ATF4 licenses C/EBPβ activity in human mesenchymal stem cells primed for adipogenesis

All the reviewers considered your studies to be well executed. However, a major concern is the biological relevance of the reported discovery, in particular the role of cell density or confluence as a “trigger” during adipogenesis in vivo. If the authors would like to make the argument that this mechanism relates specifically to adipogenesis their data are incomplete. ATF4 knockout mice have adipose tissue although they are leaner and resist diet induced obesity, but there is little evidence they have a defect in adipogenesis. It would appear from a number of studies that ATF4 plays a more important role in regulating energy metabolism than in directly affecting adipogenesis.

We appreciate the reviewers’ concerns. We have made the following modification to the second paragraph of the discussion to provide a reference point for readers: This study identifies ATF4 as a cell density sensor that links the physical and chemical requirements for adipogenesis. High cell density has long been known to be an important condition for adipocyte differentiation in vitro (Cristancho et al., 2011; Green and Kehinde, 1975; McBeath et al., 2004; Pittenger et al., 1999). It is likely that a combination of cues is required in tissue microenvironments supporting adipogenesis in vivo.

Regarding adipogenesis in ATF4 knockout mice, we agree and make the following modification to the Discussion’s third paragraph: Mice lacking ATF4 are lean and resist diet-induced obesity, although they have adipose tissue (Masuoka and Townes, 2002; Seo et al., 2009; Yang et al., 2004). Our demonstration of a role for ATF4 in adult stem cell adipocyte differentiation suggest that this regulatory mechanism could be especially relevant in adult adipose tissue, consistent with the recent demonstration that adipogenesis occurs via distinct cell lineages during development versus adulthood (Jiang et al., 2014).

In fact, the authors never directly assess adipogenesis but rather lipid accumulation, which can be affected by altering cellular metabolism. The authors should attempt to distinguish between the two: it would be relatively straightforward to examine the cells metabolically, for instance flux analysis, metabolomics, simple glycolysis assays. Without some attempt to distinguish between a differentiation phenotype and a cellular metabolic phenotype many of the conclusions about adipogenesis are not supported by the data. Furthermore, in addition to measuring oil droplet accumulation a qPCR survey of dynamic adipocyte differentiation marker expression should be conducted to elucidate the block in greater detail.

We thank the reviewers for raising this point; clarifying how ATF4 affects lipid accumulation in response to adipogenic signals improves the study. To directly address adipogenesis, we measured the expression of key differentiation markers in hMSCs with normal and reduced levels of ATF4. Transcriptional activation of PPARγ and C/EBPα, both encoding adipose-determining transcription factors, is significantly decreased in hMSCs depleted for ATF4 in response to adipogenic signals. Several downstream gene targets important for adipocyte differentiation also have reduced expression, suggesting that ATF4 affects lipid accumulation by promoting adipogenesis. These data have been added to Figure 4, and referred to in the subsection headed “Loss of ATF4 Attenuates Adipogenesis and Cell-Density-Dependent Transcription” as follows: In parallel, early markers of adipogenesis were decreased 3-4-fold in ATF4 knockdown cells (Figure 4C), including the mRNAs encoding the lineage-determining TFs PPARγ2 and C/EBPα, as well as several of their downstream transcriptional targets. This deficit in adipogenic gene expression became more pronounced over time (Figure 4–figure supplement 1), and together, these data suggest that ATF4 expression is important for adipogenesis.

A previous study by Yu et al. (cited in the manuscript) has suggested that ATF4 controls expression of C/EBPβ and PPARγ through promoter binding in a 3T3 model. The current manuscript suggests a model where ATF4 binding at enhancer elements is critical. Can the authors distinguish these two models experimentally, e.g. through rescue of adipogenesis in ATF knockdown cells through C/EBPβ overexpression?

Our data do not indicate that ATF4 directly regulates C/EBPβ or PPARγ expression. Both mRNA and protein for C/EBPβ appear unchanged in hMSCs depleted of ATF4 (Author response image 1). It should be noted that Yu et al. report ATF4 binding by ChIP to cyclic AMP response elements (CREs) in the Cebpβ (-152 to 0) and Pparγ (-360 to -58) promoters as evidence for direct regulation of these genes, yet ATF4 displays little or no interaction with the CRE motif in unbiased genome-wide studies from mouse embryonic fibroblasts (Nature Cell Biology 2013. doi:10.1038/ncb2738) and hMSCs (this work).

Author response image 1

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Furthermore, we do not observe binding at either region in hMSCs (Author response image 2). A single ATF4 peak resides at the PPARγ locus, 40 kb downstream of the PPARγ2 TSS, yet it lacks enrichment for either RNAPII or H3K27ac in high-density-seeded cells, suggesting little or no enhancer activity under these conditions

Author response image 2

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We appreciate the suggestion to distinguish between conflicting models. However, rather than restoring function, forced expression of C/EBPβ under ATF4 knockdown conditions will yield abnormally high C/EBPβ levels, limiting the interpretation of any potential effects on adipogenesis. Forced C/EBPβ expression has been shown by the Spiegelman and Farmer labs to trigger adipogenesis in NIH-3T3 cells, likely by overwhelming and/or overriding the system and obviating the need for pre-adipocyte-specific transcription. Thus, we do not believe that this experiment can resolve how ATF4 contributes to adipogenic commitment.

The authors describe 1451 ATF4 ChIP-seq peaks (Figure 3) that are found in HD MSC cells. Presumably, the peaks fall mainly within cluster 10-11 from Figure 1A since there is an enrichment of the hybrid motif, but it is hard to follow if the ATF4 binding events also exhibit a certain GO enrichment which could clarify the biological context ATF4 binding (or absence thereof) might have. The manuscript might greatly benefit from a clearer analysis and display of ATF4-binding events/hybrid motif usage.

We thank the reviewers for these suggestions, as they have led to an improved study. GO results derived from mapping ATF4-binding sites to nearby genes are now included in Figure 3–figure supplement 1B. They are similar to the GO results obtained from the HD-primed RNAPII sites. To provide a clearer display of the relationship between ATF4 and RNAPII genomic occupancy, we determined the fraction of ATF4 or C/EBPβ sites that co-localize with RNAPII, and present the results as pie charts in Figure 3–figure supplement 1C. Description of these data has been added the following to the second paragraph of the subsection headed “ATF4 Is a Density-Dependent Factor that Targets HD-primed Enhancers as a Heterodimer with C/EBPβ”: “Genomic occupancy of ATF4 is associated predominantly with genes […] in response to high-cell-density seeding of hMSCs.”

We have also expanded the third paragraph of the Discussion based on these results as follows: “It is of interest to note that ATF4 plays a central role […] demand for protein synthesis during adipogenesis.”