Ittai Eres' paper selection for Journal Club

Ittai Eres

Second year graduate student, Human Genetics

            I presented “Enhancer Divergence and cis­-Regulatory Evolution in the Human and Chimp Neural Crest” (Prescott et al. 2015) for the Human Genetics Journal Club (11/11/15). This paper presents an intriguing examination of human-chimpanzee divergence via gene regulatory profiling of iPSC-derived cranial neural crest cells from both species. How gene regulation has diverged between humans and chimpanzees is of particular interest because the two species have a high degree of protein-coding sequence similarity, suggesting that regulatory element divergence may be a large driver of phenotypic divergence between the species (King and Wilson, 1975).

            While I initially judged this to be a good paper, I noted some limitations. Among the most important were that only two individuals of each species were characterized and that the chimpanzee iPSCs utilized in the paper were retroviral rather than episomal transformants, suggesting their pluripotency was not completely endogenous. The former issue may complicate downstream analyses because such a small sample size could limit the broader significance of the findings, and because of the high level of polymorphism amongst chimpanzees (Kaessmann et al., 1999), could potentially confound a comparative study by falsely designating particular alleles as fixed differences between the species. The authors acknowledge this issue and suggest a conservative false discovery rate (FDR) of 15% for identifying true fixed inter-species differences in their data. This does not actually resolve the issue, although it does suggest that the majority (~85%) of fixed inter-species differences they find are real. The latter issue – retrovirally transformed chimpanzee iPSCs ­– is problematic because exogenous retroviral sequences have been integrated into the genomes of these cell lines, possibly in locations deleterious to endogenous genes and/or regulatory networks.

            Despite these issues, I went in to the Journal Club meeting feeling that this paper was quite good. During the course of our discussion, however,  a number of potential problems with the paper emerged. Many individuals in the discussion had quite a few questions about some of the findings presented in figure 4. Why, for instance, in 4A, is the baseline sequence conservation of species-biased enhancers so much lower than that of invariant enhancers even a full kb away from the enhancer center? Certainly, one may expect lower sequence conservation at the species-biased enhancers themselves in comparison to invariant enhancers, but one would not necessarily expect this lower conservation to extend a kb in either direction. Such a large range of reduced conservation might suggest that some proximal elements, other than the enhancers, are what natural selection is actually acting on, or that there is some other force entirely (e.g. recombination hotspots) that is driving down conservation in the species-biased enhancers. Either way, this anomaly, and the authors’ failure to address it, weakens their assertion that primary DNA sequence is the driver behind enhancer divergence. In a similar vein, in 4D, why does H3K27ac variance not increase as Levensthein distance increases within species? Presumably this increase seen in H3K27ac between the species is meant to show the functional importance of the sequences in question, but one would also expect a similar, albeit less pronounced, increase within species. Both issues, from figure 4A and 4D, might not necessarily have easy and satisfactory answers, but are important for contextualizing their findings.

            Another section of the paper that elicited discussion among the Journal Club participants was the discovery of the “coordinator” motif. Although I found this portion of the paper incredibly interesting, I concur with many of the concerns brought up in discussion. Initial discovery of the 17-bp motif was made within the set of putative CNCC specific enhancers identified in the paper, but the authors never specify exactly how it was discovered. They go on to find that this motif is enriched in their species-biased enhancers as compared to invariant enhancers, although they never specify how many instances of this motif were found in total—only the relative enrichment at species-biased sites in 4F. They also found that it is enriched in LTR9 retroelements, although these retroelements are not enriched in species-biased enhancers (figure 4C). If the coordinator motif is enriched in species-biased enhancers, and it is enriched in LTR9 retroelements, then one might anticipate these retroelements are also enriched in species-biased enhancers. This may not be the case for any number of different reasons, but identifying those reasons may provide new insight into the function of this motif. Overall, while the coordinator motif discussion was very interesting, much of the analysis on it felt somewhat cyclical and self-referential, and, without some further functional validations, I believe its significance to be limited.

            I truly enjoyed presenting this paper at Journal Club this month, and got a lot more out of discussing it with colleagues than I did simply reading it on my own and preparing a presentation. You never know what interesting angles other individuals may approach a paper from, and there will always be questions others have that you may have never thought to ask. After having presented and discussed the paper, I still find it quite interesting, but I am now able to look at it with an even more critical eye and understand what some of its more problematic limitations may be. I look forward to presenting at Journal Club once again in the near future, perhaps on a slightly shorter paper from a slightly less verbose journal.

Editor: Vincent Lynch, Assistant Professor, Dept of Human Genetics

Papers Cited

Prescott, Sara L., Rajini Srinivasan, Maria Carolina Marchetto, Irina Grishina, Iñigo Narvaiza, Licia Selleri, Fred H. Gage, Tomek Swigut, and Joanna Wysocka. "Enhancer Divergence and Cis-Regulatory Evolution in the Human and Chimp Neural Crest." Cell 163.1 (2015): 68-83.

King, M., and A. Wilson. “Evolution at Two Levels in Humans and Chimpanzees.” Science 188.4184 (1975): 107-16.

Kaessmann, H., V. Wiebe, and S. Pääbo. "Extensive Nuclear DNA Sequence Diversity Among Chimpanzees." Science 286.5442 (1999): 1159-162.