Details

Autor(en) / Beteiligte

• Hunter, Craig P
• Yanai, Itai
• Baugh, L Ryan
• Smith, Jessica J
• Roehrig, Casey
• Shen-Orr, Shai S
• Claggett, Julia M
• Hill, Andrew A
• Slonim, Donna K

Titel

Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression

Ist Teil von

• Molecular systems biology, 2008, Vol.4 (1), p.163-n/a

Ort / Verlag

Chichester, UK: John Wiley & Sons, Ltd

Erscheinungsjahr

2008

Quelle

Wiley-Blackwell Journals

Beschreibungen/Notizen

• Biological networks are inherently modular, yet little is known about how modules are assembled to enable coordinated and complex functions. We used RNAi and time series, whole‐genome microarray analyses to systematically perturb and characterize components of a Caenorhabditis elegans lineage‐specific transcriptional regulatory network. These data are supported by selected reporter gene analyses and comprehensive yeast one‐hybrid and promoter sequence analyses. Based on these results, we define and characterize two modules composed of muscle‐ and epidermal‐specifying transcription factors that function together within a single cell lineage to robustly specify multiple cell types. The expression of these two modules, although positively regulated by a common factor, is reliably segregated among daughter cells. Our analyses indicate that these modules repress each other, and we propose that this cross‐inhibition coupled with their relative time of induction function to enhance the initial asymmetry in their expression patterns, thus leading to the observed invariant gene expression patterns and cell lineage. The coupling of asynchronous and topologically distinct modules may be a general principle of module assembly that functions to potentiate genetic switches. Synopsis In the present work, we investigate a Caenorhabditis elegans transcriptional regulatory network that controls development of an embryonic cell lineage that produces muscle and skin cells. Previously, we identified a set of transcription factors (TF) induced in temporal waves during expansion and segregation of this lineage into muscle‐ and skin‐specific branches (Baugh et al, 2005a). To investigate how these TFs specify two mutually exclusive cell fates, we used RNAi to perturb each TF and examined the consequences with microarrays. From our analysis, we identify two small sets of TFs that function as cell fate‐specifying modules. The regulatory interactions among the TFs within each module form strikingly different topologies with distinct regulatory consequences. Furthermore, the microarray data and reporter gene analysis show that these two modules repress each other. We propose that these modules function together to enhance the precision of the asymmetric signal that patterns their expression within the lineage. Such coupling of topologically distinct modules to potentiate genetic switches may be a general principle of module assembly. The C. elegans embryo is an established experimental platform for systems biology. The invariant order of cell cleavage and position of each daughter cell is known (Sulston, 1983), making it possible to define discrete gene expression states for each cell in the developing embryo. This descriptive information coupled with powerful experimental tools including thousands of genetic mutations, whole‐genome RNAi libraries, modern genomic tools, and the genomic sequences of closely related nematodes that follow indistinguishable cell lineages, makes the C. elegans system ideal for investigating gene regulatory networks. The gene regulatory network we are studying is initiated by a TF called PAL‐1, which specifies the development of the C blastomere (Hunter and Kenyon 1996). The C blastomere is born at the eight‐cell stage and produces 32 muscle cells, 13 epidermal (skin) cells, 2 neurons, and 1 programmed cell death. PAL‐1 is a master regulator for the C lineage: embryos that lack PAL‐1 function fail to specify a C blastomere, whereas embryos in which PAL‐1 is active in all cells produce multiple C blastomeres (Draper et al, 1996; Hunter and Kenyon, 1996). Approximately 400 putative PAL‐1 target genes, including 13 TFs, were identified in microarray experiments using mutant embryos that contained no C blastomere or that were composed nearly entirely of C blastomeres (Baugh et al, 2005a). Expression of these TFs is induced in the C lineage in successive temporal waves corresponding to successive cell divisions. Furthermore, several of the TFs are expressed in either muscle or epidermal precursors. Although this study identified and characterized the core parts of the TF network, it did not provide insight into how a single TF (PAL‐1) can specify and pattern multiple, mutually exclusive cell fates in a single‐cell lineage. The premise of the current work is that determining the regulatory topology of interactions between these TFs would provide insight into the logic of the developmental program. We used RNAi to perturb the function of each TF and recorded effects using whole‐genome microarrays. The effect of each TF knockdown on the mRNA abundance of each TF was visualized by a graph matrix (Figure 2) of the activation or repression dependencies among the TFs. Inspection of this graph revealed two tissue‐specific TF modules expressed in epidermal and muscle precursors. To provide additional evidence for the apparent regulatory interactions among these TFs, we performed a yeast‐one‐hybrid analysis among the TFs and their promoter regions. We also used the genomic sequence of two related nematodes to identify conserved regulatory sequences and mapped the location of known TF‐binding sequences within these conserved domains. Collectively, these data allowed us to infer the regulatory topology of the two modules (Figure 4). The proposed epidermal module is controlled by a GATA factor called ELT‐1, which functions as an epidermal master regulator in C. elegans; previous experiments demonstrated that embryos lacking ELT‐1 fail to produce epidermal cells, whereas forced early expression of ELT‐1 transforms all embryonic cells into epidermal cells (Page et al, 1997; Gilleard and McGhee, 2001). In the C lineage, ELT‐1 induces the expression of three TFs. We present evidence that these three targets, in addition to regulating epidermal differentiation genes, negatively regulate ELT‐1 expression. This two‐step topology has the effect of stabilizing expression among the ELT‐1 target genes and may function to delay commitment to the epidermal fate. In contrast, the proposed muscle module is composed of three TFs that are induced in the same cell cycle, and that appear to subsequently sustain each others expression by nested positive feedback loops. This topology produces a module that once activated, rapidly becomes self‐sustaining and commits cells irreversibly to the muscle fate. Our data also support the notion that these modules repress one another and compete to specify cell fate, thus ensuring that either a muscle or epidermal fate is specified. How are these mutually antagonistic PAL‐1‐dependent modules reliably activated in sister cells? Essential to this process is an asymmetric signal that distinguishes the sister cells from each other. However, our data show that the asymmetric signal is not sufficient to restrict muscle module activity to the muscle precursors, because in elt‐1 mutants the epidermal precursors express the muscle module. We propose that ELT‐1 expression in all C cells initiates the two‐step epidermal module sufficiently early to be well established in the epidermal precursors when the muscle module is activated following the cell cycle. Thus, in the epidermal precursors, the epidermal module can repress low levels of muscle module activity, while high levels of muscle module activity dominate in the muscle precursors. One can imagine that if the two modules were more equally matched in their regulatory robustness, then they would need to be expressed simultaneously to avoid one dominating the other, and consequently the asymmetric signal would need to be much more precise to avoid errors. Thus, the pairing of topologically distinct modules enhances the effective precision of the signal; such an asymmetric pairing may be a general principle of module assembly. Topology of a developmental gene regulatory network was inferred from multiple, integrated data types including (1) genome‐wide mRNA expression analysis of precisely staged, RNAi treated embryos, (2) yeast‐one‐hybrid analysis, (3) computational analysis, and (4) in vivo analyses. This developmental gene regulatory network is minimally composed of two tissue‐specific, cell‐fate specifying modules. These two tissue‐specific modules are characterized by distinct topologies and repress each other's activity. The pairing of competing, topologically distinct modules enhances a switch‐like patterning decision and suggests an organizational principle for module assembly.