Tead1 reciprocally regulates adult β-cell proliferation and function to maintain glucose homeostasis

Reviewer #1 (Public Review):

Summary:

Hippo pathway activity is required for pancreas morphogenesis, but its role in endocrine pancreas function remains elusive. The author aims to study the function of the TEAD1 gene in b-cells.

Strengths:

The authors generated TEAD1 conditional knockout animals by crossing the TEAD1f/f mice with three Cre strains (RIP-Cre, Ins1-Cre, and MIP-CreERT). In all of them, the KO animals showed progressive loss of insulin secretion with normal beta cell mass. Further characterization of the animals indicated glucose-induced insulin secretion defect and increased beta cell proliferation rate. RNA-Seq and ChIP-Seq experiments identified Pdx1, MafA, and Glut2, etc. as direct targets of TEAD1, which might be responsible for the insulin secretion defect in the animals. Of interest, the authors also uncovered the cell cycle-related gene p16 as a direct target of TEAD1. Reduction of p16 is likely to drive the beta cell proliferation in the TEAD1 knockout model. Thus, they proposed that TEAD1 is a regulator of the proliferative quiescence process in beta cells. Overall, the evidence provided by the authors is highly relevant and supports their conclusion.

Weaknesses:

(1) The authors don't explicitly mention that some results appeared in a previous publication (https://doi.org/10.1093/nar/gkac1063) from them.

(2) The authors begin their story by introducing TEAD1 as part of the Hippo pathway. They showed Taz expression data in Figure 1. Did they do any experiments to detect Taz in their TEAD1 model? Did the authors detect any expression changes in CTGF following TEAD1 knockout? I could not see this changed. The phenotype characterization data presented here contrasts with what has been shown in TAZ b-cell knockout mice (https://doi.org/10.1101/2022.05.31.494216). Based on the data presented here, Hippo is not involved, which should at least be discussed in length.

(3) Figure 1B - TAZ staining looks different in the three-month age group.

(4) TEAD ChIP-seq data doesn't look very convincing to me. It's hard to tell whether those highlighted regions in Figures 3A and 5J were signals or background noise. Although the authors also performed ChIP-qPCR in MIN6, it's unclear whether these binding events occur in vivo. The analysis of ChIP-seq dataset is limited as well. How many peaks called? What proportion of differentially expressed genes are bound by TEAD1? Was TEAD1 also detectable at NGN3 and NEUROD1 gene regions? If acquiring enough cells is not possible, the authors could try CUT&RUN or CUT&Tag to improve the data quality.

(5) The authors should perform RNA-seq or gene expression studies in MIP-CreERT to confirm, which could help narrow down the actual targets of TEAD1 as well.

(6) Figure 6 - the experiment lacks a control: Ezh2 beta cell KO. In addition to p16, Ezh2, and PRC2 have other targets in beta-cells, the authors could not rule out the contribution of those to the phenotype, so the implication of this experiment is vague.

Reviewer #2 (Public Review):

In this manuscript, Lee et al. assessed the role of Tead1 in mouse beta cells using three Cre-driver lines: Rip-Cre, Ins-Cre, and Mip-CreERT. The authors demonstrate that loss of TEAD1 during development and in mature beta cells leads to increased cell-autonomous beta cell proliferation and reduced insulin secretion. The phenotype of Tead1 knockout is not surprising, given that it is a key player in the Hippo pathway - a well-characterized pathway controlling cell proliferation. However, as the authors suggested, the phenotype observed in Tead1 might be through other non-Hippo pathway factors as well. The authors further convincingly established PDX1 and p16 as the target of Tead1 in controlling beta cell function and proliferation correspondingly. I have the following specific comments:

(1) As the authors mentioned, there are concerns over the usage of some Cre transgenic lines. Another useful control would be the naive Cre line that is not bred to floxed mutant, in addition to the floxed mice used by the authors in the manuscript here.

(2) The logic to rely on the deletion of Ezh2 to restore p16 in the Tead1 knockout mice is unclear. Ezh2 has so many more targets than p16. Why not a direct rescue experiment by overexpression of p16?

(3) The observed correlation of PDX1 and TEAD1 in expression in human islets is intriguing. But does this correlation translate to beta cell proliferation and function? Does TEAD1 knockout in human islets elicit a similar proliferation versus function response?

(4) The argument of Tead1 only controls maturation but not differentiation and that maturation function versus proliferation phenotype is independently controlled is weak. It appears that this conclusion is only based on that "many disallowed genes...were not altered in Tead1-deficient islets". Perhaps the authors can perform a formal comparison between the transcriptomic changes of Tead1 knockout and Myc overexpressing/Notch gain of function beta cells and show that these two processes are different. In addition, what are the signatures of genes that are upregulated in Tead1 knockout compared with controls?