CHD7 is a novel interactor of LSD1
To gain deeper insights into the role of LSD1 in stem cell biology, we performed immunoprecipitation (IP) of endogenous LSD1 using an anti-LSD1 antibody, followed by LC–MS/MS analysis (Fig. 1A and B). Thirteen high-confidence LSD1-interacting proteins meeting the inclusion criteria of fold change > 2 and P < 0.001 were identified (Table S1). We retrieved known interactors of LSD1, such as HDAC-containing NuRD and Sin3a complexes8, ZNF21725, the CoREST/REST corepressor 1 and 3 (RCOR1/3)26,27, thereby confirming the efficacy of our approach (Fig. 1B). Additionally, our analysis revealed a novel interaction between LSD1 and CHD7, an ATP-dependent chromatin remodeler, which has not been previously reported. Gene ontology (GO) terms associated with the biological process and cellular component categories of the thirteen LSD1 interactors revealed categories related to “chromatin organization”, “transcription”, “DNA repair”, and “histone deacetylase” (Fig. 1C). Moreover, molecular function-based GO terms were highly enriched in categories related to “DNA binding” and “transcription regulation” (Fig. 1D), highlighting the central role of LSD1 and its interacting proteins in epigenetic regulation.
CHD7 interacts with LSD1 in ESCs. (A) Scheme representing the workflow for LSD1 immunoprecipitation (IP) and western blot (WB) of LSD1-IP depicting the specificity of LSD1 antibody. IgG was used as a negative control. Input corresponds to 10% of the nuclear extract used for IP. (B) STRING network of LSD1-interacting proteins retrieved from the LC–MS/MS analysis that followed LSD1-IP. Line thickness indicates the strength of data support. (C) GO analysis for cellular components and biological processes of top LSD1-interacting candidates. (D) Molecular functions of the top 13 interacting candidates of LSD1. (E) IP of LSD1 (left panel) and CHD7 (right panel) from nuclear extracts of mouse ESCs followed by WB with anti-LSD1 and anti-CHD7 antibodies. IgG was used as a negative control. Input corresponds to 10% of the nuclear extract used for IP. The represented blots are from different gels of the same biological replicate. (F) IP of LSD1 from nuclear extracts of mouse ESCs pretreated with RNase A (left panel) or DNase I (right panel), followed by WB with anti-LSD1 and anti-CHD7 antibodies. IgG was used as a negative control. Input represents 10% of the nuclear extract used for IP. The represented blots are from different gels of the same biological replicate. (G) Schematic representations of the different domains of CHD7 used for cloning in pCMV8-Flag tagged plasmids. (H) IP of c-MYC from nuclear extracts of HEK293T co-transfected with the indicated constructs from (G) and pSIN-c-MYC containing LSD1 followed by c-MYC and FLAG WB. IgG was used as a negative control. The represented blots are from the same gel of the same biological replicate. Results are one representative of n = 3 independent biological experiments (A, E, F and H) and n = 2 (B–D).
We further investigated the interaction of LSD1 with CHD7 since: (i) this interaction is novel; (ii) similar to LSD128, CHD7 binds at enhancers of pluripotency genes19; and (iii) the binding of CHD7 to DNA is influenced by H3K4me1, a histone mark that is targeted by LSD119. First, we verified the association between LSD1 and CHD7 in mouse ESCs by performing co-immunoprecipitation (co-IP) (Fig. 1E; left panel) and reverse co-IP experiments of the endogenous proteins (Fig. 1E; right panel). Notably, LSD1–CHD7 interaction was DNA and RNA-independent, as treatment of the nuclear extracts with DNase I or RNase A, respectively, did not disrupt the interaction of the two proteins (Fig. 1F).
CHD7 contains several multi-functional domains, including two N-terminal chromodomains (CD) which are required for histone mark recognition, a SWI2/SNF2-like ATPase/helicase domain (HD), a SANT domain that enhances nucleosome remodeling efficiency, and two BRK domains (BD) with uncharacterized functions. To pinpoint the specific region of CHD7 responsible for the interaction with LSD1, we engineered distinct pCMV8-FLAG-tagged constructs to express the different domains of CHD7 (Fig. 1G): (i) construct CD, encompassing the N-terminal fraction with the two CD domains; (ii) construct HD, representing the central region of CHD7 with the ATPase/helicase, SLIDE and SANT domains; and iii) the C-terminal domain with the BRK domains. The aforementioned constructs were transiently co-expressed with pSIN-c-MYC tagged full-length LSD1 in HEK293T cells. CHD7 and LSD1 interactions were next assessed by FLAG-immunoprecipitation of nuclear extracts, followed by western blotting against FLAG and MYC. Our results showed that LSD1 interacted with CHD7 through its ATPase/helicase domain and BRK domain (Fig. 1H), suggesting that LSD1 can modulate the nucleosome remodeling function of CHD7.
CHD7 colocalizes with LSD1 at distal regulatory regions
To investigate the interplay between CHD7 and LSD1, we checked CHD7 protein expression in whole cell extracts (WCE), nuclear and chromatin fractions of WT and Lsd1 KO mouse ESCs. We observed a modest decrease in the total CHD7 protein levels after Lsd1 loss; however, the expression of CHD7 was increased in the nuclear and chromatin extracts of WT and Lsd1 KO mouse ESCs. Such an increase of CHD7 in nuclear and chromatin fractions was not associated with Chd7 mRNA levels, suggesting that deletion of Lsd1 promotes CHD7 chromatin recruitment in mouse ESCs (Fig. 2A–D). Given that both CHD7 and LSD1 preferentially bind to active enhancers, we performed chromatin immunoprecipitation with massively high-throughput sequencing (ChIP-seq) of CHD7 in WT and Lsd1 KO mouse ESCs. Consistent with elevated levels of CHD7 in chromatin upon Lsd1 deletion, we identified 3281 peaks of CHD7 in Lsd1 KO mouse ESCs, whereas 1034 peaks were identified in WT mouse ESCs (Fig. 2E; Table S2). These peaks are distributed along the genome, preferentially enriched at distal regions in both WT and Lsd1 KO mouse ESCs (Fig. 2F and G). Further, to explore whether LSD1 and CHD7 bind to the same genomic regions, we intersect CHD7 ChIP-seq data with LSD1 ChIP-seq data in WT mouse ESCs from our previous study15 and we found ~ 60.7% of CHD7 binding sites co-bound by LSD1 in WT ESCs (Fig. 2H). GO analysis for biological processes of CHD7-bound regions and co-bound regions by both CHD7 and LSD1 indicated that they bind to genes related to “transcription”, “multicellular development”, and “neuron-related categories” (Fig. 2I), indicating a potential interplay between CHD7 and LSD1 in the regulation of cellular differentiation. Next, we overlapped genes bound by CHD7 in WT and Lsd1 KO mouse ESCs and found that ~ 69.5% of the genes in WT ESCs were common with Lsd1 KO ESCs (Fig. 2J). The subsets of genes exclusively bound by CHD7 in Lsd1 KO mouse ESCs (1842) were enriched for biological processes such as “stem cell maintenance”, “DNA methylation”, “axon regeneration”, among others (Fig. 2K).
ChIP-seq analysis of CHD7 and LSD1. (A–C) WB of LSD1 and CHD7 in WCE, nuclear and chromatin fractions of WT and Lsd1 KOs mouse ESCs. ACTIN, LAMIN A/C, and H3 were used as loading controls. The represented blots are from different gels of the same biological replicate. (D) Bar diagram representing RT-qPCR of Chd7 in WT and Lsd1 KO2 mouse ESCs. mRNA levels are relative to WT mouse ESCs. (E) Number of common CHD7 peaks retrieved from two independent biological replicates of CHD7-ChIP seq in WT and Lsd1 KO mouse ESCs. (F and G) Genomic distribution of CHD7 binding in the promoter (within 5 kb upstream of TSS), distal intergenic, exon, UTR, downstream and intron in (F) WT and (G) Lsd1 KO mouse ESCs. (H) Venn diagram of overlapped genes between CHD7 and LSD1 ChIP-seq in WT mouse ESCs. (I) GO analysis of biological processes of genes associated with CHD7 in WT and Lsd1 KO mouse ESCs (brown) and common LSD1 and CHD7 peaks in WT mouse ESCs (green). (J) Venn diagram depicting the genes identified from CHD7 ChIP in WT and Lsd1 KO2 mouse ESCs. (K) GO analysis of biological processes of genes associated with CHD7 ChIP in Lsd1 mouse ESCs. (L and M) Venn diagram representing overlapped genes between H3K4me1 and CHD7 ChIP-seq in (L) WT and (M) Lsd1 KO2 mouse ESCs. (N) Average signal of H3K4me1 and CHD7 ChIP-seq in co-occupied regions in WT and Lsd1 KO2 mouse ESCs. (O) Density plot representing H3K4me1 binding in CHD7 peaks (common with WT and unique to Lsd1 KO2) upon deletion of Lsd1 in mouse ESCs. (P) Occupancy of CHD7 at the enhancers in WT and Lsd1 KO2 mouse ESCs. (Q-T) CHD7 and H3K4me1 ChIP-seq signals in WT and Lsd1 KO2 mouse ESCs at the (Q) Nanog, (R) Pou5f1 (S) Foxd3 and (T) Otx2 genomic regions. Respective inputs are depicted in grey. Enhancers for Nanog and Pou5f1 are marked as black squares. Data are represented as mean ± SD, and each experiment was performed with n = 3 (D) and n = 2 (E) replicates. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data in (D) were analyzed using an unpaired Student’s t-test. Results are one representative of n = 3 independent biological experiments (A–C).
It has been previously shown that Lsd1 ablation leads to a global accumulation of H3K4me115 and that CHD7 preferentially localizes to chromatin regions enriched with H3K4me119,20. To investigate whether CHD7 recruitment to chromatin in Lsd1 KO ESCs is linked to H3K4me1 deposition, we integrated publicly available H3K4me1 and CHD7 ChIP-seq data from WT and Lsd1 KO mouse ESCs15. In WT ESCs, 34.15% of genes were co-occupied by CHD7 and H3K4me1, which increased to 64.49% in Lsd1 KO ESCs (Fig. 2L and M). Further, analysis of these co-occupied regions revealed a significant increase in the overlap between H3K4me1 and CHD7 at the center of peaks following Lsd1 deletion (Fig. 2N).
Additionally, we examined the distribution of H3K4me1 at CHD7 binding regions, including those common between WT and Lsd1 KO, as well as those unique to Lsd1 KO ESCs. We observed a heightened H3K4me1 signal in CHD7-bound regions in Lsd1 KO ESCs compared to WT (Fig. 2O). Moreover, there was increased CHD7 co-occupancy, particularly at the center and downstream regions of enhancers (Fig. 2P). Indeed, loci regulating self-renewal (e.g., Nanog and Pou5f1) and neurogenesis (e.g., Otx2 and Foxd3) exhibited increased H3K4me1 marks with elevated CHD7 occupancy in Lsd1 KO mouse ESCs compared to WT mouse ESCs. Specifically, enhancers of Pou5f1 and Nanog showed heightened co-occupancy of CHD7 and H3K4me1 following Lsd1 deletion (Fig. 2Q–T). Altogether, these results suggest that increased H3K4me1 levels upon Lsd1 deletion potentially promote CHD7 recruitment to chromatin, allowing it to bind to unique regulatory regions in Lsd1 KO mouse ESCs.
Chd7 deletion does not exacerbate proliferative defects in Lsd1 KO ESCs
To further investigate the role of CHD7 in stem cell pluripotency and differentiation, we deleted Chd7 in both WT and in Lsd1 KO mouse ESCs, with the Lsd1 KO lines having been originally generated in our previous study15. We targeted exon 5 of Chd7 employing two distinct single guide RNAs (sgRNAs). Specifically, sgRNA1 was designed to target the start codon of Chd7, whereas the sgRNA2 targeted a region 500 base pairs (bp) downstream of sgRNA1 (Fig. 3A). The absence of CHD7 protein following Chd7 gene targeting was confirmed by western blotting (Fig. 3B, C). From these clones, we selected Chd7 KO2 and Chd7/Lsd1 KO3 for further analysis, which will be referred to as Chd7 KO and Chd7/Lsd1 KO, respectively. Sanger sequencing revealed two different insertion/deletions (indels) (− 3 and − 4 bp) in Chd7 KO2, whereas four different indels (+ 1 bp, − 4 bp, − 13 bp, and − 16 bp) were detected in Chd7/Lsd1 KO3 mouse ESCs (Fig. 3D). Chd7 KO and Chd7/Lsd1 KO mouse ESCs were further validated by reverse transcription followed by quantitative PCR (RT-qPCR) (Fig. 3E and F).We assessed the phenotype of the engineered cell lines by performing proliferation, apoptosis, and cell cycle assays. We did not detect proliferation and apoptosis defects in Chd7 KO mouse ESCs. However, similar to Lsd1 KO, Chd7/Lsd1 KO mouse ESCs showed a dramatically decreased proliferation with augmented apoptosis compared to WT and Chd7 KO mouse ESCs (Figs. 3G and H). This result implied that the defective viability observed in the double KO arises predominantly from the deletion of Lsd1 in mouse ESCs. Of note, no significant difference in the cell cycle profile was detected upon combined /single deletion of Chd7 and Lsd1 (Fig. 3I). We next sought to determine whether KO clones could remain pluripotent by assessing alkaline-phosphatase (AP) staining. Chd7 KO ESCs exhibited a strong AP activity, similar to WT mouse ESCs. However, both Chd7/Lsd1 and Lsd1 KO mouse ESCs were organized as monolayers with undefined colony morphology (Fig. 3J). The quantification of AP-staining revealed an increased number of partially differentiated colonies in Chd7/Lsd1 KO compared to WT and Chd7 mouse ESCs, possibly due to generic growth defects occurring upon Lsd1 ablation (Fig. 3K). However, these double KO clones did not show a significant difference in the expression of OCT4 and SSEA1 proteins compared to WT and Chd7 KO mouse ESCs through western blotting and immunoassay staining, respectively (Fig. 3L and M). Overall, our data suggests that ablation of Chd7 and Lsd1 does not affect the pluripotency of ESCs; however, the combined deletion of Chd7 and Lsd1 exhibited growth defects that mirrored Lsd1 KO phenotype.
Assessment of the phenotype of Chd7 and Chd7/Lsd1 KO mouse ESCs. (A) Schematic representation of target sites in the genomic DNA of Chd7. The sgRNAs and PAM sequences are highlighted in red and blue, respectively. (B) Western blot of LSD1 and CHD7 on WCE from selected clones. ACTIN is used as loading control. The represented blots are from different gels. (C) Full western blot of CHD7 on WCE on WT, Chd7 KO2 and Chd7/Lsd1 KO3 mouse ESCs. ACTIN is used as a loading control. (D) Sanger sequencing analysis of Chd7 KO2 (top panel) and Chd7/Lsd1 KO3 mouse ESCs (bottom panel). Deletions and insertions are represented as dashes and in yellow, respectively. (E and F) RT-qPCR of (E) Lsd1 in WT, Lsd1 KO, Chd7 KO, and Chd7/Lsd1 KO ESCs and (F) Chd7 in WT, Chd7 KO and Chd7/Lsd1 KO mouse ESCs. mRNA levels are relative to WT mouse ESCs. (G) Proliferation rate of WT, Lsd1 KO, Chd7 KO, and Chd7/Lsd1 KO mouse ESCs at indicated time points relative to day 0. (H) Percentages of live (Annexin V-) and apoptotic cells (Annexin V +) in WT, Lsd1 KO, Chd7 KO, and Chd7/Lsd1 KO mouse ESCs. (I) Bar diagram depicting the percentages of cells relative to WT at G0/G1, S, and G2/M phases in Lsd1 KO, Chd7 KO, Chd7/Lsd1 KO mouse ESCs. (J) AP staining of WT, Lsd1 KO, Chd7 KO, and Chd7/Lsd1 KO mouse ESCs and (K) Percentages of undifferentiated (UD), partially differentiated (PD), and differentiated (D) colonies from cells analyzed in (K). Scale bars: 20 μm. (L) Western blots of LSD1, CHD7 and OCT4 on the WCE of WT, Lsd1 KO, Chd7 KO and Chd7/Lsd1 KO mouse ESCs. β-ACTIN is used as the loading control. (M) Representative immunofluorescence images of SSEA1 in WT, Lsd1 KO, Chd7 KO and Chd7/Lsd1 KO mouse ESCs. DAPI was used as the nuclear marker. Scale bars, 20 μm. Data are represented as mean ± SD, and each experiment was performed with n = 3 replicates. ns- non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001. Data in (G, H, and K) were analyzed using an unpaired Student’s t-test, (E, F and I (ns)) analyzed with two-way ANOVA. Each dot in the bar graphs represents independent biological replicates. Statistical comparison for (G, H and I): WT vs Lsd1 KO, Chd7 KO and Chd7/Lsd1 KO; Lsd1 KO vs Chd7 KO and Chd7/Lsd1 KO; and Chd7 KO vs Chd7/Lsd1 KO mouse ESCs. Statistical comparison for K (WT vs Lsd1 KO, Chd7 KO and Chd7/Lsd1 KO is represented in black, Lsd1 KO vs Chd7 KO and Chd7/Lsd1 KO in blue and Chd7 KO vs Chd7/Lsd1 KO mouse ESCs in brown, respectively). Results are one representative of n = 3 independent biological replicates (C, J, L and M).
Chd7 deletion leads to transcriptional dysregulation related to neuronal development
To interrogate the impact of Chd7 deletion on the ESCs transcriptome, we conducted RNA-seq analysis in WT, Chd7 KO, and Chd7/Lsd1 KO mouse ESCs (Figs. 4A and ; Table S3). Loss of Chd7 resulted in 1852 downregulated transcripts, whereas combined Lsd1 and Chd7 ablation led to 2395 downregulated transcripts (fold change (FC) > 1.5; P < 0.05) (Fig. 4A and B). In contrast, Chd7 KO and Lsd1/Chd7 KO mouse ESCs exhibited 3466 and 4588 upregulated transcripts, respectively (FC > 1.5; P < 0.05) (Fig. 4A and B). We then generated a heatmap to visualize the expression patterns of differentially expressed genes in Chd7 KO and Chd7/Lsd1 KO mouse ESCs, integrating our RNA-seq data from Lsd1 KO mouse ESCs as reported in our previous study15. Hierarchical clustering of the heatmap, based on the top differentially expressed genes in Chd7/Lsd1 KO ESCs, revealed that the transcriptomes of Lsd1 and Chd7/Lsd1 KO mouse ESCs clustered more closely together than those of Chd7 and Chd7/Lsd1 KO mouse ESCs (Fig. 4C).
CHD7 regulates transcription of neuronal genes. (A and B) Volcano plots showing the distribution of differentially expressed transcripts in (A) Chd7 KO and (B) Chd7/Lsd1 KO compared to WT mouse ESCs. Red dots represent upregulated genes, blue dots represent down-regulated genes (P < 0.05; fold-change, FC > 1.5). (C) Heatmap depicting upregulated (red) and downregulated (blue) genes retrieved from the RNA-seq data of WT, Lsd1 KO, Chd7 KO and Chd7/Lsd1 KO mouse ESCs. (D) Venn diagram of common downregulated transcripts between Chd7 KO and Chd7/Lsd1 KO ESCs. (E) Biological processes-based GO analysis of downregulated genes in Chd7 KO and Chd7/Lsd1 KO mouse ESCs. (F) Venn diagram of common upregulated transcript between Chd7 KO and Chd7/Lsd1 KO mouse ESCs. (G) Gene ontology analysis of biological processes related to the upregulated genes of Chd7 KO and Chd7/Lsd1 KO compared to WT mouse ESCs. (H and I) Overlap of RNA-seq data of (H) Chd7 KO and (I) Chd7/Lsd1 KO with CHD7 ChIP in WT mouse ESCs showing the downregulated and upregulated genes bound by CHD7. Downregulated and upregulated genes that are bound by CHD7 are depicted in blue and red, respectively.
Notably, ~ 47% of downregulated transcripts in Chd7 KO were common with Chd7/Lsd1 KO mouse ESCs (Fig. 4D). The enriched GO terms for these commonly downregulated genes included “protein transport”, “splicing”, and “tRNA methylation”, as well as categories related with ectoderm differentiation such as “dendrite development” and “neuronal projection development” (Fig. 4E). Indeed, prior studies have demonstrated that CHD7 remodels the chromatin accessibility to activate genes associated with the specification of neuronal identity and the transition of neuronal progenitors cells to mature neurons29,30. Likewise, LSD1 acts as a positive regulator of neuron differentiation and functions31,32. In contrast, only ~ 28% of upregulated genes in Chd7 KO were similar to Chd7/Lsd1 KO mouse ESCs (Fig. 4F). Analysis of upregulated genes in both Chd7 KO and Chd7/Lsd1 KO ESCs revealed enrichments related to “nervous system development”, and “chromatin organization”, amongst others (Fig. 4G). Next, to evaluate the impact of co-occupancy of LSD1 and CHD7 on transcription regulation of their target genes, we overlapped the differentially expressed genes in Chd7 and Chd7/Lsd1 KO mouse ESCs with CHD7 ChIP-seq data. We found that ~ 18% and 22.59% of genes that were repressed or activated upon deletion of Chd7, and both Chd7 and Lsd1 were bound by CHD7 (Fig. 4H). Altogether, this data shows that CHD7 can act either as a repressor or activator of gene expression and that, although Chd7/Lsd1 double ablation leads to a more profound effect in transcriptional changes, loss of Chd7 possesses a unique regulatory signature.
Enhanced impairment of EB formation with combined Chd7 and Lsd1 ablation
To investigate the impact of double ablation of both Chd7 and Lsd1 on differentiation, we generated embryoid bodies (EBs) from WT, Lsd1 KO, Chd7 KO, and Chd7/Lsd1 KO mouse ESCs. EBs derived from Chd7 KO mouse ESCs displayed normal morphology and size (Fig. 5A and B). However, both Lsd1 KO and Chd7/Lsd1 KO EBs exhibited defective differentiation, and Chd7/Lsd1 KO EBs being half the size of WT-derived EBs, did not survive beyond day 6 of differentiation (Fig. 5A and B), suggesting that the combined loss of both Chd7 and Lsd1 leads to more severe impairment in EB formation compared to the loss of Lsd1 alone.
CHD7 is dispensable for EB differentiation. (A) Representative brightfield images of EBs and (B) measurement of EBs size derived from WT, Lsd1 KO, Chd7 KO, and Chd7/Lsd1 KO mouse ESCs at the indicated time points. Scale bars: 200 µm. (C–F) RT-qPCR analysis of (C) pluripotency (Oct4), (D) endodermal (Sox17 and Foxa2), (E) mesodermal (T and Msx1), and (F) ectodermal (Sox11) markers in the EBs generated from WT, Lsd1 KO, Chd7 KO, and Chd7/Lsd1 KO mouse ESCs at the indicated days after differentiation. mRNA levels are relative to the expression of WT mouse ESCs at day D0. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. Data in (B–F) were analyzed using an unpaired Student’s t-test. Each dot in the bar graphs represents independent biological replicates. Statistical comparison for (C–F): Day 6 (WT vs Lsd1 KO, Chd7 KO and Chd7/Lsd1 KO; Lsd1 KO vs Chd7 KO and Chd7/Lsd1 KO; Chd7 KO vs Lsd1 KO and Chd7/Lsd1 KO mouse ESCs) and Day 8 (WT vs Lsd1 KO, and Chd7 KO mouse ESCs).
Further analysis of gene expression of pluripotency factors and lineage-specific markers revealed that Chd7 KO EBs silenced Oct4 expression and induced the expression of the endodermal markers Sox17 and Foxa2, and the mesodermal factor Brachyury (T) similar to WT control cells (Fig. 5C–E). Yet, they failed to upregulate Msx1 (mesoderm), and the expression of Sox11 (ectodermal) was significantly lower than in WT-derived EBs (Figs. 5E, F), indicating that CHD7 is required for the selective induction of lineage-specific markers but not for the viability of EBs. In contrast, Chd7/Lsd1 KO and Lsd1 KO mouse EBs were unable to abolish Oct4 expression (Fig. 5C) and showed significant downregulation of endodermal (Sox17, Foxa2), mesodermal (T, Msx1), and ectodermal (Sox11) markers compared to WT-derived EBs along the course of differentiation (Figs. 5D–F). This data suggests that while Lsd1 depletion primary contributes to the impaired EB formation observed in Chd7/Lsd1 KO EBs, the combined absence of both Chd7 and Lsd1 exacerbates this phenotype.
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- Source: https://www.nature.com/articles/s41598-024-78920-3