SETD6-mediated methylation of PPARγ establishes a transcriptional feedback circuit promoting lipid accumulation in liver-derived cells

Reviewer #1 (Public review):

Summary:

In this manuscript from the Levy lab, the authors investigate whether SETD6 regulates hepatic lipid accumulation through direct methylation of PPARγ. They show that SETD6 binds and mono-methylates PPARγ at K170, and provide evidence that this modification enhances PPARγ occupancy at target promoters, promotes expression of lipid metabolism genes, as well as facilitates lipid droplet accumulation in HepG2 cells. The authors also find a positive feedback loop or circuit in which PPARγ activates SETD6 transcription in a methylation-dependent manner, thereby reinforcing this lipogenic program. Overall, the work presents a novel SETD6-PPARγ regulatory axis linking lysine methylation to transcriptional control of lipid storage genes, with possible relevance to NAFLD-associated biology.

In all, I find this to be an important paper that describes and advances a new regulatory pathway that has significance to human health and disease. It would also be of interest to a broad audience. That said, there are also some concerns that the authors should address, as outlined below.

Major concerns (pertains to rigor - highest priority)

(1) Overall, the work presented is of high quality, and the data nicely support the conclusions; however, a few panels should be strengthened that have missing controls or information:
a. The co-IP panel in Figure 1B lacks a lane where HA SETD6 is expressed without PPARγ. This control is needed to verify that the SEDT6-HA signal depends on PPARγ.
b. In Figure 1C, the authors should show that the co-IP works in both directions (include IP for PPARγ/blot for SETD6). I am a bit confused also over the labeling with IP on the left and on top of the panel next to the beads label. More importantly, the data would be stronger if the authors took advantage of a deletion line to validate that the co-IP is specific to the presence of both.
c. The same IP labeling issue exists for Figure 3B (label is on the same and on top).
d. Antibody information (e.g., where the pan-methyl Ab comes from and at what dilutions they are used at) is missing.

Nice to have experiments (medium priority - strongly consider)

(2) A missing gap is how K170me1 contributes to DNA binding and gene transcription. One possibility is that methylation enhances the DNA-binding activity of PPARγ. Given that the authors have all of the reagents, it would be possible to perform a gel shift assay (or other approach) with and without SETD6-mediated methylation. Is DNA binding affected/enhanced?

(3) Along these lines, I wonder if there is another possibility: could SETD6-mediated methylation of PPARγ drive SETD6-PPARγ interaction? In other words, in the K170R, is SETD6 still even associated with PPARγ, and this interaction is required for promoter recruitment? Alternatively, would a catalytic dead version of SETD6 fail to associate with PPARγ? Currently, no experiments test the impact of an unmethylatable version of PPARγ or a catalytic dead version of SETD6 on SETD6-PPARγ interaction or SETD6 recruitment to promoters.

Minor concerns (text and figure display)

(4) The text has multiple typos and grammatical errors, and there are some issues with the figure display.

Reviewer #2 (Public review):

Summary:

In this work, the authors investigated the regulation of the transcription factor PPARγ by the post-translational modification lysine methylation. The data demonstrate that the lysine methyltransferase SETD6 targets PPARγ for methylation using biochemical and cell-based assays. Methylation of PPARγ occurs in its DNA binding domain, and the authors demonstrate that loss of methylation limits PPARγ chromatin binding, particularly to lipid storage and metabolism gene promoters. As a physiological output, the authors demonstrate that deletion of SETD6 and loss of PPARγ methylation also disrupt lipid droplet accumulation in hepatocytes. In addition, the authors uncover a positive feedback loop in which SETD6 methylation of PPARγ also regulates its binding to the SETD6 promoter and expression of the gene.

Strengths:

One of the key strengths of this manuscript is the novelty of the findings in terms of identifying a new mode of regulation of PPARγ that modulates its chromatin association in cells and thereby regulates lipid metabolism genes. The authors nicely combine biochemical studies of SETD6 activity with cell-based assays investigating PPARγ and SETD6 function in regulating lipid storage. Data supporting this conclusion is largely convincing, and frequently, multiple assays are used to provide sufficient support to the conclusions. This work therefore expands regulatory modes of PPARγ and identifies a new target for SETD6, an enzyme that targets a number of other transcription factors. Furthermore, the regulatory loop that controls SETD6 expression via PPARγ methylation is likely important for understanding SETD6 function in different cell types that have high levels of lipid accumulation or regulation. The gene expression and lipid accumulation assays are useful for testing the physiological outcome of loss of SETD6 activity or PPARγ methylation directly.

Weaknesses:

The data presented in the manuscript are largely convincing in support of the authors' conclusions; however, there are some errors in the presentation of the figures and some issues in the text that would benefit from editing. Furthermore, there are some important questions not fully addressed in the results or discussion. It would be great if the authors could speculate more on the diverse roles of SETD6 in methylated transcription factors and/or provide more context regarding the conditions that are likely to support methylation of PPARγ by SETD6. Also, while a potential cross-talk between methylation and phosphorylation is described in the discussion, it would be great to provide more structural insight into how this might regulate DNA binding of PPARγ and/or discuss whether there are other possibilities given the location of the target lysine in the DNA binding domain.