Carm1-arginine methylation of the transcription factor C/EBPα regulates transdifferentiation velocity
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eLife assessment
This important manuscript describes how methylation of a single arginine residue in a transcription factor, C/EBPα, can alter dynamics of cell fate transition. The study provides one of the most striking examples of the transcription factor regulation by methylation and is well-executed, with compelling evidence to support the authors' claims.
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Abstract
Here, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPα R35A ) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPα R35A , initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme’s perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell’s differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.
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Author Response
Reviewer #1 (Public Review):
This paper establishes a strong case for the post-translational modification of C/EBPalpha to play a strong role in its effects, in this case, to promote macrophage differentiation in collaboration with PU.1. The cellular system being used for most of the experiments here takes advantage of the dual roles of PU.1 in B cells, which normally do not express C/EBP family factors, and in myeloid cells, which normally do express C/EBP family factors. The authors and others have previously shown that PU.1 and C/EBPalpha are very powerful collaborators, both needed to establish a macrophage identity. Thus, the title of the paper provocatively implies that the C/EBP modification that keeps it from being methylated on Arg35 works by increasing the re-distribution of PU.1 from B cells to myeloid gene …
Author Response
Reviewer #1 (Public Review):
This paper establishes a strong case for the post-translational modification of C/EBPalpha to play a strong role in its effects, in this case, to promote macrophage differentiation in collaboration with PU.1. The cellular system being used for most of the experiments here takes advantage of the dual roles of PU.1 in B cells, which normally do not express C/EBP family factors, and in myeloid cells, which normally do express C/EBP family factors. The authors and others have previously shown that PU.1 and C/EBPalpha are very powerful collaborators, both needed to establish a macrophage identity. Thus, the title of the paper provocatively implies that the C/EBP modification that keeps it from being methylated on Arg35 works by increasing the re-distribution of PU.1 from B cells to myeloid gene sites in combination with C/EBP. Indeed, the authors show proximity ligation data to show that PU.1-C/EBPalpha juxtaposition is more frequent in the nucleus if C/EBPalpha cannot be Arg-methylated. The paper also shows careful and thorough characterization of the B to myeloid lineage conversion gene expression changes and the mapping of the Arg residues in C/EBPalpha that are most important to keep demethylation. Similarly, the paper provides strong evidence that it is Carm1, and not another protein arginine methyltransferase, that is responsible for the regulatory modification. This is a valuable and well-characterized demonstration of a mechanism that should be considered more generally as a regulator of transcription factor action.
The mechanism proposed by the authors is that C/EBPalpha relocates PU.1 to macrophage sites and that C/EBPalpha R35A binds and relocates PU.1 more efficiently than wildtype, and this seems likely and appealing. However, it is not as strongly supported by data within the paper itself as the other points in the paper are. There is a puzzling gap in the data: no direct evidence is shown that C/EBPalpha is really relocating PU.1 from B cell to macrophage regulatory elements at all. Despite the figure titles (Fig. 4 and Fig. S4), there is no ChIP-seq data to show PU.1 binding sites before and after interaction with either wildtype or R35A mutant C/EBPalpha, just accessibility data. There is also a question of whether such a redistribution would occur fast enough to account for the impressive speed of the R35A mutant's other effects. These questions seem fairly straightforward to address. If relevant data could be added, it would greatly increase the impact and generality of the paper. The paper could be published with this claim converted to a suggestion, based on the current data, or it could be published in a higher-impact form if additional data could be provided to demonstrate the relocation more directly. The authors would be more expert about the logistics of the experiment, but it seems that a direct ChIP-seq-based comparison should be feasible and powerful for the argument of the paper.
We have now included PU.1 and C/EBPa ChIP-seq experiments, using C/EBPaWT and C/EBPaR35A- induced cells, replacing the virtual ChIP-seq experiments. Integrating the data obtained with our dynamic ATACseq data, the new findings largely support the previously proposed PU.1 redistribution (‘theft’) model. To make the data easier to understand, we now first show the PU.1 and C/EBPa binding to distinct B cell- and macrophage- restricted GREs contained in a single genomic fragment (new Fig. 5). The findings nicely visualize how PU.1 becomes redistributed from B-GREs to M-GREs, in a C/EBPa mutant-accelerated manner. We were also happy to see that a genome-wide analysis of the data again shows the accelerated redistribution of PU.1 by C/EBPaR35A (new Fig. 6). Finally, the comparison of the ChIP-seq and ATAC-seq data also added more mechanistic detail, such as by revealing that chromatin remodeling of lineage restricted GREs can be uncoupled from the regulation of associated genes.
Finally, the effect of the mutation is assumed to be only on the interface for interaction between C/EBPalpha and PU.1 (or other co-factors). However, C/EBPalpha is such a short-lived protein that any modification that slightly increased its half-life could increase its potency. It seems important to present some quantitative protein staining evidence to clarify whether the steady-state level of C/EBPalpha in C/EBPalpha R35A-expressing cells is really unchanged from C/EBPalpha wild-type-expressing cells.
We agree that this is an important issue and have therefore now performed a cycloheximide experiment with 3T3 cells expressing inducible forms of the two proteins. The data in Figure S4C show that C/EBPaR35A exhibits a similar stability than wild type protein and is expressed at 20-30% lower levels under steady-state conditions in uninduced cells. They also show that C/EBPa is surprisingly stable. These new findings are in line with the comparison of the two proteins by Western blots of mutant and wild type transfected 293T cells and of infected B cells, which also show similar levels of the two proteins (Fig. 7C and D). Therefore, the finding that expression of C/EBPaR35A is similar or slightly lower than that of the wild type argues against the possibility that an elevated expression level of the mutant could explain the effects observed.
Finally, although not requested by the reviewer, we have now addressed the possibility that that the effect of the alanine replacement of R35 is mostly due to a change from a charged to a non-charged hydrophobic residue. This is not the case, as a replacement of arginine 35 by the charged amino acid lysine still leads to an accelerated BMT induction (Figure S7).
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eLife assessment
This important manuscript describes how methylation of a single arginine residue in a transcription factor, C/EBPα, can alter dynamics of cell fate transition. The study provides one of the most striking examples of the transcription factor regulation by methylation and is well-executed, with compelling evidence to support the authors' claims.
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Reviewer #1 (Public Review):
This paper establishes a strong case for the post-translational modification of C/EBPalpha to play a strong role in its effects, in this case, to promote macrophage differentiation in collaboration with PU.1. The cellular system being used for most of the experiments here takes advantage of the dual roles of PU.1 in B cells, which normally do not express C/EBP family factors, and in myeloid cells, which normally do express C/EBP family factors. The authors and others have previously shown that PU.1 and C/EBPalpha are very powerful collaborators, both needed to establish a macrophage identity. Thus, the title of the paper provocatively implies that the C/EBP modification that keeps it from being methylated on Arg35 works by increasing the re-distribution of PU.1 from B cells to myeloid gene sites in …
Reviewer #1 (Public Review):
This paper establishes a strong case for the post-translational modification of C/EBPalpha to play a strong role in its effects, in this case, to promote macrophage differentiation in collaboration with PU.1. The cellular system being used for most of the experiments here takes advantage of the dual roles of PU.1 in B cells, which normally do not express C/EBP family factors, and in myeloid cells, which normally do express C/EBP family factors. The authors and others have previously shown that PU.1 and C/EBPalpha are very powerful collaborators, both needed to establish a macrophage identity. Thus, the title of the paper provocatively implies that the C/EBP modification that keeps it from being methylated on Arg35 works by increasing the re-distribution of PU.1 from B cells to myeloid gene sites in combination with C/EBP. Indeed, the authors show proximity ligation data to show that PU.1-C/EBPalpha juxtaposition is more frequent in the nucleus if C/EBPalpha cannot be Arg-methylated. The paper also shows careful and thorough characterization of the B to myeloid lineage conversion gene expression changes and the mapping of the Arg residues in C/EBPalpha that are most important to keep demethylation. Similarly, the paper provides strong evidence that it is Carm1, and not another protein arginine methyltransferase, that is responsible for the regulatory modification. This is a valuable and well-characterized demonstration of a mechanism that should be considered more generally as a regulator of transcription factor action.
Some weaknesses:
1. The mechanism proposed by the authors is that C/EBPalpha relocates PU.1 to macrophage sites and that C/EBPalpha R35A binds and relocates PU.1 more efficiently than wildtype, and this seems likely and appealing. However, it is not as strongly supported by data within the paper itself as the other points in the paper are. There is a puzzling gap in the data: no direct evidence is shown that C/EBPalpha is really relocating PU.1 from B cell to macrophage regulatory elements at all. Despite the figure titles (Fig. 4 and Fig. S4), there is no ChIP-seq data to show PU.1 binding sites before and after interaction with either wildtype or R35A mutant C/EBPalpha, just accessibility data. There is also a question of whether such a redistribution would occur fast enough to account for the impressive speed of the R35A mutant's other effects. These questions seem fairly straightforward to address. If relevant data could be added, it would greatly increase the impact and generality of the paper.
2. Also, there is evidence presented that the mutant C/EBPalpha still binds PU.1 at least as well as wildtype in co-immune precipitation and that the bands co-immune precipitated by the mutant may be about twofold stronger. However, this important interaction experiment is not done under quantitative titration conditions that would give confidence about the magnitude of the differences seen.
3. Finally, the effect of the mutation is assumed to be only on the interface for interaction between C/EBPalpha and PU.1 (or other co-factors). However, C/EBPalpha is such a short-lived protein that any modification that slightly increased its half-life could increase its potency. It seems important to present some quantitative protein staining evidence to clarify whether the steady-state level of C/EBPalpha in C/EBPalpha R35A-expressing cells is really unchanged from C/EBPalpha wild-type-expressing cells.
In summary, the authors have demonstrated an exciting and precise mechanism for modulating the effects of C/EBPalpha, but more direct evidence would be needed before concluding that this mechanism operates primarily by exposing a stronger interaction interface to speed up the relocation of PU.1 from B cell sites to macrophage sites.
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Reviewer #2 (Public Review):
The manuscript by Torcal Garcia et al. shows that the mutation of a single arginine residue in a transcription factor, C/EBPα is able to accelerate the kinetics of B-cell to macrophage transdifferentiation without impacting the characteristics of the fully reprogrammed cell state. The authors delve into the mechanism underlying this phenotype and demonstrate that R35A mutation increases the affinity of C/EBPα for another transcription factor PU.1, and accelerates chromatin accessibility changes that accompany the transition from B-cell to macrophage program. The authors subsequently demonstrate that R35 is a methylation site for the arginine methyltransferase Carm1. Through Carm1 gain- and loss-of-function experiments, authors recapitulate R35me2/R35A effects on transdifferentiation. Overall, this is an …
Reviewer #2 (Public Review):
The manuscript by Torcal Garcia et al. shows that the mutation of a single arginine residue in a transcription factor, C/EBPα is able to accelerate the kinetics of B-cell to macrophage transdifferentiation without impacting the characteristics of the fully reprogrammed cell state. The authors delve into the mechanism underlying this phenotype and demonstrate that R35A mutation increases the affinity of C/EBPα for another transcription factor PU.1, and accelerates chromatin accessibility changes that accompany the transition from B-cell to macrophage program. The authors subsequently demonstrate that R35 is a methylation site for the arginine methyltransferase Carm1. Through Carm1 gain- and loss-of-function experiments, authors recapitulate R35me2/R35A effects on transdifferentiation. Overall, this is an interesting and well-executed study that provides one of the most striking examples of transcription factor regulation by methylation and documents the profound impact it can have on the kinetics of cell fate transition. Data are of high quality, experiments are rigorous, and deeply probe into the mechanism. Overall, the study sheds light on an under-appreciated level of transcription factor regulation.
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