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CisRegulation Info
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Gene expression is controlled via transcription factors that bind to cis-regulatory sites in a gene’s promoter. Variant instances of a transcription factor binding motif, which differ at non-consensus positions, may cause differential regulation. We have systematically characterized the condition-specific activity of motif variants by analyzing the relationship between transcription factor binding motif variants and gene expression. For the genomes considered from four species, ~29% to ~39% of transcription factors have target genes that are differentially expressed in a condition-specific manner according to the nucleotide at variant motif positions. We have found good correspondence between our results and cases in the experimental literature, suggesting that transcription factor binding variants may often be functionally distinct and may play an important role in the evolution of condition-specific gene regulation.

Example: The function of variant nucleotides at position 10 of the Rap1 binding motif is conserved in S. cerevisiae, S. paradoxus, S. mikatae, and S. kudriavzevii. In all four species, genes associated with the “C” variant (blue) are more highly expressed than genes associated with the “T” variant (red) in starvation conditions (glycerol), and the opposite relationship is apparent during nitrogen starvation.

Ongoing and future work:
-Experimentally validate the functional effect of variants.
-Explicitly account for combinatorial interactions among multiple binding sites when looking for functional variants.
-Determine interactions between different functional variants in the same binding motif.

We will identify the distinct phenotypic role of binding site variants in specific environments or developmental stages. We will study variants that cause condition, tissue, or strain-specific gene expression profiles and control genes involved in pathways or processes such as ribosomal biogenesis, the cell cycle, pathology, development and tissue differentiation.

We will identify functional TF binding site variants that have contributed to regulatory novelty. Regulatory novelty is an important source of variation enabling adaptation to new environments. However, trans regulatory changes can have pleiotropic effects, and gain/loss of TF binding sites alone offers a limited range of variation. Changes in functional variants may provide a source of cis-regulatory variation for fine-tuning gene expression profiles. We will identify substitutions between species that have caused changes in a gene’s expression profile, and identify changes in functional variants between duplicate genes that facilitated their regulatory divergence, perhaps accommodating the evolution of novel cellular pathways.

Computational
We will characterize TF binding site nucleotide variants that cause expression profile differences in target genes and determine the mechanism. Since gene regulation is biologically complex and poorly understood, we will develop methods to robustly account for multiple binding sites, degeneracies, expression noise, and multiple comparisons. These additional factors will be integrated using statistical methods such as rough set theory.

Experimental
In yeast, we will alter nucleotides at functionally variant binding site positions with site-directed mutagenesis to validate predicted expression changes. We will evaluate mechanisms of regulatory differences between variants, considering both differences in the TF’s affinity for the variant binding sites, and differences in the allosteric effect of variants on the TF and resulting interaction with different sets of cofactors.