TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation

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1. 

   eLife

   Nov 5, 2020

   Summary: In this well done manuscript, the authors examine the bHLH transcription factor TWIST1 and its interacting proteins in neural crest cell development using an unbiased screen. Given the important role of neural crest cells in craniofacial and cardiac developmental defects, the data are both useful and important.

   The major problem is the claim that the regulation reported here is important for neural crest specification / induction. This cannot be the case, as Twist 1 starts to be expressed in mouse only during the delamination step according to published single cell data. The premigratory Zic/Msx positive neural crest shows no expression of Twist1 before EMT markers kick in. The authors need to deal with this. It would be important to show in vivo expression data analysis and bring the conclusions in line with the timing in …

   Summary: In this well done manuscript, the authors examine the bHLH transcription factor TWIST1 and its interacting proteins in neural crest cell development using an unbiased screen. Given the important role of neural crest cells in craniofacial and cardiac developmental defects, the data are both useful and important.

   The major problem is the claim that the regulation reported here is important for neural crest specification / induction. This cannot be the case, as Twist 1 starts to be expressed in mouse only during the delamination step according to published single cell data. The premigratory Zic/Msx positive neural crest shows no expression of Twist1 before EMT markers kick in. The authors need to deal with this. It would be important to show in vivo expression data analysis and bring the conclusions in line with the timing in neural crest development.

   Reviewer #1:

   This excellent study is focused on the mechanisms of action of Twist1 in the neural crest cells and on the identification of core components of the Twist1 network. The authors performed an in-depth experimental study and sophisticated analysis to identify Chd7/8 as the key partners of Twist 1 during NCC development. This identification and corresponding predictions later appeared consistent with experimental in vivo data including single and combinatorial gene knockout mouse models with phenotypes in the cranial neural crest. Overall, this study is important for the field. However, I disagree with some secondary interpretations the authors give to their results. At the same time, the major conclusions stay solid. Below I discuss the most critical points.

   1. Chd7, Chd8 and Whsc1 are ubiquitously expressed. Thus, the specificity of regulation is achieved via interactions with other, more cell type- and stage-specific, factors. This would be good to mention.

   2. The authors suggest: "The phenotypic data so far indicate that the combined activity of TWIST1-chromatin regulators might be required for the establishment of NCC identity. To examine whether TWIST1- chromatin regulators are required for NCC specification from the neuroepithelium and to pinpoint its primary molecular function in early neural differentiation, we performed an integrative analysis of ChIP-seq datasets of the candidates".

   • This is a strange assumption, given that Twist1 is expressed only starting from the NCC delamination stage in mouse cranial neural crest (Soldatov et al., 2019). It does not seem to correlate with premigratory NCC identity and the situation inside of the neural tube. The authors conclude: "Therefore, combinatorial binding sites for TWIST1, CHD7 and CHD8 may confer specificity for regulation of patterning genes in the NECs." Or, alternatively, they may confer the control of mesenchymal phenotype, downstream migration and fate biasing etc. I do not think the authors have good arguments to bring up induction or patterning of NCCs at the level of the neural tube.
   • I have a good suggestion for the authors: I would extract the regulons from Soldatov et al. single cell data and run the binding site proximity check for the individual genes belonging to the gene modules /regulons specific to delamination and early NCC migration stages. I am curious, if the proximity of binding sites of Twist1-related crowd would rather correlate with genes from these specific regulons as compared to randomly selected regulons from the entire published single cell dataset. Randomization/bootstrapping analysis are welcomed. So far, being an excellent study, this paper does not solve a problem of downstream (of Twist1) gene expression program in the neural crest cells. At the same time, this is what the author can try to obtain with their DNA binding data in combination with published single cell data. Repression of Sox2 and upregulation of Pdgfra (reported in Figure 4) might be a part of this downstream program being in line with the published single cell gene expression data (Soldatov et al., 2019).
   • The authors conclude the paragraph: "Therefore, combinatorial binding sites for TWIST1, CHD7 and CHD8 may confer specificity for regulation of patterning genes in the NECs". Again, this is not a good or plausible explanation based on specificity of expression of suggested patterning genes (or visualized genes are poorly selected). Additionally, although I believe the obtained results are important and of a good quality, I would not call them "developmentally equivalent to ectomesenchymal NCCs" or other NCCs. This is because the in vitro system will never reflect the embryonic in vivo development with high accuracy (especially when it comes to patterning and positional identity). This might explain that some prominent binding positions and interpretations the authors give do not correspond to the gene expression logic during neural crest development. Besides, Twist1 and Chd7/8 are naturally expressed in many other cell types and might target non-NCC genes (Vegfa?). This does not reduce the value of the data, but it is good to mention for the community.

   1. Figure 2: Twist1-/+ Chd8-/+ is repeated two times in panel B (but the embryos look differently), although the authors most likely meant to show Twist1-/+ Chd7-/+ in the second case. If this is indeed the case, the authors should also show a phenotype of Chd7 KO.

   2. The authors write: "Impaired motility in Twist1, Chd8 and Whsc1 knockdowns was accompanied by reduced expression of EMT genes (Pdgfrα, Pcolce, Tcf12, Ddr2, Lamb1 and Snai2) (Figure 6D, S3D) and ectomesenchyme markers (Sox9, Spp1, Gli3, Klf4, Snai1), while 375 genes that are enriched in the sensory neurons located in the dorsal root ganglia (Ishii et al., 2012) were upregulated (Sox2, Sox10, Cdh1, Gap43; Figure 6E).

   • From the list of genes characterizing EMT, I can agree only on Pdgfra and Snai2, the rest is unspecific for EMT, and appears rather ubiquitous or specific to different cell populations (non-EMT).
   • From the list of suggested ectomesenchyme markers, I cannot pick any gene that would be a bit specific for ectomesenchyme (within neural crest lineage) except for Snai1. Sox9 is broadly expressed also in the trunk neural crest, Spp1 and Klf4 are not expressed in early mouse ectomesenchyme, Gli3 is too broad and non-selective. I suggest to select other gene sets (check the expression with online PAGODA app from Soldatov et al): http://pklab.med.harvard.edu/cgi-bin/R/rook/nc.p63-66.85-87.dbc.nc/index.html
   • The choice of DRG genes is also non-optimal, as Sox10 is pan-NCC, Sox2 is expressed in early migrating crest and satellite glial cells of DRG and Schwann cell precursors, Gap43 and Cdh1 are not specific enough. These genes clearly suggest the beginning of neuro-glial fates or trunk neural crest bias. To be more precise and for claiming sensory neurons, the authors should come up with pro-neuronal genes such as neurogenins, NeuroD, Isl1, Pou4f1, Ntrk and many others.

   Still, overall, I agree with the author's main conclusions.

   1. The authors write: "The genomic and embryo phenotypic data collectively suggest a requirement of TWIST1- chromatin regulators in the establishment of NCC identity in heterogeneous neuroepithelial 403 populations". Again, I do not think the authors can claim anything related to the establishment of NCC identity. NCC identity, in broad sense, includes NCC induction within the neural tube, at both trunk and cranial levels. In mice, Twist1 is not expressed in trunk NCCs at all. At a cranial level, Twist1 is expressed too late to be a NCC inducing or patterning gene. As I mentioned earlier, it comes up during delamination.

   2. Figure 7G only partly corresponds to the positioning of the NCC markers in a mouse embryo. Id1 and Id2 are broadly expressed throughout all phases of NCC development and in the entire dorsal neural tube beyond the NC region. Mentioning Otx2 as a NCC specifier is strange. At the same time, Msx1, Msx2, Zic1 are excellent genes! Tfap2 is a bit too late, but still ok. Please keep in mind, Msx1/2, Zic1 are expressed before Twist1, and, thus, Twist1 can be downstream of this gene expression program. Also, these genes become downregulated quite soon upon delamination, whereas Twist1/Chd7/8 expression stays (in vivo). Expression pattern of Tfap2a better corresponds to Twist1, although Tfap2a comes a bit before Twist1, and, besides, Tfap2a is expressed independently of Twist1 in trunk NCC. Despite such gene expression divergence, Twist1-based networks might provide positive feedback loops stabilizing the expression of other transcriptional programs that were originally induced by other factors. It might be good mentioning this to the readers. This "stabilizing role" of the Twist1 network can be a really important one. Given the incremental and combinatorial nature of the phenotype in vivo - this is most likely the case. I believe these points are important to reflect in the discussion section.

   Reviewer #2:

   This manuscript, by Fan et. al, is a comprehensive look into the bHLH protein TWIST1 and its interacting proteins in neural crest cell differentiation. The study employs an unbiased screen where a TWIST1-BirA fusion is used in conjunction with biotin linking to collect Twist protein transcriptional complexes. (BioID-Proximity-labeling, TWIST1-CRMs). The work appears carefully done and the data and impact of this study are high given the nature of NCCs being involved as key players in craniofacial and cardiac developmental defects. The association of TWIST1 with the chromatin helicases CHD7 & 8 is important to understand as numerous TWIST1 loss-of-function studies indicate that its role in NCCs clearly is required for normal NCC function.

   The NCC cell line O9-1 is used to collect the data. Genetic interactions between TW1, Chd7, Chd8 and Whsc1 are tested in genome edited ESCs. Overall, this is a well-executed, interesting and important study.

   Reviewer #3:

   Using BioID, the authors identified more than 140 proteins that potentially interact with transcription factor Twist1 in a neural crest cell line. Most of these 140 Twist1-interactomes do not overlap with the 56 known Twist1 binding partners during neural crest cell development (see below). By focusing on several strong Twist1 binding partner candidates (particularly a novel candidate CHD8), the authors found:

   1. Twist1 interacts with these proteins via its N-terminal protein domain as demonstrated by co-IP.

   2. Compound heterozygous mutation of Chd8, Chd7 or Whsc and Twist1 displayed more severe phenotype compared to heterozygous mutation of Twist1 alone, for example, more significant reduction of the cranial nerve bundle thickness.

   3. ChIPseq analysis of Twist1 and CHD8 and key histone modifications revealed that the binding of Chd8 strongly correlates with those of Twist1, to active enhancers that are also labeled by H3K4me3 and H3K27ac.

   4. The binding of CHD8 requires the binding of Twist1, but not vice versa.

   5. Twist1-Chd8 regulatory module represses neuronal differentiation, and promotes neural crest cell migration, and potentially their differentiation into the non-neuronal cell types.

   The authors use an impressive array of different techniques, both in vitro and in vivo, and yield consistent results. The manuscript is nicely written. The findings are nuanced, but the major conclusions are largely expected.

   Critiques:

   • As the title states, the three key TWIST interacting factors that most of the study focuses on are chromatin regulators. However, the consequence of mutating these factors at the epigenetic level was not directly addressed, including the level of active histone modification, the accessibility of the Twist1/CDH co-bound promoters/enhancers, and the position of nucleosomes.
   • CRISPR-generated ESCs and chimera technology were used effectively to generate mutants. In comparison, the analysis of the phenotypes was rather cursory and can benefit from more in-depth molecular analysis. The altered genes found in mutant NEC and NCC in the last section of the study, especially, should be validated in mutants.
   • Across the manuscript, there were jumps from NCC to NEC and back. It will be important to justify why a certain cell type is selected for each analysis, focusing on the biological question at hand.
   • Using BioID, the authors detected 140 different proteins that interact with Twist1. However, only 4 of them overlap with the 56 known Twist1 partners (Figure 1A). This result suggests that BioID identified almost a distinct set of Twist1-interacting proteins, compared to the published results. The authors need to discuss the discrepancy, and the underlying reasons.
   • The authors show that Twist1 colocalizes with Cdh8, and is required for the binding of Cdh8, thus suggesting that Twist1-Cdh8 form a regulatory module. Given the degenerate nature of bHLH factor binding motifs, it is likely that the binding of Twist1, and subsequently the binding of Cdh8, are dictated by other transcription factors. Therefore, a motif enrichment analysis should be done among the Twist1/Cdh8 co-binding sites, and compare those motifs enriched in Twist1-only and Cdh8-only binding sites.
   • The increasing expression of DRG neurons genes in Twist1/Cdh8 mutants suggests a possible transition from cranial NC to trunk NC. Therefore, the authors should examine the expression of marker genes accordingly.

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2. 

   Version published to 10.1101/2020.09.06.285387 on bioRxiv

   Sep 7, 2020