1. Author Response:

    We thank the editors and reviewers for their assessment of our manuscript on the instructive role of enhancer activity in the probability of gene allele activation and random monoallelic expression, and the associated helpful comments.

    Concerning the possibility that our findings apply to gene types other than hematopoietic-related genes, we believe that answer is yes. In fact, the first documented examples of enhancers regulating the probability of target gene expression were in nonhematopoietic cells: the non-hematopoietic cell lines CV-1 and HeLa (Weintraub PNAS 1988 PMID: 3045805; Walters et al. PNAS 1995 PMID: 7624382). Furthermore, the characteristic constitutive accessibility of enhancers at RME loci regardless of expression of the gene, which is suggestive of probabilistic effects, is shared by hematopoietic and non-hematopoietic (neural lineage) cells (Xu et al. Nat. Genet. 2017 PMID: 28112738). Together with our study, the available evidence argues for a unified role of enhancer activity in determining gene expression probabilities across cell types.

    Concerning the thoughtful review from reviewer 1, these dynamics are not limited to genes encoding cell surface receptors. Recent work that we cited in our manuscript showed that a distal enhancer regulates the expression probability of the gene encoding the transcription factor Bcl11b which determines the T cell fate (Ng et al. eLife 2018 PMID: 30457103).

    In summary, while we focused on the NK cell receptor genes and genes encoding other cell surface proteins in various hematopoietic cell types due to experimental tractability, we believe it is unlikely that our findings will be restricted to specific cell types or to receptor genes.

    Reviewer #1:

    This study investigates the role of enhancer activity in the regulation of stable random monoallelic expression (RME) using the Ly49 and Nkg2 receptor gene families expressed in natural killer (NK) cells, as models of RME genes. The authors show that, unlike promoters of RME genes, enhancer are accessible on both alleles and display histone marks of active enhancers. Moreover, they show that weakening enhancer activity, via CRISPR-mediated deletion, can lower the frequency of gene expression or lead to variegated expression patterns, that are reminiscent of RME. The manuscript is clearly written and the data presented are compelling. This study takes advantage of previously-characterised allele-specific antibodies for various genes expressed in NK cells, a powerful tool allowing the analysis of random monoallelic expression (RME) at the protein and single-cell level within a population. The use of these antibodies allows the investigation of in vivo cell population and circumvents the analysis at the RNA level, which is limited by expression bursts and transcript levels. The authors also substantiate their model using examples of receptor genes expressed in other cell types from the hematopoietic lineage. One question that remains is whether this model applies to other developmentally regulated stable RME genes, that are 1-not expressed at the cell surface (such as transcription factors) and 2- expressed in other cell lineages? It is also unclear what defines the strength of an enhancer upstream of the RME genes studied, e.g. what is the difference between a weak enhancer for Ly49 genes and strong enhancer. These points should be of broad interest for the readers and could be discussed further in the discussion part of the manuscript.

    We thank the Reviewer 1 for very thoughtful comments.

    First, we would like to address the reviewer’s question concerning whether our findings apply to genes that encode transcription factors or other proteins that are not on the cell surface. This indeed IS the case based on recent work we cited showing that a distal enhancer regulates the expression probability of the gene encoding the transcription factor Bcl11b, which determines the T cell fate (Ng et al. eLife 2018 PMID: 30457103). That our findings also apply to genes expressed in non-hematopoietic cells is addressed in the response above to the evaluation summary.

    We also welcome the opportunity to elaborate on enhancer “strength”, albeit somewhat speculatively. Enhancer activity acting upon a locus varies quantitatively in a context-dependent manner. The strength of enhancer activity is likely a function of several factors including (but not limited to) A) the collective (nonredundant) effects of multiple enhancers in genes that have more than one; B) the concentration of enhancer-binding transcription factors (TFs) in the nucleus; C) the affinity of those factors for the target DNA sequences; D) interactions of the relevant transcription factors with each other and with other components of the transcriptional machinery; E) interactions of the enhancer with the specific promoters; and F) the distance between an enhancer and a promoter. Concerning A), our work suggests that where multiple enhancers are present, elimination of one of them reduces overall enhancer strength/activity, resulting in a lower frequency of gene expression. Relevant to B) is work from several groups showing that Ly49 expression frequencies change when relevant TF expression levels are experimentally altered (Held et al. Immunity 1999 PMID: 10549625; Ohno et al. Int. Immunol. 2008 PMID: 18003603; Bezman et al. J. Exp. Med 2011 PMID: 22124110); those results suggest that one means by which enhancer activity may be increased is by increasing the concentration of available TFs. Relevant to F), enhancer-promoter distance may play a role in determining enhancer “strength”, as recent work has shown a distance-dependent binary effect of enhancers on gene expression in integrated reporters (Rinzema et al. BioRxiv 2021 https://doi.org/10.1101/2021.10.05.463209). Fully fleshing out the definition of enhancer strength in the context of RME gene expression will likely accompany a better understanding of how enhancers work generally, a subject of intense current study in the field. Finally, we do not exclude are role for the promoter, which may possess varying levels of intrinsic “competence” to be activated by the collective enhancer activity acting upon it.

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  2. Evaluation Summary:

    In this manuscript by Kissiov et al. the authors show that enhancers can play an instructive role in controlling stable random monoallelic expression (RME). In order to do so, they initially focus on a limited set of natural killer (NK) receptor genes that are subject to RME, which they investigate using several in vivo genetic models. Furthermore, they also show that RME can be considerably more prevalent than previously thought and that enhancer strength and/or number might influence the extent of RME for different genes. One remaining question may be whether this model may apply to other gene types than hematopoietic-related genes. Overall, this is a highly relevant manuscript with major implications in gene regulation and enhancer biology and, thus, of broad scientific interest.

    (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 and Reviewer #2 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    This study investigates the role of enhancer activity in the regulation of stable random monoallelic expression (RME) using the Ly49 and Nkg2 receptor gene families expressed in natural killer (NK) cells, as models of RME genes. The authors show that, unlike promoters of RME genes, enhancer are accessible on both alleles and display histone marks of active enhancers. Moreover, they show that weakening enhancer activity, via CRISPR-mediated deletion, can lower the frequency of gene expression or lead to variegated expression patterns, that are reminiscent of RME. The manuscript is clearly written and the data presented are compelling. This study takes advantage of previously-characterised allele-specific antibodies for various genes expressed in NK cells, a powerful tool allowing the analysis of random monoallelic expression (RME) at the protein and single-cell level within a population. The use of these antibodies allows the investigation of in vivo cell population and circumvents the analysis at the RNA level, which is limited by expression bursts and transcript levels. The authors also substantiate their model using examples of receptor genes expressed in other cell types from the hematopoietic lineage. One question that remains is whether this model applies to other developmentally regulated stable RME genes, that are 1-not expressed at the cell surface (such as transcription factors) and 2- expressed in other cell lineages? It is also unclear what defines the strength of an enhancer upstream of the RME genes studied, e.g. what is the difference between a weak enhancer for Ly49 genes and strong enhancer. These points should be of broad interest for the readers and could be discussed further in the discussion part of the manuscript.

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  4. Reviewer #2 (Public Review):

    In this manuscript, Kissiov et al. investigate the role of enhancers in the control of stable random monoallelic expression (RME). In order to do so, they initially focus on a limited set of natural killer (NK) receptor genes that are subject to RME, which they investigate using several in vivo genetic models. Once they convincingly showed that the expression of these NK receptor genes is controlled by bona fide enhancers, they perform a number of allele specific analyses to convincingly show that enhancer deletions can lead to a clear increase in the number of cells showing mitotically stable RME. Lastly, the authors also show that RME seems to be considerably more prevalent than previously estimated, which is in agreement with the proposed importance of enhancers in RME and the variability of enhancer landscapes (strength and number) among different genes.

    At a mechanistic level, the authors show that enhancers remain active in both cells expressing or not expressing the target gene alleles, which, nevertheless differ in their promoter state (active vs inactive). Since in the investigated loci the enhancers are quite proximal to their target genes, it is rather intriguing and still an open question why the enhancers fail to activate the gene alleles subject to RME. Therefore, the molecular mechanisms behind RME and of how the interplay between genes and enhancers control this process remain unknown.

    Overall, the presented work, which is based on an impressive battery of in vivo mouse models, has major implications for the gene regulation field. Briefly, the presented work provides novel insights into how RME can be regulated and extends previous observations that indicate that enhancers work in a binary manner controlling the probability rather than the level of gene expression.

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  5. Reviewer #3 (Public Review):

    In this article, the authors investigate in detail several regulatory elements of the NKR locus, located on mouse Chr6 and that include several Ly49 and NKg2 genes. More specifically they perform a series of analyses and genetic experiments to test the idea that regulatory elements potentially regulating gene expression of the NKR genes are random mono-allelic enhancers. Based on their results, they propose that the analyzed genes obey a binary model of activation in which the enhancers control gene expression through a probabilistic rather than through an increase of gene expression. This article is interesting and provides a rich set of data useful for the community. It will also feed the debate about the role and mode of action of enhancers in random mono-allelic gene expression (RME) and provides clues about some possible roles of enhancer redundancy. One general criticism is that the work is not simple to read for a general audience that is not familiar with immunology and the RME questions. Some of the statements in the beginning of the manuscript are also too strong and need to be moderated. The presentation of the genomic datasets used in the study could also be improved.

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