Young transposable elements rewired gene regulatory networks in human and chimpanzee hippocampal intermediate progenitors

Sruti Patoori, Samantha Barnada, Marco Trizzino

Preprint posted on 29 November 2021 https://www.biorxiv.org/content/10.1101/2021.11.24.469877v2.full

Article now published in Development at http://dx.doi.org/10.1242/dev.200413

The evolution of human cognition - new research suggests young transposable elements caused major changes in neurodevelopmental gene expression within the human hippocampus.

Selected by Julia Grzymkowski

Categories: developmental biology, evolutionary biology, genetics

Updated 14 October 2022 with a postLight by Julia Grzymkowski

In looking at both the preprint of this manuscript and the final version, which was recently published in Development (http://dx.doi.org/10.1242/dev.200413), not a lot changed between the two versions. The main analyses and conclusions of the preprint were valid and kept as is.

The only thing that changed is Figure 1. The authors decided to conduct a more robust characterization of their human and chimpanzee hpIPCs that were generated from iPSCs. To do this, they added scRNA sequencing to their repertoire of sequencing methods. They performed scRNA-seq during the differentiation period of iPSCs to hpIPCs to determine whether there are any differences or similarities between humans and chimpanzees. They used the scRNA-seq data to show the expression of TBR2, PAX6, and OTX2, which they had originally done using immunohistochemistry. They also made the interesting observation that the differentiation trajectory of chimpanzee hpIPCs occurred quicker than in humans, but their overall transcriptome profiles were the same at day 5, which is the time point used for all subsequent experiments.

The addition of scRNA-seq has allowed the authors to report some more interesting observations about the differentiation of human and chimpanzee hpIPCs and has offered more convincing data on the reliability of comparing hpIPCs between the two species.

While the final published version of the paper does not address the questions posed in my preLight, this does not take anything away from it. Those questions were outside the scope of the manuscript and may be discussed in a later study.

Background Information

The human mind is capable of complex thought processes and reasoning, with the ability to understand language, acquire and disseminate knowledge, and remember things long past. These capabilities are attributable to the hippocampus, and the recent hippocampal volume expansion seen in the human lineage (1). Researchers believe this expansion may have been caused by an increase in the proliferative potential of intermediate progenitor cells (IPCs), a specific class of neuronal progenitors (2). In addition, changes in the regulation of gene expression networks are thought to have caused the divergence of humans from our closest relative, chimpanzees (3).
The main downside of the human hippocampal expansion is that this brain region is greatly affected by neurodegenerative diseases such as Alzheimer’s Disease, which is thought to be unique to humans (4). However, the mechanisms of the evolution of the human hippocampus and its susceptibility to neurodegenerative diseases are understudied. The best way to enhance our knowledge would be to compare the development of the human hippocampus to that of chimpanzees. With this preprint, the authors utilized human and chimp induced pluripotent stem cell (iPSC)-derived hippocampal progenitors to study key genetic differences between the human and chimp hippocampus during neurodevelopment.

Key Findings

RNA-Seq reveals key differences in neurodevelopmental pathways in human and chimpanzee hpIPCs

For their study, the authors generated hippocampal intermediate progenitor cells (hpIPCs) from human and chimpanzee iPSCs. Subsequent experiments were performed with hpIPCs that were differentiated for 5 days because nearly all cells at this time point were positive for three crucial neurodevelopmental markers: Pax6, Tbr2, and Otx2.
Bulk RNA-Seq revealed a total of 2,588 differentially expressed genes, with 65.1% being upregulated in human hpIPCs and 34.9% upregulated in chimpanzee hpIPCs. Ingenuity Pathway Analysis of the differentially expressed genes revealed top upstream pathway regulators, where three of the top five were transcription factors (TFs) with known roles in neurodevelopment: Creb1, Foxa2, and Tbr2. Furthermore, transcriptional targets of these TFs were found within the original dataset of differentially expressed genes and they showed species-specific expression.

Neurodevelopmental genes in human and chimpanzee hpIPCs are differentially regulated by young transposable elements

ATAC-Seq was conducted to identify cis-regulatory differences between human and chimpanzee hpIPCs. Data analysis was conducted in both a human-centric and chimpanzee-centric manner. Human-centric analysis found 82,235 ATAC-Seq human peaks that were reproducible across cell lines with orthologs in the chimp genome, whereas chimp-centric analysis found 72,211 chimp peaks reproducible across cell lines with orthologs in the human genome. Within both data sets, ~3,000 regions were differentially accessible between species, and these were found to more significantly overlap with transposable elements (TE) than non-differentially accessible regions, and were more likely to be TE-derived. In the human-centric data, more TE-derived differentially accessible peaks were located close to a differentially expressed gene. In addition, these TE’s were enriched for long terminal repeats (LTRs), specifically endogenous retroviruses (ERVs) and SINE-Vntr-Alus (SVAs). Moreover, distinct differentially expressed genes with known roles in neurodevelopment were found near the chimp- and human-enriched LTRs.

Given that a large percentage of SVAs are human-specific, the authors performed motif analysis on these SVAs and found that the binding motif for Tbr2 was the most enriched. Pathway analysis of all genes within 50 kb from each human specific SVA revealed an enrichment for melatonin and nicotine degradation pathways, which are known to be strongly activated in the human hippocampus. Furthermore, Phf8 was found to be the top upstream regulator of these pathways and mutations in this gene are known to cause cognitive impairment and intellectual disability (5). ChIP-Seq was used to profile Tbr2 binding in the SVAs that were accessible in human hpIPCs, with ~40% of this retrotransposon family showing Tbr2 signal and half of Tbr2-bound regions were human-specific and located near 37 of the genes differentially expressed between human and chimp hpIPCs.

Finally, repression of the active SVAs using doxycycline-inducible CRISPR-interference revealed 5,795 differential expressed genes between cells with active and inactive SVAs. 677 of these genes were previously identified as different between chimpanzees and humans, and a third of the genes that were near a Tbr2-bound human-specific SVA lost expression upon SVA repression. These results reveal human specific SVA transposons as key regulators of gene networks important for hippocampal development.

Why is this paper interesting?

With this preprint, the authors provide the first comparison of the transcriptome of human and chimpanzee hpIPCs. Moreover, the analysis of the genome is thorough and robust, by looking at not just the transcriptome, but also chromatin accessibility and gene regulation through RNA-, ATAC-, and ChIP-Seq, in addition to functional analyses with CRISPR-interference. Observations made from this study, and the extensive collection of raw data, will pave the way for further research into this evolutionary phenomenon to help us better understand the origin of human cognition and neurodegenerative diseases.

Questions to the authors

• Did you perform a pathway analysis on just the “Human UP” genes? Did you, or would you expect to see an enrichment for pathways involved in cell proliferation that may explain the increase in size of the human hippocampal region in comparison to chimpanzees?
• Can you touch on some future directions for this work in your lab?

References

1. Barger, N., Hanson, K.L., et al. 2014. Evidence for evolutionary specialization in human limbic structures. Front. Hum. Neurosci. 8, p.277.
2. Martínez-Cerdeño, V., Noctor, S. C. and Kriegstein, A. R. 2006. The role of intermediate progenitor cells in the evolutionary expansion of the cerebral cortex. Cereb Cortex. 16 Suppl 1, i152–161.
3. Agoglia, R. M., et al. 2021. Primate cell fusion disentangles gene regulatory divergence in neurodevelopment. Nature. 592, 421–427.
4. Walker, L. C. and Jucker, M. (2017). The Exceptional Vulnerability of Humans to Alzheimer’s Disease. Trends. Mol. Med. 23, 534–545.
5. Chen, X., et al. 2018. Phf8 histone demethylase deficiency causes cognitive impairments through the mTOR pathway. Nat Commun. 9, 114.

Tags: brain, chimp, evolution, human cognition

Posted on: 16 December 2021 , updated on: 19 December 2022

doi: https://doi.org/10.1242/prelights.31177

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Author's response

Sruti Patoori shared

We haven’t yet performed separate pathway analyses on the “Human UP” and “Chimp UP” genes, but it would be interesting to see what comes out. We would certainly like to see an enrichment for cell cycle or cell proliferation pathways, since cell cycle plays such a large role in the control of neurogenesis.
However, in our pathway analysis of all 2,588 genes, cell cycle and proliferation pathways were listed as the fifteenth candidate network out of 25 networks. Additionally, the cell proliferation genes involved may be differentially expressed in either direction. Repression of certain genes may cause an increase in proliferation as well. Given all of this, we do not necessarily expect to see a greater enrichment for cell cycle/proliferation genes when analyzing just the “Human UP” genes. However, this does not rule out the involvement of cell cycle regulators or proliferation genes in the differences between the human and chimpanzee hippocampus

• Can you touch on some future directions for this work in your lab?

We are very interested in conducting some more functional assays of the transposon-derived regulatory elements we identify in this pre-print. We are interested in conducting CRISPR-based assays to determine the impact of SVA transposons and LTR transposons on developmental gene expression in hippocampal cells. Other possible directions are to select a few of the transposon-derived enhancers and conduct reporter assays to determine whether they are sufficient to drive gene expression on their own, or to overexpress some of the transcription factors such as CTCF and determine their effect on gene expression in hpIPCs.

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Young transposable elements rewired gene regulatory networks in human and chimpanzee hippocampal intermediate progenitors

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