DNA methylation insulates genic regions from CTCF loops near nuclear speckles

Reviewer #1 (Public review):

Summary
Roseman et al. use a new inhibitor of the maintenance DNA methyltransferase DNMT1 to probe the role of methylation on binding of the CTCF protein, which is known to be involved chromatin loop formation. As previous reported, and as expected based on our knowledge that CTCF binding is methylation-sensitive, the authors find that loss of methylation leads to additional CTCF binding sites and increased loop formation. By comparing novel loops with the binding of the pre-mRNA splicing factor SON, which localizes to the nuclear speckle compartment, they propose that these reactivated loops localize to near speckles. This behavior is dependent on CTCF whereas degradation of two speckle proteins does not affect CTCF binding or loop formation. The authors propose a model in which DNA methylation controls the association of genome regions with speckles via CTCF-mediated insulation.

Strengths
The strengths of the study are 1) the use of a new, specific DNMT1 inhibitor and 2) the observation that genes whose expression is sensitive to DNMT1 inhibition and dependent on CTCF (cluster 2) show higher association with SON than genes which are sensitive to DNMT1 inhibition but are CTCF insensitive, is in line with the authors' general model.

Weaknesses
There are a number of significant weaknesses that as a whole undermine many of the key conclusions, including the overall mechanistic model of a direct regulatory role of DNA methylation on CTCF-mediated speckle association of chromatin loops.

(1) The authors frequently make quasi-quantitative statements but do not actually provide the quantitative data, which they actually all have in hand. To give a few examples: "reactivated CTCF sites were largely methylated (p. 4/5), "many CTCF binding motifs enriched..." (p.5), "a large subset of reactivated peaks..."(p.5), "increase in strength upon DNMT1 inhibition" (p.5); "a greater total number....." (p.7). These statements are all made based on actual numbers and the authors should mention the numbers in the text to give an impression of the extent of these changes (see below) and to clarify what the qualitative terms like "largely", "many", "large", and "increase" mean. This is an issue throughout the manuscript and not limited to the above examples.
Related to this issue, many of the comparisons which the authors interpret to show differences in behavior seem quite minor. For example, visual inspection suggests that the difference in loop strength shown in figure 1E is something like from 0 to 0.1 for K562 cells and a little less for KCT116 cells. What is a positive control here to give a sense of whether these minor changes are relevant. Another example is on p. 7, where the authors claim that CTCF partners of reactivated peaks tend to engage in a "greater number" of looping partners, but inspection of Figure 2A shows a very minor difference from maybe 7 to 7.5 partners. While a Mann-Whitney test may call this difference significant and give a significant P value, likely due to high sample number, it is questionable that this is a biologically relevant difference.

(2) The data to support the central claim of localization of reactivated loops to speckles is not overly convincing. The overlap with SON Cut&Tag (figure 2F) is partial at best and although it is better with the publicly available TSA-seq data, the latter is less sensitive than Cut&Tag and more difficult to interpret. It would be helpful to validate these data with FISH experiments to directly demonstrate and measure the association of loops with speckles (see below).

(3) It is not clear that the authors have indeed disrupted speckles from cells by degrading SON and SRRM2. Speckles contain a large number of proteins and considering their phase separated nature stronger evidence for their complete removal is needed. Note that the data published in ref 58 suffers from the same caveat.

(4) The authors ascribe a direct regulatory role to DNA methylation in controlling the association of some CTCF-mediated loops to speckles (p. 20). However, an active regulatory role of speckle association has not been demonstrated and the observed data are equally explainable by a more parsimonious model in which DNA methylation regulates gene expression via looping and that the association with speckles is merely an indirect bystander effect of the activated genes because we know that active genes are generally associated with speckles. The proposed mechanism of a regulatory role of DNA methylation in controlling speckle association is not convincingly demonstrated by the data. As a consequence, the title of the paper is also misleading.

(5) As a minor point, the authors imply on p. 15 that ablation of speckles leads to misregulation of genes by altering transcription. This is not shown as the authors only measure RNA abundance, which may be affected by depletion of constitutive splicing factors, but not transcription. The authors would need to show direct effects on transcription.

Reviewer #2 (Public review):

Summary:
CTCF is one of the most well-characterized regulators of chromatin architecture in mammals. Given that CTCF is an essential protein, understanding how its binding is regulated is a very active area of research. It has been known for decades that CTCF is sensitive to 5-cystosine DNA methylation (5meC) in certain contexts. Moreover, at genomic imprints and in certain oncogenes, 5meC-mediated CTCF antagonism has very important gene regulatory implications. A number of labs (eg, Schubeler and Stamatoyannopoulos) have assessed the impact of DNA methylation on CTCF binding, but it is important to also interrogate the effect on chromatin organization (ie, looping). Here, Roseman and colleagues used a DNMT1 inhibitor in two established human cancer lines (HCT116 [colon] and K562 [leukemia]), and performed CTCF ChIPseq and HiChIP. They showed that "reactivated" CTCF sites-that is, bound in the absence of 5meC-are enriched in gene bodies, participate in many looping events, and intriguingly, appear associated with nuclear speckles. This last aspect suggests that these reactivated loops might play an important role in increased gene transcription. They showed a number of genes that are upregulated in the DNA hypomethylated state actually require CTCF binding, which is an important result.

Strengths:
Overall, I found the paper to be succinctly written and the data presented clearly. The relationship between CTCF binding in gene bodies and association with nuclear speckles is an interesting result. Another strong point of the paper was combining DNMT1 inhibition with CTCF degradation.

Weaknesses:
The most problematic aspect of this paper in my view is the insufficient evidence for the association of "reactivated" CTCF binding sites with nuclear speckles needs to be more diligently demonstrated (see Major Comment). One unfortunate aspect was that this paper neglected to discuss findings from our recent paper, wherein we also performed CTCF HiChIP in a DNA methylation mutant (Monteagudo-Sanchez et al., 2024 PMID: 39180406). It is true, this is a relatively recent publication, although the BioRxiv version has been available since fall 2023. I do not wish to accuse the authors of actively disregarding our study, but I do insist that they refer to it in a revised version. Moreover, there are a number of differences between the studies such that I find them more complementary rather than overlapping. To wit, the species (mouse vs human), the cell type (pluripotent vs human cancer), the use of a CTCF degron, and the conclusions of the paper (we did not make a link with nuclear speckles). Furthermore, we used a constitutive DNMT knockout which is not viable in most cell types (HCT116 cells being an exception), and in the discussion mentioned the advantage of using degron technology:

"With high-resolution techniques, such as HiChIP or Micro-C (119-121), a degron system can be coupled with an assessment of the cis-regulatory interactome (118). Such techniques could be adapted for DNA methylation degrons (eg, DNMT1) in differentiated cell types in order to gauge the impact of 5meC on the 3D genome."

The authors here used a DNMT1 inhibitor, which for intents and purposes, is akin to a DNMT1 degron, thus I was happy to see a study employ such a technique. A comparison between the findings from the two studies would strengthen the current manuscript, in addition to being more ethically responsible.