CARM1 methylates MED12 to regulate its RNA-binding ability

CARM1 methylates MED12 at arginine 1899 to generate a TDRD3 binding site, which in turn regulates the ability of mediator to interact with activating ncRNAs and modulate gene expression.

The reviewers provide constructive input and outline where further clarifications are needed (reviewers 1-3). A control to show loss of ncRNA binding for the R1899K mutant should be included, too (reviewer #1). The further reaching insight on the effects on the whole Mediator complex (reviewer #3) does not need to get experimentally provided for acceptance here.
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Thank you for this interesting contribution to Life Science Alliance. We are looking forward to receiving your revised manuscript.  This manuscript reports the development of tools to identify the substrates of the arginine methylase CARM1 and to apply these tools to characterize one of them (MED12). In the end, it is a very interesting story of the methylation dependence of the recruitment of a non-coding RNA for gene activation. Although many CARM1 substrates had been described before, this more comprehensive approach led to entirely new insights regarding the impact of the methylation of one of its substrates. Overall, this manuscript is a no-brainer, but it still requires some clarifications and at least one experiment.
(2) Results, 1st paragraph, last sentence: "The four polyclonal antibodies..." -> since a cocktail of peptides was used, it isn't clear what "four" refers to. Indeed, which one was used for the IP-MS experiments does not seem to be indicated anywhere. Assuming it was all four, this should be explicitly stated.
(3) Fig. 4A: with the labels as shown (and a cryptic legend), this experiment makes no sense at all. (4) Fig. 4D: there is a strange disconnect between what's claimed in the text and what is actually shown in this figure. There is no evidence whatsoever that R1912 is critical. It wasn't tested by itself and the triple mutant is barely different from the single mutant R1899K. Reviewer #2 (Comments to the Authors (Required)): Growing evidence suggests the biological importance of the regulatory role of of arginine side chain methylation. A major regulatory arginine-methyltransferase with broad functionality in signal transfer and chromatin regulation is CARM1, yet knowledge about the range of CARM1 targets is scarce. Because of the diversity in substrate specificity and lack of consensus CARM1 methylation target sites the authors used a mixed methylated peptide immunization strategy to raise antibodies against various known CARM1 methylation sites on several cellular proteins, speculating that these antibodies may also display extended immunoreactivity with other targets because of related but hidden structural similarities/constraints. Affinity purified antibodies obtained from 4 rabbits were then compared for substrate recognition and specificity in WT/KO cells of CARM1 and PRMT1 as a control. Results indicated that 4 antibody preparation may contain novel CARM1 targets, in addition to targets methylated by other PRMTs. IP-MS identified 112 novel CARM1 target proteins, 10 were selected and confirmed as antigens specifically recognized by at least one of the anti-CARM1 methylation specific substrate antibodies, using WT and conditional CARM1 knockdown cells and IP-protein blotting. The authors then focused on the MED12 protein and a CARM1 R-methylation site at position R1899. IP-blotting experiments using WT/mutant MED12 confirmed methylation at R1899 by CARM1 and probably transient interaction with MED12, but no effect on integration of MED12 methylation into the MED complex, suggesting that R1899 is a regulatory site. The authors went on to determine whether known Rme-binding Tudor proteins may interact differentially with a MED12 R1899+/-methylation peptide and identified TDRD3 as a MED12 interacting protein affected by R1899 methylation. Peptide binding and IP of TDRD3 from WT but not from CARM1 knockdown cells co-IPed MED12 and out of three Rme-sites located in the vicinity of R1899 (R1862, R1912), two (R1899, 1892) appear to be critical for MED12-TDRD3 interaction. Genome wide analysis of MED12 and CARM1 is complicated by the cross-reactivity of the available antibodies. Careful comparison of results obtained using different antibody sources nevertheless convincingly revealed canonical ERalpha enhancer sites and G-rich sites as important CARM1 MED12 targets. Four of the ERalpha target genes were further explored and found repressed in CARM1 KO MCF7 but not in WT MCF7 cells. Further, a MED12 MCF7 KO was generated and expression of a ER-target gene panel could be activated with WT but not with R1862/1899/1912 K mutant MED12 constructs. Finally, the MED complex interaction with lnc/ncRNAs was examined by IP/RT-qPCR and members of ncRNA that interact with WT but less with R1899 modification dependent pull-down were identified. Knockdown of one selected ncRNA that maps to the vicinity of the ER target gene GREB1 was shown to suppress GREB1 expression.
The authors provide several sets of convincing data that extend the number of known CARM1 targets. The authors describe in depth a novel connection between CARM1 mediated methylation of MED12 and the regulation of ER target genes in connection to CARM1-MED12 methylation, TDRD3 recruitment and functional connection to the regulatory role of ncRNAs in conjunction with MED12. In addition to an earlier publication where the Bedford lab has revealed cross reactivity between FLAG-antibodies with PRMT5 as a source of misinterpretation of experimental data by other labs they now also show that seemingly H3R17me2a specific antibodies react with a selection of CARM1 target proteins, which may lead to misinterpretation of ChIP data. Although this is just an additional observation, it reflects the careful conduction and interpretation of experimental data. The authors present an important study and I do see only a few shortcomings in the manuscript:

Major:
The mechanistic evidence for the functional CARM1 depending TDRD3-MED12 interaction via MED12 R1899 methylation remains indirect and is currently not entirely convincing, as Figure 4 D still shows TDDR3 binding with the MED12 methylation site mutants (second last and last lanes; R to K mutants # 1899, 1912). Is this due to a technical problem ? Why did the authors not attempt to examine leucine or phenylalanine (up-) mutants of MED12 for compensation of CARM1 deficiency / upregulation of enhancer ncRNAs / ER target gene expression ? Why did they not include / compare genome wide analysis of TDRD3 binding in MED12 and/CARM1 deficient cells ? Although these experimental setups might be difficult to achieve the authors should at least discuss why they omitted such experiments and state that final prove of proposed/implied mechanisms remains an open issue that still needs to be addressed, just in case they can not solve this issue experimentally.
Minor: 1. analysis of CARM1, MED12 ...." the cell type (MCF7) should be mentioned in the text (not only in the Figure Legend). 2. In addition to ER binding sites, SP1 (in the results section and discussion) and C/EBP1 binding sites (discussion only) are mentioned. The SP1 consensus is shown in S3, but information about C/EBP is missing and should be included.

Reviewer #3 (Comments to the Authors (Required)):
Mark Bedford and his colleagues in the manuscript titled "CARM1 methylates MED12 to regulate its RNA binding ability" provides detailed further characterization on the MED12 methylation and its impact on transcription. The biochemical analysis were comprehensive and resourceful, the findings are truly interesting and significant. This reviewer has only a few questions here.
1. MED12 is a subunit of the Mediator complex which consists of 30 subunits or so in mammalian cells, however, MED12 seems to be treated as a single protein in this study. It is not clear whether methylation on MED12 affects functions of MED12 alone or the whole complex throughout the whole manuscript. 2. Since MED12 is a subunit within the CDK8 kinase submodule, does MED12 methylation affects the CDK8 kinase submodule structure and functions, especially the kinase activity? When MED12 was knockout, what happened to the rest of Mediator complex? 3. It is an important finding that MED12/ncRNA interaction is dependent on MED12 methylation by CARM1. A previous finding (Lai et al. 2013) seemed to demonstrate that disease-related mutations within the MED12 subunit disrupts the interaction between MED12/ncRNA. One wonders if there is any connection between these findings. 4. Figure 4A panel seems to have mistake in labeling.

Point-by-point rebuttal
We would like to thank the reviewers for there constructive criticism. As a result of the reviewer's suggestions and requests the manuscript is substantially improved.

Reviewer #1:
Overall, this manuscript is a no-brainer, but it still requires some clarifications and at least one experiment.
To clarify, we have changed "its activity" to "the methyl mark it deposits.
(2) Results, 1st paragraph, last sentence: "The four polyclonal antibodies..." -> since a cocktail of peptides was used, it isn't clear what "four" refers to. Indeed, which one was used for the IP-MS experiments does not seem to be indicated anywhere. Assuming it was all four, this should be explicitly stated.
To remove any confusion, we added "using a cocktail of the four ADMA CARM1 antibodies" on page 7 in the first sentence of the second paragraph, which talks about the IP-MS experiment.
(3) Fig. 4A: with the labels as shown (and a cryptic legend), this experiment makes no sense at all. This is indeed a mistake. Our (+) and (-) signs were scrambled at some point during the figure assembly. It is now clear that the unmethylated peptide can act as a substrate for recombinant CARM1, but the methylated peptide cannot because the methyl acceptor site is already occupied.
(4) Fig. 4D: there is a strange disconnect between what's claimed in the text and what is actually shown in this figure. There is no evidence whatsoever that R1912 is critical. It wasn't tested by itself and the triple mutant is barely different from the single mutant R1899K.
We agree with the reviewer on this point and have changed to wording as follows: "These co-immunoprecipitation experiments revealed that the R1862 and R1912 are not critical for this interaction, but that the R1899 is important." (5) Description of Fig. 5A: the text says "....CARM1, MED12, and CARM1....". The second CARM1 should probably be H3R17me2a.
(6) Fig. 7A/B: the experiments on the left side of these two graphs seem to be the same (of the same type) and yet, the values and relative binding are remarkably different.
We thank the review for his/her keen eye. There are two "issues" with Figure  7A/B. First, we had listed MCF-7 as the control line in the A and B panels. This is not totally correct, because they actually represent stable selected MCF-7 lines that harbor the Doc-inducible knockdown vectors for CARM1 (A) and TDRD3 (B). They are thus different lines that were both derived from MCF7 cells, and we have re-labeled the figure to highlight this difference. This may also explain why there are slight differences between the relative binding patterns in the controls for panel A and B.
Second, after viewing our data, we found that we had used different pair of primers for ncRNA-a7 in panel A and B. The Nature manuscript from the Shiekhattar lab that describes ncRNA-a7 used two different primer sets to detect this RNA by RT-qPCR. The bars for ncRNA-a7 in Fig. 7A came from data using primer1, and the bars of ncRNA-a7 in Fig.7B and Fig.S6A Moreover, the legend says nothing about the number of replicates or the statistical analyses.
To address this issue we have now added the following sentence in the Figure  7A/B legend "Error bars represent standard deviation based on replicates (n=3)". We have also added a new "Statistical Analysis" section to the "Materials and Methods" to address the statistical analysis that was used for all the qPCR experiments.
(7) Fig. 7: a final experiment, which is missing, is to show that the R1899K mutant fails to bind the ncRNA. This is indeed a critical experiment. We had actually performed this experiment, but it was presented in the Supplementary Figure section  (8) ChIP-seq experiments: I could not find any indication about negative controls and replicates. This information must be included.
For the ChIP-seq experiments the negative controls are input DNA. This information is now provided in the "Peak Calling and Gene Annotation" section in the "Materials and Methods". There are no replicates.

Reviewer #2:
The authors present an important study and I do see only a few shortcomings in the manuscript:

Major:
The mechanistic evidence for the functional CARM1 depending TDRD3-MED12 interaction via MED12 R1899 methylation remains indirect and is currently not entirely convincing, as Figure 4 D still shows TDDR3 binding with the MED12 methylation site mutants (second last and last lanes; R to K mutants # 1899, 1912). Is this due to a technical problem?
We agree that there is still a weak interaction between MED12 and TDRD3 even in the triple mutant. We think that MED12 may have additional CARM1 methylation sites that could interact weakly with TDRD3. What is clear is that the MED12 R1899me2a site is the primary binding site for TDRD3.
Why did the authors not attempt to examine leucine or phenylalanine (up-) mutants of MED12 for compensation of CARM1 deficiency / upregulation of enhancer ncRNAs / ER target gene expression?
The notion of whether or not the phenylalanine mutation accurately mimics arginine methylation is the subject of debate. Phenylalanine only mimicked the hydrophobic characteristic of methylated arginine by not positive charge. Only a few published papers have used this mimic replacement approach, and none have presented evidence that this replacement will facilitate an interaction with the aromatic cage of a Tudor domain.
Why did they not include / compare genome wide analysis of TDRD3 binding in MED12 and/CARM1 deficient cells? Although these experimental setups might be difficult to achieve the authors should at least discuss why they omitted such experiments and state that final prove of proposed/implied mechanisms remains an open issue that still needs to be addressed, just in case they can not solve this issue experimentally. This is an important suggestion, and we are currently planning to perform TDRD3 ChIP-seq analysis in MED12 and CARM1 deficient cells as the fellow-up story.