human ES cells[17–20], but the transcriptional regulators Oct4 and Nanog are required in ES cells of both species[21–23]. Recent studies indicate that transcription factors other than Oct4 and Nanog are also important for maintenance of the ES cell state[24, 25]. A major goal will be to obtain a complete description of the transcriptional regulatory networks of ES cells. The increasing availability of whole genome sequences and high-throughput experimental methods, such as microarrays, have led to the development of systematic approaches for deciphering transcriptional regulation. Such analyses generally lead to the identification of sets of genes whose expression is coregulated. It has been shown that genes within a coregulated set often share common cis-regulatory motifs, corresponding to transcription factor binding sites, in their upstream genomic sequences( for reviews see[26, 27]). A number of computational algorithms have been developed to identify such regulatory motifs. These algorithms include enumeration of overrepresented substrings or regular expression patterns, local multiple sequence alignment, or sequence segmentation to decompose the DNA sequence into the most plausible set of motifs[28–32]. The strategy of identifying clusters of coregulated genes by expression profiling followed by a computational search for regulatory motifs has been successfully applied to a number of questions, mostly in lower eukaryotes such as yeast. For mammalian species, the problem is much more challenging[33], as the genomes are more complex and regulation often involves combinatorial action of transcription factors[34]. Examples of computational de novo motif discovery followed by experimental validation in mammalian species are scarce. One experimentally validated case recently reported using motif discovery to identify mouse transcription factors that regulate oxidative phosphorylation[35]. Recent algorithms targeted at higher eukaryotes use interspecies comparisons to identify functional motifs in orthologous promoters[36–38], because functional elements are subject to selective pressure and tend to evolve more slowly than nonfunctional sequences[34]. These algorithms typically use conserved blocks of DNA sequence that can be aligned to reduce the background noise. However, alignment-based approaches can miss important sequence motifs as many regulatory sequences do not fall into conserved regions[39, 40]. In this paper, we used a combination of gene expression profiling with computational genomic analyses and biochemical assays to systematically identify novel cis-regulatory sequences that control gene expression in pluripotent stem cells. To gain insight into the transcriptional regulatory networks of pluripotent cells, we compared the gene expression profiles of ES cells and PGCs to embryonic and adult somatic cell types. We identified clusters of genes upregulated in ES cells and PGCs, which include several known markers of pluripotency. To identify regulatory motifs that control gene expression within these clusters, we developed a novel algorithm, CompMoby. This algorithm combines the strategies of comparative genomics with DNA sequence segmentation to identify sets of motifs in the upstream regions of coregulated genes. Using CompMoby, we identified motifs that are statistically overrepresented in genes upregulated in pluripotent cells and highly conserved across multiple mammalian species. We demonstrate that several of the predicted motifs are novel regulatory elements of gene expression in mouse and human ES cells. Finally, we show that the transcription factor NF-Y binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation.
Discussion:
In this study we report a systematic approach that combines comprehensive expression analysis of coregulated genes, computational de novo motif prediction, biochemical validation of cis-regulatory elements, and identification of transcription factors that bind to those elements in pluripotent stem cells. Our methodology can be used with any set of coregulated genes, and, as such, is broadly applicable to the characterization of transcriptional regulatory networks. The approach we describe compares favorably to the standard experimental method to identify regulatory sequences, which relies on time-consuming dissection of large noncoding regions of a single gene. When compared to other methods to identify cis-regulatory elements, like ChIP in combination with microarrays( ChIP-chip) or paired-end ditag sequencing( ChIP-PET), our approach has two principal advantages: it does not require prior knowledge of the critical transcription factors whose targets are to be investigated, and it is not limited by the number of cells available for analysis. In particular, we have been able to generate reliable expression data from as low as 500–1, 000 cells( unpublished data), whereas current ChIP-chip and ChIP-PET methods require several million cells[23, 74, 75]. Thus, we envision that the approach described here will be particularly useful for the characterization of transcriptional networks that regulate cell fate decisions during embryonic development and stem cell differentiation. We identified short DNA sequence motifs that are highly active in undifferentiated ES cells but not in differentiated cells( Figure 3B, motifs 1 and 2). Importantly, the level of activity of these motifs is significantly higher than that of the Oct4/Sox2 element in the Nanog promoter( Figure 3B, compare motifs 1a, 1b, 2a, and 2c to Nng). These results indicate that we identified enhancer elements that are bound by transcriptional factors preferentially active in undifferentiated mouse and human ES cells. The availability of EMSAs for motifs 1 and 2 and of mutated versions that highly reduce or abolish motif activity( Figures 4, 5, and 6A) should facilitate the unbiased identification of the transcription factors that bind to these motifs. An important validation of our systematic analysis of cis-regulatory elements active in ES cells is the identification of NF-Y as a transcription factor that binds specifically to one of those elements and regulates ES cell proliferation. In support of our findings, the NF-Y binding site was detected as overrepresented in genomic regions bound by Oct4 and Sox2 in human ES cells( Qing Zhou and Wing Wong, personal communication). It is possible that NF-Y contributes to the regulation of the peculiar cell cycle pattern of ES cells, with a short G1 phase and insensitivity to the Rb pathway( reviewed in[76]). NF-Y had previously been shown to regulate cell proliferation in other experimental paradigms[69, 70], but its role in early embryonic development remains poorly understood. The strong upregulation of subunits of NF-Y in oocytes[66] and the ICM[67], and the early arrest of NF-YA mutant embryos[70], indicates that NF-Y plays important roles during early embryogenesis. It is also worth noting the dramatic difference in expression of NF-YA isoforms during ES cell differentiation( Figure 7A). Both NF-YA isoforms contain a glutamine-rich region that is reduced in the short isoform of NF-YA[68]. The glutamine-rich region of NF-YA has been shown to activate transcription[68, 77, 78] and it is also a protein–protein interaction domain[79]. The functional significance of the two NF-YA isoforms remains to be elucidated, although recent data indicate that NF-YA( short) promotes self-renewal of hematopoietic stem cells[80]. Future studies will address the specific contribution of NF-Y and its different subunits, in particular NF-YA( short), in ES cells. ES cells may be governed at the molecular level by the action of cell-specific transcription factors, such as Oct4 and Nanog, and factors that are also expressed in other cell types, such as NF-Y, c-Myc[50], and Stat3[11]. Interestingly, NF-Y binds to the promoter of Sall4( Figure 6C), an essential ES cell regulator[24]. It will be important to identify the target genes that are regulated by NF-Y in ES cells. We expect that the combination of ChIP-chip and expression profiling will reveal the contribution of NF-Y to the transcriptional program of ES cells. In summary, we report here the identification of clusters of genes upregulated in pluripotent cells, the development of a novel algorithm for discovery of short cis-acting regulatory motifs, the validation of the activity of several novel motifs in mouse and human pluripotent stem cells, and the identification of transcription factor NF-Y as a regulator of gene expression in ES cells that is required for their proliferation. Genetic and biochemical approaches should allow the identification of other transcription factors that bind to the motifs. Our results provide a basis for understanding the transcriptional regulatory networks that underlie early mammalian embryogenesis and ES cell self-renewal and pluripotency.
Embryonic stem cells have two remarkable properties: they can proliferate very rapidly , and they can give rise to all of the body's cell types . Understanding how gene activity is regulated in embryonic stem cells will be an important step towards therapeutic applications . The activity of genes is regulated by proteins called transcription factors , which bind to stretches of DNA sequences that act as on or off switches . We identified genes that are active in mouse embryonic stem cells but not in differentiated cells . We reasoned that if these genes have similar patterns of activity , they may be regulated by the same transcription factors . We therefore developed a computational approach that takes information on gene activity and predicts DNA sequences that may act as switches . Using this approach , we discovered new DNA switches that regulate gene activity in mouse and human embryonic stem cells . Furthermore , we identified a transcription factor that binds to one of these DNA switches and is important for the rapid proliferation of embryonic stem cells . Our approach sheds light on the genetic regulation of embryonic stem cells and will be broadly applicable to questions of how gene activity is regulated in other cell types of interest .