Cell type-specific cis-regulatory divergence in gene expression and chromatin accessibility revealed by human-chimpanzee hybrid cells

Reviewer #1 (Public Review):

This study aims to identify gene expression differences exclusively caused by cis-regulatory genetic changes by utilizing hybrid cell lines derived from human and chimpanzee. While previous attempts have focused on specific tissues, this study expands the comparison to six different tissues to investigate tissue specificity and derive insights into the evolution of gene expression.

One notable strength of this work lies in the use of composite cell lines, enabling a comparison of gene expression between human and chimpanzee within the same nucleus and shared trans factors environment. However, a potential weakness of the methodology is the use of bulk RNA-seq in diverse tissues, which limits the ability to determine cell-type-specific gene expression and chromatin accessibility regions.

Another concern is the use of two replicates derived from the same pair of individuals. While the authors produced cell lines from two pairs of individuals in a previous study (Agloglia et al., 2021), I wonder why only one pair was used in this study. Incorporating interindividual variation would enhance the robustness of the species differences identified here.

Furthermore, the study offers the opportunity to relate inter-species differences to trends in molecular evolution. The authors discovered that expression variance and haploinsufficiency score do not fully account for the enrichment of divergence in cell-type-specific genes. The reviewer suggests exploring this further by incorporating external datasets that bin genes based on interindividual transcriptomics variation as a measure of extant transcriptomics constraint (e.g., GTEx reanalysis by Garcia-Perez et al., 2023 - PMID: 36777183). Additionally, stratifying sequence conservation on ASCA regions, which exhibit similar enrichment of cell-type-specific features, using the Zoonomia data mentioned also in the text (Andrews et al., 2023 -- PMID: 37104580) could provide valuable insights.

Another potential strength of this study is the identification of specific cases of paired allele-specific expression (ASE) and allele-specific chromatin accessibility (ASCA) with biological significance. Prioritizing specific variants remains a challenge, and the authors apply a machine-learning approach to identify potential causative variants that disrupt binding sites in two examples (FABP7 and GAD1 in motor neurons). However, additional work is needed to convincingly demonstrate the functionality of these selected variants. Strengthening this section with additional validation of ASE, ASCA, and the specific putative causal variants identified would enhance the overall robustness of the paper.

Additionally, the authors support the selected ASE-ASCA pairs by examining external datasets of adult brain comparative genomics (Ma et al., 2022) and organoids (Kanton et al., 2019). While these resources are valuable for comparing observed species biases, the analysis is not systematic, even for the two selected genes. For example, it would be beneficial to investigate if FABP7 exhibits species bias in any cell type in Kanton et al.'s organoids or if GAD1 is species-biased in adult primate brains from Ma et al. Comparing these datasets with the present study, along with the Agoglia et al. reference, would provide a more comprehensive perspective.

The use of the term "human-derived" in ASE and ASCA should be avoided since there is no outgroup in the analysis to provide a reference for the observed changes.

Finally, throughout the paper, the authors refer to "hybrid cell lines." It has been suggested to use the term "composite cell lines" instead to address potential societal concerns associated with the term "hybrid," which some may associate with reproductive relationships (Pavlovic et al., 2022 -- PMID: 35082442). It would be interesting to know the authors' perspective on these concerns and recommendations presented in Pavlovic et al., given their position as pioneers in this field.

Reviewer #2 (Public Review):

In this paper, Wang and colleagues build on previous technical and analytical achievements in establishing tetraploid human-chimpanzee hybrid iPSCs to investigate the cell type-specificity of allele-specific expression and allele-specific chromatin accessibility across six differentiated cell types (here, "allele-specific" indicates species differences with a cis-regulatory basis). The combined body of work is remarkable in its creativity and ambition and has real potential for overcoming major challenges in understanding the evolutionary genetics of between-species differences. The present paper contributes to these efforts by showing how differentiated cells can be used to test a long-standing hypothesis in evolutionary genetics: that cis-regulatory changes may be particularly important in divergence because of their potential for modularity.

In my view, the paper succeeds in making this case: allele (species)-specific expression (ASE) and allele-specific chromatin accessibility (ASCA) are enriched in genes asymmetrically expressed in one cell type, and many cases of ASE/ASCA are cell type-specific. The authors do an excellent job showing that these results are robust across a set of possible analysis decisions. It is somewhat less clear whether these enrichments are primarily a product of relaxed constraint on cell type-specific genes or primarily result from positive selection in the human or chimp lineage. While the authors attempt to control for constraint using several variables (variance in ASE in humans and the sequence-based probability of haploinsufficiency score, pHI), these are imperfect proxies for constraint. For the pHI scores, enrichments for ASE also appear to be strongest in the least constrained genes. Overall, the relative role of relaxation of constraint versus positive selection is unresolved, although the manuscript's language leans in favor of an important role for selection.

The remainder of the manuscript draws on the cell type-specific ASE/ASCA data to nominate candidate genes and pathways that may have been important in differentiating humans and chimpanzees. Several approaches are used here, including comparing human-chimp ASE to the distribution of ASE observed in humans and investigating biases in the direction of ASE for genes in the same pathway. The authors also identify interesting candidate genes based on their role in development or their proximity to human accelerated regions (where many changes have arisen on the human lineage in otherwise deeply conserved sequence) and use a deep neural network to identify sequence changes that might be causally responsible for ASE/ASCA. These analyses have value and highlight potential strategies for using ASE/ASCA and hybrid cell line data as a hypothesis-generating tool. Of course, the functional follow-up that experimentally tested these hypotheses or linked sequence/expression changes in the candidate pathways to organismal phenotype would have strengthened the paper further- but this is a lot to ask in an already technically and analytically challenging piece of work.

As a minor critique, the present paper is very closely integrated with other manuscripts that have used the hybrid human-chimp cell lines for biological insight or methods development. Although its contributions make it a strong stand-alone contribution, some aspects of the methods are not described in sufficient detail for readers to understand (even on a general conceptual level) without referencing that work, which may somewhat limit reader understanding.

Reviewer #3 (Public Review):

The authors utilize chimpanzee-human hybrid cell lines to assess cis-regulatory evolution. These hybrid cell lines offer a well-controlled environment, enabling clear differentiation between cis-regulatory effects and environmental or other trans effects.
In their research, Wang et al. expand the range of chimpanzee-human hybrid cell lines to encompass six new developmental cell types derived from all three germ layers. This expansion allows them to discern cell type-specific cis-regulatory changes between species from more pleiotropic ones. Although the study investigates only two iPSC clones, the RNA- and ATAC-seq data produced for this paper is a valuable resource.

The authors begin their analysis by examining the relationship between allele-specific expression (ASE) as a measure of species divergence and cell type specificity. They find that cell-type-specific genes exhibit more divergent expression. By integrating this data with measures of constraint within human populations, the authors conclude that the increased divergence of tissue-specific genes is, at least in part, attributable to positive selection. A similar pattern emerges when assessing allele-specific chromatin accessibility (ASCA) as a measure of divergence of cis-regulatory elements (CREs) in the same cell lines.

By correlating these two measures, the authors identify 95 CRE-gene pairs where tissue-specific ASE aligns with tissue-specific ASCA. Among these pairs, the authors select two genes of interest for further investigation. Notably, the authors employ an intriguing machine-learning approach in which they compare the inferred chromatin state of the human sequence with that of the chimpanzee sequence to pinpoint putatively causal variants.

Overall, this study delves into the examination of gene expression and chromatin accessibility within hybrid cell lines, showcasing how this data can be leveraged to identify potential causal sequence differences underlying between-species expression changes.

I have three major concerns regarding this study:

1. The only evidence that the cells are indeed differentiated in the right direction is the expression of one prominent marker gene per cell type. Especially for the comparison of conservation between the differentiated cell types, it would be beneficial to describe the cell type diversity and the differentiation success in more detail.

2. Check for a potential confounding effect of sequence similarity on the power to detect ASE or ASCA.

3. In the last part the authors showcase 2 examples for which the log2 fold changes in chromatin state scores as inferred by the machine learning model Sei are used. This is an interesting and creative approach, however, more sanity checks on this application are necessary.