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Regulatory Sequence Function and Evolution
Summary: Michael Eisen is interested in understanding how genome sequences specify when and where genes will be expressed, and in unraveling the molecular mechanisms that link changes in the sequences that control gene expression to changes in organismal form and function, including human disease.
As animal embryos develop, cells acquire distinct identities and functions by deploying different collections of genes in a carefully choreographic process of differentiation. The times and places in which a gene is expressed are encoded in its DNA. Mutations in the sequences that control gene expression during development underlie many human diseases and contribute significantly to evolutionary change. Yet, it is remarkable how poorly we understand the relationship between genome sequences and gene expression patterns.
Of the many ways in which protein abundance can be controlled, regulated mRNA synthesis (transcription) plays a particularly important role during development. The amount of transcript produced from a given promoter is modulated by the activity of transcription factors, proteins that bind to DNA in a sequence-specific manner and interact with each other and hundreds of other proteins to recruit or repel the RNA polymerase machinery.
My lab focuses on the sequences that control gene expression in the early embryo of the fruit fly Drosophila melanogaster and its relatives. We study the binding and activity of the 50 transcription factors involved in pattern formation in the early embryo in the context of the entire transcriptional network. We couple this experimental work with evolutionary comparisons that exploit the signature of regulatory sequence function revealed in the record of natural selection. Our ultimate goals are to determine the expression pattern of a gene from its surrounding sequences, predict the consequences of variation in regulatory sequences, and design regulatory sequences de novo to produce an arbitrary pattern of expression.
Modeling the Regulatory Network in the D. melanogaster Blastoderm
Over the past five years, we have developed high-throughput, quantitative techniques to measure the protein concentration of each transcription factor and the mRNA concentration of their targets in each of the 6,000 nuclei present immediately prior to gastrulation, catalog the potential target sequences for each factor and determine the affinity of each protein-DNA interaction, and survey the locations bound by each transcription factor and relevant chromatin proteins in vivo. We are using these systematic data, along with the genome sequences of D. melanogaster and 20 other Drosophila species, to develop models that simultaneously predict the genome-wide binding of transcription factors and the transcriptional output of known regulatory sequences based only on the DNA sequence and the known concentration of factors in each nucleus. We are validating these models with transgenic manipulation of D. melanogaster regulatory sequences and the evaluation of regulatory networks in mutant D. melanogaster embryos and other Drosophila species.
Comparing Regulatory Networks Within and Between Species
The sequencing of 11 additional Drosophila species and numerous strains of many species, and the development of functional genomic assays based on high-throughput sequencing, are allowing us to expand our experimental studies beyond laboratory strains of D. melanogaster. Comparison of single-cell resolution expression patterns between species points to precise conservation of spatial patterns of gene expression, with less conservation of expression levels. Although expression patterns are remarkably conserved between species, we find notable differences in the expression patterns of transcriptional regulators. These observations suggest that small perturbations in embryo morphology and developmental timing can generate new combinations of transcription factors—which we call transcriptional niches—that may represent a potent force for the generation of evolutionary novelty.
We are finding that the binding of transcription factors to important regulatory sequences is highly conserved, but that elsewhere in the genome, transcription factor binding varies significantly, even between closely related species. This supports our earlier proposal—based on analyses of D. melanogaster alone—that much of the genome-wide binding of transcription factors does not contribute significantly to gene expression or organismal fitness.
Constraints on Regulatory Sequence Evolution
Although Drosophila regulatory sequences are highly conserved, individual transcription factor–binding sites within regulatory sequences are gained and lost at a surprisingly high rate. Elegant experiments from Martin Kreitman's lab (University of Chicago) have shown that such "binding site turnover" can occur without altering regulatory activity. These data suggest the binding sites within regulatory sequences are being constantly rearranged, with natural selection keeping only those that function properly. We have recently shown that essential regulatory sequences from fly species that diverged from Drosophila more than 100 million years ago have essentially no binding sites in the same place as they are in Drosophila. And yet, these sequences drive patterns that are identical to their Drosophila orthologs in D. melanogaster embryos.
Our analysis of the shared features of these regulatory sequences with different binding site organizations but the same regulatory output suggests that regulatory sequences consist of multiple "mini modules" containing a small number of binding sites whose proximity to each other is essential for activity, but that these modules can be rearranged relative to each other. We are currently expanding this work, taking advantage of inexpensive DNA sequencing to sequence the genomes of dozens of fly species at different distances from D. melanogaster.
This work was initially supported by grants from the National Human Genome Research Institute, the National Institute of General Medical Sciences, and the Pew Scholars Program in the Biomedical Sciences.
Last updated January 21, 2009
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