Mediator Subunit MED16 Collaborates with UBP1-TFCP2 to Control Transcriptional Activation or Repression via Promoter Positional Specificity

Reviewer #1 (Public Review):

Summary:

Characterization of a dissociable Mediator subunit implicated in cellular pathways, particularly lung alveolar function, and HIV latency is conceptually interesting.

Strengths:

The strengths of this study are:

(1) Demonstration of MED16 dissociation from the core Mediator complex and formation of a subcomplex containing MED16, upstream-binding protein 1 (UBP1), and transcription factor cellular promoter 2 (TFCP2) by elegant biochemical fractionation and immunoblotting analysis.

(2) Defining nine N-terminal WD-40 repeats (WDRs) of MED16 as a Mediator-incorporating module and the C-terminal ⍺β-domain (157 amino acids) important for interaction with the UBP1-TFCP2 heterodimeric complex.

(3) Illustration of a weak hydrophobic interaction between MED16 and the Mediator core that could be disrupted by 1,6-hexanediol, but not by its 2,5-hexanediol isomer nor by high salt (500 mM NaCl) disruption.

(4) Classification of UBP1-upregulated cellular genes typically containing binding sites flanking the transcription start site (TSS) in contrast to UBP1-downregulated genes often containing a TSS-overlapping UBP1-binding site

(5) Presenting evidence for Mediator complex-dissociated free MED16-repressed HIV promoter activity through functional association with UBP1 and showing bromodomain-containing protein 4 (BRD4) inhibitor JQ1 that potentially disrupts BRD4-inhibited HIV-1 transcription elongation could lead to reversal of HIV-1 latency.

Weaknesses:

Nevertheless, foreseeable weaknesses include:

(1) No clear demonstration of MED16-UBP1-TFCP2 indeed forming a trimeric core subcomplex in regulating cellular gene transcription and HIV-1 promoter inhibition

(2) No validation of transcriptomic datasets and pathways identified.

(3) Use of mostly artificial reporter gene constructs and non-HIV host cells (e.g., human 293T embryonic kidney cells, human HeLa cervical cancer cells, and mouse HT pancreatic cancer cells) for examining MED16/UBP1-regulated HIV transcription.

(4) Inconsistent use of 293T and HeLa cells in the characterization of dissociated MED16 interaction with UBP1 and TFCP2.

(5) In vitro transcription using immobilized DNA templates was not performed to a high standard, thus failing to convincingly show MED16/UBP1-inhibited HIV-1 transcription preinitiation complex formation.

Reviewer #2 (Public Review):

Summary:

The article from Zheng et al. proposes an interesting hypothesis that the Med16 subunit of Mediator detaches from the complex, associates with transcription factor UBP1, and this complex activates or represses specific sets of genes in human cells. Despite my excitement upon reading the abstract, I was concerned by the lack of rigor in the experimental design. The only statement in the abstract that has some experimental support is the finding that Med16 dissociates from the Mediator and forms a subcomplex, but the data shown remain incomplete.

Strengths:

The authors have preliminary evidence that a stable Med16 complex may exist and that it may regulate specific sets of genes.

Weaknesses:

The experiments are poorly designed and can only infer possible roles for Med16 or UBP1 at this point. Furthermore, the data are often of poor quality and lack replication and quantitation. In other cases, key data such as MS results aren't even shown. Instead, we are given a curated list of only about 6 proteins (Figure S1), a subset of which the authors chose to pursue with follow-up experiments. This is not the expected level of scientific process.

(1) The data supporting the Med16 dissociation and co-association with UBP1 are incomplete and not convincing at this stage. According to the Methods and text, the gel filtration column was run with "un-dialyzed HeLa cell nuclear extract" and eluted in 300mM KCl buffer. The extracts were generated with the Dignam/Roeder method according to the text. Undialyzed, that means the extract would be between 0.4 - 0.5M NaCl. Under these high salt conditions (not physiological), it's possible and even plausible that Mediator subunits could separate over time. This caveat is not mentioned or controlled for by the authors. Because a putative Med16 subcomplex is a foundational point of the article, this is concerning.

The data are incomplete because a potential Med16 complex is not defined biochemically. The current state suggests a smaller Med16-containing complex that may also contain UBP1 and other factors, but its composition is not determined. This is important because if you're going to conclude a new and biologically relevant Med16 complex, which is a point of the article, then readers will expect you to do that.

Equally concerning are the IP-western results shown in Figure 1. In my opinion, these experiments do nothing to support the claims of the authors. The authors use hexanediols at 5% or 10% in an effort to disrupt the Mediator complex. Assuming this was weight/volume, that means ~400 to 800mM hexanediol solution, which is fairly high and can be expected to disrupt protein complexes, but the effects haven't been carefully assessed as far as I'm aware. The 2,5 HD (Figure 1B) experiments appear to simply contain greater protein loading, and this may contribute to the apparent differential results. In fact, in looking at the data, it seems that all MED subunits probed show the same trend as Med16. They are all reduced in the 1,6HD experiment relative to the 2,5 HD experiment. But it's hard to know, because replicates weren't completed and quantitation was not done. There aren't even loading controls. Other concerns about the IP-Western experiments are outlined in point 2.

(2) At no point do the authors apply rigorous methods to test their hypothesis. Instead, methods are applied that have been largely discredited over time and can only serve as preliminary data for pilot studies, and cannot be used to draw definitive conclusions about protein function.

a) IP-westerns are fraught with caveats, especially the way they were performed here, in which the beads were washed at relatively low salt and then eluted by boiling the beads in loading buffer. This will "elute" bound proteins, but also proteins that non-specifically interact with or precipitate on the beads. And because Westerns are so sensitive, it is easy to generate positive results. It's just not a rigorous experiment.

b) Many conclusions relied on transient transfection experiments, which are problematic because they require long timeframes, during which secondary/indirect effects from expression/overexpression will result. This is especially true if the proteins being artificially expressed/overexpressed are major transcription regulators, which is the case here. It is simply impossible to separate direct from indirect effects with these types of experiments. Another concern is that there was no effort to assess whether the induced protein levels were near physiological levels. Protein overexpression, especially if the protein is a known regulator of pol2 transcription (e.g., UBP1 or Med16), will create many unintended consequences.

c) Many conclusions were made based upon shRNA knockdown experiments, which are problematic because they require long timeframes (see above point), which makes it nearly impossible to identify effects that are direct vs. indirect/secondary/tertiary effects. Also, shRNA experiments will have off-target effects, which have been widely reported for well over a decade. An advantage of shRNA knockdowns is that they prevent genetic adaptation (a caveat with KO cell lines). A minimal test would be to show phenotypic rescue of the knockdown by expressing a knockdown-resistant Med16 (for example), but these types of experiments were not done.

d) Many experiments used reporter assays, which involved artificial, non-native promoters. Reporters are good for pilot studies, but they aren't a rigorous test of direct regulatory roles for Med16 or other proteins. Reporters don't even measure transcription directly. In fact, no experiment in this study directly measures transcription. An RNA-seq experiment was done with overexpressed or Med16 knockdown cells, but these required long timeframes and RNA-seq measures steady-state mRNA, which doesn't test the potential direct effects of these proteins on nascent transcription.

e) The MS experiments show promise, but the data were not shown, so it's hard to judge. The reader cannot compare/contrast the experiments, and we have no indication of the statistical confidence of the proteins identified. How many biological replicate MS experiments were performed?

(3) The data are over-interpreted, and alternative (and more plausible) hypotheses are ignored. Many examples of this, some of which are alluded to in the points above. For example, Med16 loss or overexpression will cause compensatory responses in cells. An expected result is that Mediator composition will be disrupted, since Med16 directly interacts with several other subunits. Also in yeast, the Robert, Gross, and Morse labs showed that loss of Med16/Sin4 causes loss of other tail module subunits, and this would be expected to cause major changes in the transcriptome. The authors also mention that yeast Med16/Sin4 "alters chromatin accessibility globally" and this would be expected to cause major changes in the transcriptome, leading to unintended consequences that will make data analysis and identification of direct Med16 effects impossible. The unintended consequences will be magnified with prolonged disruption of MED16 levels in cells (e.g., longer than 4h). These unintended consequences are hard to predict or define, and are likely to be widespread given the pivotal role of Mediator in gene expression. One unintended consequence appears to be loss of pol2 upon Med16 over-expression, as suggested by the western blot in Figure 8B. I point this out as just one example of the caveats/pitfalls associated with long-term knockdowns or over-expression.

Reviewer #3 (Public Review):

Summary:

There are two major flaws that fundamentally undermine the value of the study. First, nearly all the central conclusions drawn here rely on the unfounded assumption that the effects observed are direct. No rigorous cause-and-effect relationships are established to support the claims. Second, the quality of the experimental data is substandard. Collectively, these concerns significantly limit any advances that might be gained in our understanding of the UBP1 pathway or Mediator function.

Weaknesses:

(1) The decrease in 1,6-hexanediol-treated cells of MED16 is modest, variable, not quantified, and internally inconsistent. For example, in Figure 1A, 1,6-hexanediol treatment should not have an impact on the level of the protein being directly IP. For MED12 (and CDK8 and MED1 to a lesser extent), 1,6-hexanediol treatment alters the level of the target protein in the IP. Along these lines, Figure 1A shows a no 1,6H-D dependent decrease in MED1 or MED12 levels in the CDK8 IP, whereas Figure 1B does show a decrease. Figure 1A shows no 1,6H-D dependent decrease in CDK8 levels in the MED1 IP, whereas Figure 1B shows a dramatic decrease. MED24 levels in the MED12 IP increase upon 1,6H-D in Figure 1A, but decrease in Figure 1B. Internal inconsistencies of this nature persist in the other Figures.

(2) Undermining the value of Figure 1E/F, UBP1 and TFCP2 may also associate with the small amount of MED16 in the 2MDa fractions. This is not tested, and therefore, the conclusion that they just associate with the dissociable form of MED16 is not supported.

(3) Domain mapping studies in Figure 2 are overinterpreted. Since the interactions could be indirect, it is not accurate to conclude "Therefore, the N-terminal WDR domain of MED16 is crucial for its integration into the Mediator complex, while the C-terminal αβ-domain is essential for interacting with UBP1-TFCP2. "

(4) A close examination of Figure 2C undermines confidence in the association studies. The bait protein in lanes 5-8 should be equal. Also, there is significant binding of GST to UBP1 and TFCP2, in roughly the same patterns as they bind to GST-MED16 αβ. The absence of input samples makes the results even more difficult to interpret.

(5) The domain deletion mutants are utilized throughout the manuscript as evidence of the importance of the UBP1-MED16 interaction. However, in Figure 2F lanes 7 and 8, the delta-S mutant binds MED16 as well as full-length UBP1. This undermines much of the subsequent data and conclusions about specificity.

(6) Even if the delta-S mutant were defective for MED16 binding, the result in Figure 3B does not "confirm that MED16 is required for the transcriptional activity of UBP1,". Removal of that domain may have other effects.

(7) As Mediator is critical for the activation of many genes, it is not accurate to assume that the impact of its deletion in Figure 3E/F demonstrates a direct requirement in UBP1-driven transcription. This could easily be an indirect effect.

(8) Without documenting the relative protein expression levels in Figure 3G/H, conclusions cannot be drawn about the titration experiments, nor the co-expression experiments. These findings are likely the result of squelching or some form of competition that is not directly related to the UBP1-mediated transcription. A great deal of validation would be required in order to support the model that these effects are a result of MED16 overexpression sequestering UBP1 away from holo-Mediator.

(9) The lack of any documentation of expression levels for the various ectopic proteins in the majority of Figures, renders mechanistic claims meaningless (Figures 3, 4, 5, 6, 7, S2, S3). This is particularly relevant since the model presented for many of the results invokes concentration-dependent competition.