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  1. Evaluation Summary:

    This manuscript will be of interest to geneticists seeking to establish rules that govern gene regulation. To explain why a sequence enhances, rather than silences, gene transcription the authors draw our attention away from the binding of a single transcription factor, to focus instead on the number and diversity of transcription factor molecules that bind to it. Using a relatively simple metric called sequence information content they appear to be able to improve the prediction of enhancer over silencer sequences. A concern is whether the silencers are true silencers, or whether they only act as such in this specific experimental paradigm.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Why can silencers be enhancers in other cell types? Why is it that active chromatin epigenetic marks or binding of a single transcription factor do not reliably predict active enhancers? These are thorny issues in genomics because they hinder our mechanistic understanding of gene transcription regulation.

    In this well-written submission the authors go beyond their previous publications using the same experimental system (White et al., 2013, 2016). They use MPRA for the CRX transcription factor (TF) in explanted mouse retinas to show that epigenetically indistinguishable sequences are classified more accurately as enhancers or silencers by the number and diversity of lineage-specific transcription factor binding motifs that they contain. They separate enhancers from silencers by enhancers' more diverse collection of TF motifs. This distinction is captured in a metric called sequence information content calculated from both TF motif count and diversity. This single metric is slightly worse at predicting strong enhancers over silencers than a model considering the PWMs for 8 TFs.

    Two issues require a response:
    1. Whether the authors observe a bias in the linear arrangement of these TFs' motifs that might assist in distinguishing enhancers from silencers?

    2. p10 The choice of the 8 lineage-defining TFs was somewhat arbitrary because of the arbitrary nature of PWM significance thresholds. Please justify their choice and number, and comment on how well the model performs when this TF set is altered?

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  3. Reviewer #2 (Public Review):

    The authors state that enhancers and silencers often have the same epigenomic profiles and attempt to identify sequence-based information to differentiate between the two types of elements. They use massively parallel reporter assays to test elements that bind CRX for activity in retinal explants. The authors then look for differences in motif content between the elements that act as silencers vs. those that act as enhancers of gene expression from a basal promoter. They find that although enhancers and silencers have motifs for the same transcription factor - CRX, the number of sites and diversity of other TF sites is greater within enhancers. They suggest motif content is a way to distinguish between the two types of elements. I'm not convinced that anything can be determined about silencers using this experimental design.

    Strengths:
    The authors test many putative enhancers in mouse retinas and identify elements whose function requires CRX sites.

    Interestingly, different behaviors of functional elements could not be predicted based on differences in DNA accessibility or ATAC-seq peak or CRX occupancy. This is a nice systematic example of how difficult it is to predict an enhancer strength or activity based on differences in epigenomic data and highlights the need for sequence-based approaches to identify the specific activity of an element.

    They do a nice analysis of the inert vs. weak and strong enhancers. The data and analysis of these experiments could be really informative for understanding why not all regions that bind CRX and are within open chromatin are active enhancers.

    Weaknesses:

    I'm concerned that the silencers they detect could be an artifact of the experimental design. The promoter contains CRX sites and NRL sites, so there is some level of basal expression; the silencers are enriched in repressors, so is it just that the elements containing a repressor are silencing the basal transcription? Moreover, what does this mean relative to the elements in the endogenous locus, if an endogenous promoter doesn't have CRX or NRL sites within its promoter or basal transcription does this mean the silencers as described in this assay are not really silencers within the genome. I don't think it is possible to make conclusions about a cis-reg element's silencer capacity based on these experiments.

    In line with this, they find that the silencers bind CRX in combination with a repressive TF. Would they find that enhancers as they define them bind a combination of transcriptional activators and that silencers bind some activators such as CRX in combination with a transcriptional repressor expressed in the cell type where the element acts as a silencer?

    I am also not convinced that silencers and enhancers are different things. If a genomic element controls the time and location of gene expression, then it is an enhancer - enhancers can bind activators and repressors and restrict expression to only particular cell types. I think trying to call things enhancers, and silencers makes things overly complex, especially considering the fact the authors point out that the same element can be an enhancer in one tissue type and a silencer in another. I am also concerned about this in relation to my previous comments on the experimental design and the issues demonstrating that a silencer really works this way within the genome.

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