A developmentally regulated long-range enhancer-promoter contact mediates human neural development
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eLife Assessment
In this important study, Bready et al. investigate how a highly conserved long-range enhancer mediates neural-specific SOX2 regulation during neural differentiation using human neural stem cells. This study has broad appeal to developmental neuroscience; however, the data remain incomplete given the need for homozygous enhancer knockouts and biological replicates in the scRNAseq assays.
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Abstract
SOX2 is a core pluripotency factor in human embryonic stem cells (hESCs), but upon differentiation to the three germ layers, its expression is preserved selectively in neuroectoderm. The mechanisms regulating SOX2 transcription in distinct developmental stages remain incompletely understood. Here, we demonstrate that a distant enhancer 550 kb from the human SOX2 locus is selectively activated in neural stem cells (NSCs) and establishes long-range contact with the SOX2 gene. CRISPR-Cas9 excision of the enhancer has no effect in hESCs but reduces SOX2 transcription in NSCs and impairs neuroectodermal differentiation and forebrain specification in teratomas and cerebral organoids. CRISPR excision of a CTCF recognition motif adjacent to the enhancer does not affect enhancer activation in neuroectoderm but reduces chromatin looping and SOX2 transcription to partially reproduce phenotypes seen with enhancer deletion. Our findings indicate that the development of the human nervous system depends on a developmentally regulated long-range contact between a distant enhancer and the SOX2 locus.
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eLife Assessment
In this important study, Bready et al. investigate how a highly conserved long-range enhancer mediates neural-specific SOX2 regulation during neural differentiation using human neural stem cells. This study has broad appeal to developmental neuroscience; however, the data remain incomplete given the need for homozygous enhancer knockouts and biological replicates in the scRNAseq assays.
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Reviewer #1 (Public review):
Summary:
In this study, the authors examine how a developmentally regulated cis-regulatory element controls SOX2 expression during neural differentiation of human stem cells. The results suggest that this highly conserved long-range enhancer mediates neural-specific SOX2 regulation and offer insight into the role of promoter-enhancer contacts in this process. Although the findings are interesting, several limitations need to be addressed.
Strengths:
A central question in developmental biology is how genes are regulated in a context-dependent manner. SOX2, a major pluripotency factor, is expressed in diverse tissues during development, and therefore understanding the mechanisms that control its spatiotemporal expression is critical. This study addresses this important question by examining the functional …
Reviewer #1 (Public review):
Summary:
In this study, the authors examine how a developmentally regulated cis-regulatory element controls SOX2 expression during neural differentiation of human stem cells. The results suggest that this highly conserved long-range enhancer mediates neural-specific SOX2 regulation and offer insight into the role of promoter-enhancer contacts in this process. Although the findings are interesting, several limitations need to be addressed.
Strengths:
A central question in developmental biology is how genes are regulated in a context-dependent manner. SOX2, a major pluripotency factor, is expressed in diverse tissues during development, and therefore understanding the mechanisms that control its spatiotemporal expression is critical. This study addresses this important question by examining the functional relevance of a neural-specific, developmentally regulated SOX2 enhancer and its associated promoter-enhancer contacts in driving gene expression during human neural development. Using multiple model systems and techniques, the authors test the requirement of this enhancer by analyzing SOX2 expression in mutant lines, providing evidence for its role in this process.
Weaknesses:
A key limitation of the study is the absence of data from homozygous SOX2 enhancer deletion, which leaves the analysis incomplete and tempers the conclusions that can be drawn. Furthermore, the suitability of teratomas as a model system is questionable, given their limited capacity to recapitulate the spatial patterning, regional specification, and organized developmental processes characteristic of the human forebrain. Finally, the manuscript remains largely descriptive with little mechanistic insight.
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Reviewer #2 (Public review):
Summary:
The authors use a combination of genomics, genome conformation assays, and CRISPR-mediated deletion to study the transcriptional regulation of the SOX2 gene in human neural stem cells (hNSCs).
Strengths:
The authors show that two distal elements, located ~550kb downstream of the SOX2 gene, are important for SOX2 transcription in hNSC. They investigate both the deletion of these elements in established hNSCs and in hNSCs generated by differentiation of human pluripotent stem cells, suggesting these elements are important in both the establishment and maintenance of SOX2 expression in hNSCs.
Weaknesses:
Homologous elements have been studied in the mouse genome and have conserved function in mouse NSCs, yet these findings are not mentioned. Inclusion of biological replicates for the scRNA-seq and …
Reviewer #2 (Public review):
Summary:
The authors use a combination of genomics, genome conformation assays, and CRISPR-mediated deletion to study the transcriptional regulation of the SOX2 gene in human neural stem cells (hNSCs).
Strengths:
The authors show that two distal elements, located ~550kb downstream of the SOX2 gene, are important for SOX2 transcription in hNSC. They investigate both the deletion of these elements in established hNSCs and in hNSCs generated by differentiation of human pluripotent stem cells, suggesting these elements are important in both the establishment and maintenance of SOX2 expression in hNSCs.
Weaknesses:
Homologous elements have been studied in the mouse genome and have conserved function in mouse NSCs, yet these findings are not mentioned. Inclusion of biological replicates for the scRNA-seq and replicate CRISPR-deleted clones would strengthen the study.
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Author Response:
eLife Assessment
In this important study, Bready et al. investigate how a highly conserved long-range enhancer mediates neural-specific SOX2 regulation during neural differentiation using human neural stem cells. This study has broad appeal to developmental neuroscience; however, the data remain incomplete given the need for homozygous enhancer knockouts and biological replicates in the scRNAseq assays.
We thank the expert reviewers and eLife editors Drs. Eade and White for complementing our work and deeming it an “important study” of “broad appeal to developmental neuroscience”. We also acknowledge some of the limitations of our work, including the lack of homozygous deletion of the enhancer element. As we detail below, we tried tirelessly to identify human embryonic stem cell (hESC) clones with homozygous deletions …
Author Response:
eLife Assessment
In this important study, Bready et al. investigate how a highly conserved long-range enhancer mediates neural-specific SOX2 regulation during neural differentiation using human neural stem cells. This study has broad appeal to developmental neuroscience; however, the data remain incomplete given the need for homozygous enhancer knockouts and biological replicates in the scRNAseq assays.
We thank the expert reviewers and eLife editors Drs. Eade and White for complementing our work and deeming it an “important study” of “broad appeal to developmental neuroscience”. We also acknowledge some of the limitations of our work, including the lack of homozygous deletion of the enhancer element. As we detail below, we tried tirelessly to identify human embryonic stem cell (hESC) clones with homozygous deletions but were unable to. As we speculate in the discussion, this failure may represent a biological property of the enhancer element (possibly an essentiality manifested even in hESCs), or a technical limitation related to the large size (2.7 kb) of the genomic element targeted for deletion. We also clarify that every scRNAseq assay included cells from multiple teratomas.
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this study, the authors examine how a developmentally regulated cis-regulatory element controls SOX2 expression during neural differentiation of human stem cells. The results suggest that this highly conserved long-range enhancer mediates neural-specific SOX2 regulation and offer insight into the role of promoter-enhancer contacts in this process. Although the findings are interesting, several limitations need to be addressed.
Strengths:
A central question in developmental biology is how genes are regulated in a context-dependent manner. SOX2, a major pluripotency factor, is expressed in diverse tissues during development, and therefore understanding the mechanisms that control its spatiotemporal expression is critical. This study addresses this important question by examining the functional relevance of a neural-specific, developmentally regulated SOX2 enhancer and its associated promoter-enhancer contacts in driving gene expression during human neural development. Using multiple model systems and techniques, the authors test the requirement of this enhancer by analyzing SOX2 expression in mutant lines, providing evidence for its role in this process.
We thank the reviewer for highlighting the significance of our work in the field of developmental biology.
Weaknesses:
A key limitation of the study is the absence of data from homozygous SOX2 enhancer deletion, which leaves the analysis incomplete and tempers the conclusions that can be drawn. Furthermore, the suitability of teratomas as a model system is questionable, given their limited capacity to recapitulate the spatial patterning, regional specification, and organized developmental processes characteristic of the human forebrain. Finally, the manuscript remains largely descriptive with little mechanistic insight.
We appreciate the reviewer’s disappointment with lack of data from a homozygous SOX2 enhancer deletion. We too felt disappointed when we started genotyping our hESC clones. In fact, we spent a year screening multiple hESC clones for a homozygous deletion but were unable to find one. We performed several assays to better characterize the heterozygous clones, including Sanger sequencing, whole-genome sequencing (WGS) and fluorescent in situ hybridization (FISH). All assays pointed in the direction of hemizygous deletion. We do not understand the reasons for the absence of homozygous deletion clones. One possibility is that homozygous deletion of the enhancer is selected against in hESCs, thus preventing growth of colonies. Another possibility is the technical challenge of achieving a large deletion (2.7 kb) in hESCs. We also entertained the possibility of the excised enhancer being excised from the genome but retained as extrachromosomal (ec) DNA, thus producing the hemizygous genotype. However, several assays, such as FISH and PCR diagnostics, argued against this possibility.
The teratoma assay was chosen as an in vivo metric of spontaneous differentiation of hESCs into the three germ layers, because our overarching hypothesis was that perturbing the enhancer element and 3D chromatin loop regulating SOX2 transcription would impair specification of neuroectodermal precursors. We believe that teratomas offer an opportunity to allow pluripotent cells to declare any predilections toward germ layers in unbiased fashion. Importantly, we did not rely solely on teratomas to assess effects of our genomic perturbations on specification of neuroectoderm, but also pursued cerebral organoids as an orthogonal approach focused on the tissue of interest, the central nervous system.
Our work does not only describe an important mechanism for regulation of SOX2 transcription in the transition from pluripotency to neuroectodermal specification, but also provides mechanistic insight into the question of whether the developmentally co-regulated activation of the enhancer and formation of the 3D chromatin loop are dependent on each other. Our findings indicate that the two processes occur independently of each other, as evidenced by the fact that the enhancer is uncoupled from chromatin folding, as occurs when the adjacent CTCF motif is deleted. This finding raises the possibility that enhancer activation occurs through yet to be determined transcriptional events, and that establishment of the local 3D chromatin architecture helps fine-tune its influences in the Topologically Associating Domain (TAD) of interest.
We are further pursuing mechanisms that regulate activation of the enhancer within neuroectodermal lineages and may explain its actions on genomic elements other than the SOX2 locus within the relevant TAD. We are also investigating reasons explaining why hemizygous enhancer deletion produces stronger phenotypes than deletion of the CTCF motif that helps stabilize the 3D chromatin loop.
Reviewer #2 (Public review):
Summary:
The authors use a combination of genomics, genome conformation assays, and CRISPR-mediated deletion to study the transcriptional regulation of the SOX2 gene in human neural stem cells (hNSCs).
Strengths:
The authors show that two distal elements, located ~550kb downstream of the SOX2 gene, are important for SOX2 transcription in hNSC. They investigate both the deletion of these elements in established hNSCs and in hNSCs generated by differentiation of human pluripotent stem cells, suggesting these elements are important in both the establishment and maintenance of SOX2 expression in hNSCs.
We thank the reviewer for appreciating the importance of this regulatory mechanism in the establishment and maintenance of SOX2 expression in the human neural lineage.
Weaknesses:
Homologous elements have been studied in the mouse genome and have conserved function in mouse NSCs, yet these findings are not mentioned. Inclusion of biological replicates for the scRNA-seq and replicate CRISPR-deleted clones would strengthen the study.
We appreciate the recommendation of the reviewer to better acknowledge prior work in mouse neural development. We will ensure full acknowledgment of these studies in the revised manuscript.
We also appreciate the suggestion for biological replicates in our scRNA-seq assays. We clarify that each scRNA-seq arose from combining multiple teratomas from each experimental group, thus ensuring that findings reflect reproducible biology rather than isolated findings from single teratomas. This clarification will be emphasized in the revised manuscript.
Finally, we absolutely agree with the reviewer that more CRISPR-deleted clones would have strengthened the study. Unfortunately, we realized that characterization of each clone takes multiple years and addition of more clones would have made the study too lengthy.
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