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Title: Extensive Divergence of Transcription Factor Binding in Drosophila Embryos with Highly Conserved Gene Expression}
Article:
Abstract:
To better characterize how variation in regulatory sequences drives divergence in gene expression, we undertook a systematic study of transcription factor binding and gene expression in blastoderm embryos of four species, which sample much of the diversity in the 40 million-year old genus Drosophila: D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. We compared gene expression, measured by mRNA-seq, to the genome-wide binding, measured by ChIP-seq, of four transcription factors involved in early anterior-posterior patterning. We found that mRNA levels are much better conserved than individual transcription factor binding events, and that changes in a gene's expression were poorly explained by changes in adjacent transcription factor binding. However, highly bound sites, sites in regions bound by multiple factors and sites near genes are conserved more frequently than other binding, suggesting that a considerable amount of transcription factor binding is weakly or non-functional and not subject to purifying selection.
Introduction:
In the pursuit of the genetic basis of phenotypic evolution, researchers have favored divergence of gene expression as a major source of diversity[1],[2]. Changes in gene expression during animal development can have many origins, but it is widely assumed that the divergence of the enhancer sequences that drive complex spatial and temporal patterns of gene expression during development will play an important role in the evolution of morphology. Enhancer sequences often undergo rapid changes during evolution[3]–[6], and the binding of transcription factors( TFs) to these and other sequences has been shown to be highly divergent in related species, where, based on functional conservation, little divergence was expected[7]–[14]. While many studies have compared gene expression between species at the genomic scale and reported various degrees of divergence( e. g.[15]–[17]), the downstream effects of TF binding divergence on gene expression has received relatively little attention[18]. One limitation of many of these studies is that significant changes in DNA sequence are often accompanied by extensive changes in morphology or physiology, complicating direct comparison of molecular phenotypes like transcription factor binding and gene expression. This is not, however, the case in Drosophila embryogenesis, which is highly conserved across the 40 million-year old Drosophila genus despite accumulating sequence changes equivalent to those separating different classes of amniotes[19]. To take advantage of this morphological conservation, we measured gene expression and the genome-wide binding of four TFs, namely Bicoid( BCD), Giant( GT), Hunchback( HB) and Krüppel( KR), involved in anterior-posterior( A-P) patterning in blastoderm embryos of four fully-sequenced species- D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis – that span the genus, with divergence times between 5 and 40 million years ago[20]( Figure 1).
Discussion:
This study addresses both the genomic causes and the consequences on gene expression of TF binding divergence. We found that the genomic binding of A-P factors has changed extensively along the Drosophila tree with little effect on downstream gene expression. We identified two potential genetic sources of binding divergence. First, we found that alterations of BCD, HB, GT and KR-specific motifs drive a portion of the binding divergence. This was previously found for other TFs[7],[10],[12],[13],[44],[45], though our study is among the first to quantify this effect. We found that the A-P factors are undergoing correlated evolution among themselves( Figures 5D and 6D) and potentially with the protein Zelda( Figure 5E), pointing to the importance of cis-acting sites beyond target sites for the factors themselves in binding divergence. Given the high degree of morphological stability amongst these four species, we predicted and observed highly conserved embryonic gene expression – for both maternal and zygotic genes. Yet, conservation of gene expression was not coupled with conservation of genome-wide transcription factor binding. One relatively simple explanation is that our measurements of transcription factor binding are more subject to systematic biases and noise, since ChIP experiments are generally less robust than mRNA-seq and involve species-specific reagents. The inherent experimental noise of ChIP experiments will thus lead to overestimating binding divergence, although it is difficult to quantify its contribution. This said, we believe our conclusions are robust to ChIP noise, if only because the same conclusions were drawn from ChIPs of different qualities( GT and HB ChIPs were the best datasets whereas BCD and KR ChIPs showed lower enrichment and reproducibility, Figure S3). It is also possible that, even though we carefully optimized embryo collections to account for the different development times of the species, we may not have perfectly matched stage sampling between species. However, the divergence of TF binding we observe is in line with most genome-wide comparisons of TF binding, including in Drosophila[7]–[10],[12],[14],[46], although these studies also face the same limitations that ours does. Our recovery of motif signals for BCD, GT, HB and KR in their bound regions, and ZLD in all of them, argues against extreme levels of noise in the data. Overall some of the difference in divergence( Figure 7C) undoubtedly originates from both the different nature and means of measurement of TF binding and gene expression. Our interpretation of extensive binding divergence of A-P factors contrasts somewhat with a recent comparison of the binding of the dorsal-ventral patterning factor Twist[47]. He et al. found that 34% of the regions bound by Twist in D. melanogaster are also bound in five species ranging from the closely related D. simulans to the more distant D. pseudoobscura, leading the authors to claim that Twist binding was highly conserved. In comparison, we find that 28%, 38%, 23% and 15%, of BCD, GT, HB and KR regions bound in D. melanogaster are also bound in D. yakuba and D. pseudoobscura, which correspond to roughly similar levels of binding conservation. Thus we differ primarily in whether the finding that only one of three bound regions found in D. melanogaster are conserved constitutes a high degree of conservation. More interestingly, the Twist study confirmed our results that functional binding is more constrained than seemingly non functional binding since clustered Twist binding and binding near target genes were better conserved[47]. Perhaps the strongest argument that the overall low levels of conservation we observe is real is that we observe strong conservation for the subset of bound regions that are most likely to be functional. This suggests, unsurprisingly, that there is strong purifying selection to maintain binding in functional regions. But it also demonstrates that we can detect strong conservation when it is there, with the corollary that the regions we observe to be divergent are likely to really be divergent. That two thirds of the regions bound by any of the four TFs we examined are poorly conserved and thus are probably under weak or no purifying selection supports the emerging view that a large fraction of measureable biochemical events are not functional( in contrast to claims made by ENCODE[48]). We note, however, that strong selection to maintain binding does not require conservation of individual binding sites[45],[49]–[53] There is also evidence that even within highly conserved regulatory networks, differences in embryo size and other factors necessitate constant tuning of enhancer activities, with corresponding adjustments in the strength and organization of TF binding sites[54]. Unfortunately, the ChIP data we report here lacks sufficient resolution to see the turnover of individual binding events. New techniques like ChIP-exo[55] enable much higher resolution moving forwards, and the growing repertoire of sequenced and experimentally tractable Drosophila species should allow us to study turnover at both the sequence and binding level with much more precision.
Inter-species differences in gene expression during development are a major source of phenotypic diversity , yet the molecular origins of such differences are poorly understood . In this study we use a combination of biochemical and genomic methods to explore how an important component of the machinery of gene regulation varies between species . We mapped the genomic sites bound by four specific proteins regulating gene expression along the head to tail axis of young embryos of four diverse species of fruit flies . We were surprised at the extent of variation we observed , especially as we found far less variation in gene expression between these same species' embryos . We conclude , based on various analyses of our binding divergence data , that most of the time these regulators bind to DNA they have no effect on gene expression , and therefore natural selection does not act to preserve these interactions between species .