Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This manuscript investigates a mechanism between the histone reader protein YEATS2 and the metabolic enzyme GCDH, particularly in regulating epithelial-to-mesenchymal transition (EMT) in head and neck cancer (HNC).
Strengths:
Great detailing of the mechanistic aspect of the above axis is the primary strength of the manuscript.
Weaknesses:
Several critical points require clarification, including the rationale behind EMT marker selection, the inclusion of metastasis data, the role of key metabolic enzymes like ECHS1, and the molecular mechanisms governing p300 and YEATS2 interactions.
We would like to sincerely thank the reviewer for the detailed, in-depth, and positive response. We are committed to implementing constructive revisions to the manuscript to address the reviewer’s concerns effectively.
Major Comments:
(1) The title, "Interplay of YEATS2 and GCDH mediates histone crotonylation and drives EMT in head and neck cancer," appears somewhat misleading, as it implies that YEATS2 directly drives histone crotonylation. However, YEATS2 functions as a reader of histone crotonylation rather than a writer or mediator of this modification. It cannot itself mediate the addition of crotonyl groups onto histones. Instead, the enzyme GCDH is the one responsible for generating crotonyl-CoA, which enables histone crotonylation. Therefore, while YEATS2 plays a role in recognizing crotonylation marks and may regulate gene expression through this mechanism, it does not directly catalyse or promote the crotonylation process.
We thank the reviewer for raising this concern. As stated by the reviewer, YEATS2 functions as a reader protein, capable of recognizing histone crotonylation marks and assisting in the addition of this mark to nearby histone residues, possibly by assisting the recruitment of the writer protein for crotonylation. Our data indicates the involvement of YEATS2 in the recruitment of writer protein p300 on the promoter of the SPARC gene, making YEATS2 a regulatory factor responsible for the addition of crotonyl marks in an indirect manner. Thus, we have decided to make changes in the title by replacing the word “mediates” with “regulates”. Therefore, the updated title can be read as: “Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer”.
(2) The study suggests a link between YEATS2 and metastasis due to its role in EMT, but the lack of clinical or pre-clinical evidence of metastasis is concerning. Only primary tumor (PT) data is shown, but if the hypothesis is that YEATS2 promotes metastasis via EMT, then evidence from metastatic samples or in vivo models should be included to solidify this claim.
We appreciate the reviewer’s suggestion. Here, we would like to state that the primary aim of this study was to delineate the molecular mechanisms behind the role of YEATS2 in maintaining histone crotonylation at the promoter of genes that favour EMT in head and neck cancer. We have dissected the importance of histone crotonylation in the regulation of gene expression in head and neck cancer in great detail, having investigated the upstream and downstream molecular players involved in this process that promote EMT. Moreover, with the help of multiple phenotypic assays, such as Matrigel invasion, wound healing, and 3D invasion assays, we have shown the functional importance of YEATS2 in promoting EMT in head and neck cancer cells. Since EMT is known to be a prerequisite process for cancer cells undergoing metastasis(1), the evidence of YEATS2 being associated with EMT demonstrates a potential correlation of YEATS2 with metastasis. However, as part of the revision, we will use publicly available patient data to investigate the direct association of YEATS2 with metastasis by checking the expression of YEATS2 between different grades of head and neck cancer, as an increase in tumor grade is often correlated with the incidence of metastasis(2).
(3) There seems to be some discrepancy in the invasion data with BICR10 control cells (Figure 2C). BICR10 control cells with mock plasmids, specifically shControl and pEGFP-C3 show an unclear distinction between invasion capacities. Normally, we would expect the control cells to invade somewhat similarly, in terms of area covered, within the same time interval (24 hours here). But we clearly see more control cells invading when the invasion is done with KD and fewer control cells invading when the invasion is done with OE. Are these just plasmid-specific significant effects on normal cell invasion? This needs to be addressed.
We appreciate the reviewer for the thorough evaluation of the manuscript. The figure panels in question, Figure 2B and 2C, represent two different experiments performed independently, the invasion assay performed after knockdown and overexpression of YEATS2, respectively. We would like to clarify that both panels represent results that are distinct and independent of each other and that the method used to knockdown or overexpress YEATS2 is also different. As stated in the Materials and Methods section, the knockdown is performed using lentivirus-mediated transfection (transduction) of cells, on the other hand, the overexpression is done using standard method of transfection by directly mixing transfection reagent and the respective plasmids, prior to the addition of this mix to the cells. The difference in the experimental conditions in these two experiments might have attributed to the differences seen in the controls as observed previously(3). Hence, we would like to state that the results of figure panels Figure 2B and Figure 2C should be evaluated independently of each other.
(4) In Figure 3G, the Western blot shows an unclear band for YEATS2 in shSP1 cells with YEATS2 overexpression condition. The authors need to clearly identify which band corresponds to YEATS2 in this case.
The two bands seen in the shSP1+pEGFP-C3-YEATS2 condition correspond to the endogenous YEATS2 band (lower band, indicated by * in the shControl lane) and YEATS2-GFP band (upper band, corresponding to overexpressed YEATS2-GFP fusion protein, which has a higher molecular weight). To avoid confusion, the endogenous band will be highlighted (marked by *) in the lane representing the shSP1+pEGFP-C3-YEATS2 condition in the revised version of the manuscript.
(5) In ChIP assays with SP1, YEATS2 and p300 which promoter regions were selected for the respective genes? Please provide data for all the different promoter regions that must have been analysed, highlighting the region where enrichment/depletion was observed. Including data from negative control regions would improve the validity of the results.
Throughout our study, we have performed ChIP-qPCR assays to check the binding of SP1 on YEATS2 and GCDH promoter, and to check YEATS2 and p300 binding on SPARC promoter. Using transcription factor binding prediction tools and luciferase assays, we selected multiple sites on the YEATS2 and GCDH promoter to check for SP1 binding. The results corresponding to the site that showed significant enrichment were provided in the manuscript. The region of SPARC promoter in YEATS2 and p300 ChIP assay was selected on the basis of YEATS2 enrichment found in the YEATS2 ChIP-seq data. We will provide data for all the promoter regions investigated (including negative controls) in the revised version of the manuscript.
(6) The authors establish a link between H3K27Cr marks and GCDH expression, and this is an already well-known pathway. A critical missing piece is the level of ECSH1 in patient samples. This will clearly delineate if the balance shifted towards crotonylation.
We thank the reviewer for their valuable suggestion. To support our claim, we had checked the expression of GCDH and ECHS1 in TCGA HNC RNA-seq data (provided in Figure 4—figure supplement 1A and B) and found that GCDH showed increase while ECHS1 showed decrease in tumor as compared to normal samples. We hypothesized that higher GCDH expression and decreased ECHS1 expression might lead to an increase in the levels of crotonylation in HNC. To further substantiate our claim, we will check the abundance of ECHS1 in HNC patient samples as part of the revision.
(7) The p300 ChIP data on the SPARC promoter is confusing. The authors report reduced p300 occupancy in YEATS2-silenced cells, on SPARC promoter. However, this is paradoxical, as p300 is a writer, a histone acetyltransferase (HAT). The absence of a reader (YEATS2) shouldn't affect the writer (p300) unless a complex relationship between p300 and YEATS2 is present. The role of p300 should be further clarified in this case. Additionally, transcriptional regulation of SPARC expression in YEATS2 silenced cells could be analysed via downstream events, like Pol-II recruitment. Assays such as Pol-II ChIP-qPCR could help explain this.
Using RNA-seq and ChIP-seq analyses, we have shown that YEATS2 affects the expression of several genes by regulating the level of histone crotonylation at gene promoters globally. The histone writer p300 is a promiscuous acyltransferase protein that has been shown to be involved in the addition of several non-acetyl marks on histone residues, including crotonylation(4). Our data provides evidence for the dependency of the writer p300 on YEATS2 in mediating histone crotonylation, as YEATS2 downregulation led to decreased occupancy of p300 on the SPARC promoter (Figure 5F). However, the exact mechanism of cooperativity between YEATS2 and p300 in maintaining histone crotonylation remains to be investigated. To address the reviewer’s concern, we will perform various experiments to delineate the molecular mechanism pertaining to the association of YEATS2 with p300 in regulating histone crotonylation. Following are the experiments that will be performed:
(a) Co-immunoprecipitation experiments to check the physical interaction between YEATS2 and p300.
(b) We will check H3K27cr levels on the SPARC promoter and SPARC expression in p300-depleted HNC cells.
(c) Rescue experiments to check if the decrease in p300 occupancy on the SPARC promoter can be compensated by overexpressing YEATS2.
(d) As suggested by the reviewer, Pol-II ChIP-qPCR at the promoter of SPARC will be performed in YEATS2-silenced cells to explain the mode of transcriptional regulation of SPARC expression by YEATS2.
(8) The role of GCDH in producing crotonyl-CoA is already well-established in the literature. The authors' hypothesis that GCDH is essential for crotonyl-CoA production has been proven, and it's unclear why this is presented as a novel finding. It has been shown that YEATS2 KD leads to reduced H3K27cr, however, it remains unclear how the reader is affecting crotonylation levels. Are GCDH levels also reduced in the YEATS2 KD condition? Are YEATS2 levels regulating GCDH expression? One possible mechanism is YEATS2 occupancy on GCDH promoter and therefore reduced GCDH levels upon YEATS2 KD. This aspect is crucial to the study's proposed mechanism but is not addressed thoroughly.
The source for histone crotonylation, crotonyl-CoA, can be produced by several enzymes in the cell, such as ACSS2, GCDH, ACOX3, etc(5). Since metabolic intermediates produced during several cellular pathways in the cell can act as substrates for epigenetic factors, we wanted to investigate if such an epigenetic-metabolism crosstalk existed in the context of YEATS2. As described in the manuscript, we performed GSEA using publicly available TCGA RNA-seq data and found that patients with higher YEATS2 expression also showed a high correlation with expression levels of genes involved in the lysine degradation pathway, including GCDH. Since the preferential binding of YEATS2 with H3K27cr and the role of GCDH in producing crotonyl-CoA was known(6,7), we hypothesized that higher H3K27cr in HNC could be a result of both YEATS2 and GCDH. We found that the presence of GCDH in the nucleus of HNC cells is correlated to higher H3K27cr abundance, which could be a result of excess levels of crotonyl-CoA produced via GCDH. We also found a correlation between H3K27cr levels and YEATS2 expression, which could arise due to YEATS2-mediated preferential maintenance of crotonylation. This states that although being a reader protein, YEATS2 is affecting the promoter H3K27cr levels, possibly by helping in the recruitment of p300 (as shown in Figure 5F). Thus, YEATS2 and GCDH are both responsible for the regulation of histone crotonylation-mediated gene expression in HNC.
We did not find any evidence of YEATS2 regulating the expression of GCDH in HNC cells. However, we found that YEATS2 downregulation reduced the nuclear pool of GCDH in head and neck cancer cells (Figure 7F). This suggests that YEATS2 not only regulates histone crotonylation by affecting promoter H3K27cr levels (with p300), but also by affecting the nuclear localization of crotonyl-CoA producing GCDH. Also, we observed that the expression of YEATS2 and GCDH are regulated by the same transcription factor SP1 in HNC. We found that the transcription factor SP1 binds to the promoter of both genes, and its downregulation led to a decrease in their expression (Figure 3 and Figure 7).
We would like to state that the relationship between YEATS2 and the nuclear localization of GCDH, as well as the underlying molecular mechanism, remains unexplored and presents an open question for future investigation.
(9) The authors should provide IHC analysis of YEATS2, SPARC alongside H3K27cr and GCDH staining in normal vs. tumor tissues from HNC patients.
We thank the reviewer for their suggestion. We are consulting our clinical collaborators to assess the feasibility of including this IHC analysis in our revision and will make every effort to incorporate it.
Reviewer #2 (Public review):
Summary:
The manuscript emphasises the increased invasive potential of histone reader YEATS2 in an SP1-dependent manner. They report that YEATS2 maintains high H3K27cr levels at the promoter of EMT-promoting gene SPARC. These findings assigned a novel functional implication of histone acylation, crotonylation.
We thank the reviewer for the constructive comments. We are committed to making beneficial changes to the manuscript in order to alleviate the reviewer’s concerns.
Concerns:
(1) The patient cohort is very small with just 10 patients. To establish a significant result the cohort size should be increased.
We thank the reviewer for this suggestion. We will increase the number of patient samples to assess the levels of YEATS2 and H3K27cr in normal vs. tumor samples.
(2) Figure 4D compares H3K27Cr levels in tumor and normal tissue samples. Figure 1G shows overexpression of YEATS2 in a tumor as compared to normal samples. The loading control is missing in both. Loading control is essential to eliminate any disparity in protein concentration that is loaded.
In Figures 1G and 4D, we have used Ponceau S staining as a control for equal loading. Ponceau S staining is frequently used as an alternative for housekeeping genes like GAPDH as a control for protein loading(8). It avoids the potential for variability in housekeeping gene expression. However, it may be less quantitative than using housekeeping proteins. To address the reviewer’s concern, we will probe with an antibody against a house keeping gene as a loading control in the revised figures, provided its expression remains stable across the conditions tested.
(3) Figure 4D only mentions 5 patient samples checked for the increased levels of crotonylation and hence forms the basis of their hypothesis (increased crotonylation in a tumor as compared to normal). The sample size should be more and patient details should be mentioned.
A total of 9 samples were checked for H3K27cr levels (5 of them are included in Figure 4D and rest included in Figure 4—figure supplement 1D). However, as a part of the revision, we will check the H3K27cr levels in more patient samples.
(4) YEATS2 maintains H3K27Cr levels at the SPARC promoter. The p300 is reported to be hyper-activated (hyperautoacetylated) in oral cancer. Probably, the activated p300 causes hyper-crotonylation, and other protein factors cause the functional translation of this modification. The authors need to clarify this with a suitable experiment.
In our study, we have shown that p300 is dependent on YEATS2 for its recruitment on the SPARC promoter. As a part of the revision, we propose the following experiments to further substantiate the role of p300 in YEATS2-mediated gene regulation:
(a) Co-immunoprecipitation experiments to check the physical interaction between YEATS2 and p300.
(b) We will check H3K27cr levels on the SPARC promoter and SPARC expression in p300-depleted HNC cells.
(c) Rescue experiments to check if the decrease in p300 occupancy on the SPARC promoter can be compensated by overexpressing YEATS2.
(d) Pol-II ChIP-qPCR at the promoter of SPARC will be performed in YEATS2-silenced cells to explain the mode of transcriptional regulation of SPARC expression by YEATS2.
(5) I do not entirely agree with using GAPDH as a control in the western blot experiment since GAPDH has been reported to be overexpressed in oral cancer.
We would like to clarify that GAPDH was not used as a loading control for protein expression comparisons between normal and tumor samples. GAPDH was used as a loading control only in experiments using head and neck cancer cell lines where shRNA-mediated knockdown or overexpression was employed. These manipulations specifically target the genes of interest and are not expected to alter GAPDH expression, making it a suitable loading control in these instances.
(6) The expression of EMT markers has been checked in shControl and shYEATS2 transfected cell lines (Figure 2A). However, their expression should first be checked directly in the patients' normal vs. tumor samples.
We thank the reviewer for the suggestion. To address this, we will check the expression of EMT markers alongside YEATS2 expression in normal vs. tumor samples.
(7) In Figure 3G, knockdown of SP1 led to the reduced expression of YEATS2 controlled gene Twist1. Ectopic expression of YEATS2 was able to rescue Twist1 partially. In order to establish that SP1 directly regulates YEATS2, SP1 should also be re-introduced upon the knockdown background along with YEATS2 for complete rescue of Twist1 expression.
To address the reviewer’s concern regarding the partial rescue of Twist1 in SP1 depleted-YEATS2 overexpressed cells, we will perform the experiment as suggested by the reviewer. In brief, we will overexpress both SP1 and YEATS2 in SP1-depleted cells and then assess the expression of Twist1.
(8) In Figure 7G, the expression of EMT genes should also be checked upon rescue of SPARC expression.
We thank the reviewer for the suggestion. We will check the expression of EMT markers on YEATS2/ GCDH rescue and update Figure 7G in the revised version of the manuscript.
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