A genome-wide nucleosome-resolution map of promoter-centered interactions in human cells corroborates the enhancer-promoter looping model

Reviewer #1 (Public Review):

The authors presented a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale. With improved resolution and sensitivity, they explored the spatial connectivity of active promoters and identified the potential candidates for establishing/maintaining E-P interactions. Finally, with published CRISPRi screens, they found that most functionally verified enhancers do physically interact with their cognate promoters, supporting the enhancer-promoter looping model.

The study's experimental approach and findings are interesting. However, several issues need to be addressed.

1. The authors described that "the lack of interaction between experimentally-validated enhancers and their cognate promoters in some studies employing C-methods has raised doubts regarding the classical promoter-enhancer looping model", so it's intriguing to see whether the MChIP-C could indeed detect the E-P interactions which were not identified by C-methods as they mentioned (Benabdallah et al., 2019; Gupta et al., 2017). I agree that they identified more E-P interactions using MChIP-C, but specifically, they should show at least 2-3 cases. It's important since this is the main conclusion the authors want to draw.

2. The authors compared their data to those of Chen et al. (Chen et al., 2022), who used PLAC-seq with anti-H3K4me3 antibodies in K562 cells and standard Micro-C data previously reported for K562, concluding that "MChIP-C achieves superior sensitivity and resolution compared to C-methods based on standard restriction enzymes.". This is not convincing since they only compared their data to one dataset. More datasets from other cell lines should be included.

3. The reasons for choosing Chen's data (Chen et al., 2022) and CRISPRi screens (Fulco et al., 2019; Gasperini et al., 2019) should be provided since there are so many out there.

4. The authors identify EP300 histone acetyltransferase and the SWI/SNF remodeling complex as potential candidates for establishing and/or maintaining enhancer-promoter interactions, but not RNA polymerase II, mediator complex, YY1, and BRD4. More explanation is needed for this point since they're previously suggested to be associated with E-P interactions.

5. The limitations of the method should be discussed.

Reviewer #2 (Public Review):

Summary:
Golov et al performed the capture of MChIP-C using the H3K4me3 antibody. The new method significantly increases the resolution of Micro-C and can detect clear interactions which are not well described in the previous HiChIP/PLAC-seq method. Overall, the paper represents a significant technological advance that can be valuable to the 3D genomic field in the future.

Strengths:
1. The authors established a novel method to profile the promoter center genomic interactions based on the Micro-C method. Such a method could be very useful to dissect the enhancer promoter interaction which has long been an issue for the popular HiC method.

2. With the MChIP-C method the authors are able to find new genomic interactions with promoter regions enriched in CTCF. The author has significantly increased the detection sensitivity of such methods as PLAC-seq, Micro-C, and HiChIP.

3. The authors identified a new type of interaction between the CTCF-less promoter and the CTCF binding site. This particular type of interaction could explain the CTCF's function in regulating gene transcription activity as observed in many studies. I personally think the second stripe model of P-CTCF interaction is more likely as this has been proposed for the super-enhancer stripe model before. The author should also discuss this part of the story more.

Weaknesses:
1. The data presentation should include the contact heat map. The current data presentation makes it hard for the readers to have a comprehensive view of pair-wise interactions between promoters and the PIR. In particular, these maps may directly give answers to the proposed model of promoter-CTCF interactions by the authors in Figure 3a.

2. In Fig 3D, there seems a very limited increase of power predicting MChIP-C signal for DHS-promoter pairs beyond the addition of CTCF. This figure could be simplified with fewer factors.

3. The current method seems to have a big fraction of unusable reads. How the authors process the data should be included to allow for future reproduction. Ideally, the authors should generate a package on R or Bioconda for this processing.

Reviewer #3 (Public Review):

Summary:
This manuscript represents a technological development- specifically a micrococcal nuclease chromatin capture approach, termed MChIP-C to identify promoter-centered chromatin interactions at single nucleosome resolution via a specific protein, similar to HiChIP, ChIA-PET, etc.. In general, the manuscript is technically well done. Two major issues raise concerns that need to be addressed. First, it does not appear that novel chromatin interactions identified by MChIP-C which were missed by other approaches such as HiChIP, were validated. This is central to the argument of "improved" sensitivity, which is one of the key factors to assess sensitivity. Second is the question of resolution. Because the authors focus on a histone mark (H3K4me3) it is unclear whether the resolution of the assay truly exceeds other approaches, especially microC. These two issues are not completely supported by the data provided.

Strengths:

1. The method appears to hold promise to improve both the sensitivity and resolution of protein-centered chromatin capture approaches.

Weaknesses:

1. Specific validation experiments to demonstrate the identification of previously missed novel interactions are missing.

2. It is unclear if the resolution is really superior based on the data provided.

3. It is unclear how much advantage the approach has, especially compared to existing approaches such as HiChIP< since sequencing depth as a variable is not adequately addressed.