Silencers, Enhancers, and the Multifunctional Regulatory Genome

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Trends in Genetics

Spotlight Silencers, Enhancers, difficulty of assaying for silencers has sna-binding silencers failed to do so. resulted in a paucity of clearly defined Remarkably, all but one of the identified si- and the Multifunctional examples, and little understanding of their lencers were known, or shown, to function Regulatory Genome characteristics and mechanism of action. as enhancers, based on reporter gene fi 1,2,3,4,5, However, a recent rst-of-its-kind large- analysis, in a different tissue. Marc S. Halfon * scale screen has now led to a significant in- crease in the number of known silencers Although such ‘bifunctional’ elements have Negative regulation of gene expres- [4]. Rather than being the negative counter- been observed previously, they had ap- sion by transcriptional silencers has parts to enhancers, the data suggest that peared to be exceptions rather than the been difficult to study due to limited most, if not all, silencers are in fact en- rule. However, current results imply that all defined examples. A new study by hancers, and that activation and silencing silencers may also be enhancers. By con- Gisselbrecht et al. has dramatically are merely two sides of the same cis- trast, most known enhancers tested in [4] regulatory coin. did not show silencer activity. It remains increased the number of identified unknown whether most enhancers there- silencers and reveals that they are Gisselbrecht et al. [4] screened for silencers fore lack this ability or whether these en- bifunctional regulatory sequences by placing putative silencer sequences up- hancers have silencing activity in a tissue that also act as gene expression- stream of a reporter cassette comprising a or time point not observable in the study. promoting enhancers. ubiquitous enhancer driving GFP expression. By looking for tissue-specific (in the Could the silencing results merely be an Exquisite spatial and temporal control of Drosophila mesoderm) reductions in GFP artifact of the reporter gene assay used? gene expression is a hallmark of metazoan activity (Figure 1A), their assay was able to This was addressed for one bifunctional development. Much of this regulation detect sequences with an affirmative si- silencer via CRISPR-mediated deletion of is carried out by distal positive-acting lencing ability while ruling out nonsilencer the genomic sequence. Transcription of ‘enhancers’, a well-studied class of cis- mechanisms, such as short-range repres- the target gene was upregulated, demon- regulatory elements with a substantial sion, failure to activate transcription, and strating that the element functions as a literature exploring their function. No less insulator activity (problems that have con- silencer in its native genomic context. important, but less well-studied, is negative founded previous studies). Close to 10% However, transcript levels from the tissue gene regulation. The action of short-range of N300 assayed sequences (29) were where the element functions as an repressors bound at enhancers to keep revealed to be silencers. No particular enhancer were not assessed. This is un- them in an ‘off’ state is one common histone modification signature was asso- fortunate, because it leaves open the mechanism [1], but at least two other ciated with these sequences, consistent possibility that the enhancer activity is an forms of negative regulation have been de- with the idea that commonly cited sets artifact of the reporter gene assay. Re- scribed: Polycomb-mediated silencing and of histone marks may not in fact be suf- porter gene assays are considered a transcriptional ‘silencer’ elements. Proteins ficient to reliably identify specificregula- ‘gold standard’ for enhancer function, but of the Polycomb group (PcG) mediate tory sequences [5]. There was little it is not in fact known how often they lead chromatin-based gene silencing marked overlap with Polycomb-bound PREs, to either false-positive (ectopic activity) or by histone 3 lysine 27 trimethylation and there were no universally present false-negative results. Therefore, a combi- (H3K27me3), which often spreads broadly transcription factor-binding sites or bound nation of both reporter gene and genomic throughout the silenced locus [2]. In co-repressors (based on extensive existing deletion studies might be needed to pro- Drosophila, PcG proteins are recruited via data sets). However, 40% of the se- vide final definitive proof of bifunctionality aspecific class of negative cis-regulatory quences were co-enriched for a set of [5,6]. More comprehensive studies of element, the Polycomb response element known mesodermal repressors, includ- deleted putative bifunctional elements (PRE), whereas the method of targeted ing snail (sna), and mutation of sna bind- wouldalsobeabletoascertainwhether PcG recruitment remains unclear in mam- ing sites abrogated silencer function. silencer and enhancer functions are malian cells. By contrast, transcriptional Interestingly, the presence or absence directed to the same target gene, which silencers are the negative-regulatory coun- of sna binding appeared to divide the cannot be determined from the reporter terpart of enhancers: defined cis-regulatory identified silencers into two functional gene assay (Figure 1B,C). However, re- sequences that repress transcription from classes: Hi-C studies demonstrated that cent studies in Heliconius butterflies con- otherwise active promoters [3]. Although non-sna-binding silencers made contact firm that this is at least sometimes the initially described over 30 years ago, the with their target-gene promoters, whereas case [7]. While not seeking to define

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(A) silencers, CRISPR-based knockout of a Screen: putative regulatory sequence revealed in- Tested Ubiquitous TATA GFP sequence enhancer stances of both gained and lost expression from what appears to be a single Transgenic gene, suggesting the presence of a bi- embryos functional regulatory element. These results highlight the power and promise of CRISPR-mediated genome editing for in- terrogating putative regulatory sequences FACS and, in particular, its ability to uncover silencers and other negative regulatory sequences. As these methods become in- creasingly mainstream, we should expect that a richer diversity of regulatory ele- Validate: ments will be revealed.

Negative con- Mesoderm TATA GFP trol or silencer enhancer An important unsettled question is whether enhancer/silencer elements are Transgenic embryos truly bifunctional or whether they instead comprise adjacent silencer and enhancer sequences (Figure 1D,E). Given that the boundaries of regulatory elements, such as enhancers and silencers, are not clearly Negative control Silencer/bifunctional element demarcated based on sequence, only de- tailed functional dissections can address this question. Even then, the limits of (B) where one regulatory feature ends and X another begins can be a matter of interpre- tation, as revealed recently in conﬂicting Gene 1 Gene 1 views over pleiotropic versus functionally Tissue A Tissue B distinct enhancers [5,8]. (C) The emerging and unprecedented in- X depth view of the regulatory genome Gene 1 Gene 2 reveals both that there is a greater com- Tissue A Tissue B plexity and abundance of regulatory elements than previously had been appre- ciated, and that these regulatory elements (D) may exist more on a continuum of func- X tional types than as a series of discrete features with unique properties. The study by Gene 1 Gene 1 Gisselbrecht et al. [4] raises the possibility Tissue A Tissue B of both bifunctional and monofunctional (E) enhancers, and their identiﬁed silencers X appear to fall into at least two mechanisti- Gene 1 Gene 1 cally distinct classes (sna-bound, promoter

Tissue A Tissue B not contacted vs sna-unbound, promoter contacting). It is also tempting to speculate TrendsTrends inin GeneticsGenetics that observed differences from many (See ﬁgure legend at the bottom of the next page.) studies at known enhancer regions in,

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among others, histone modifications, the coming years as our ability to delve 2. Erceg, J. et al. (2017) Dual functionality of cis-regulatory elements as developmental enhancers and Polycomb re- transcription of enhancer RNAs (eRNA), deeply into regulatory features and mecha- sponse elements. Genes Dev. 31, 590–602 and binding of mediator or other cofactors nisms increases. 3. Ogbourne, S. and Antalis, T.M. (1998) Transcriptional control and the role of silencers in transcriptional regulation in (e.g., at ‘superenhancers’), may distinguish eukaryotes. Biochem. J. 331, 1–14 1 enhancer subclasses, perhaps with distinct Department of Biochemistry, University at Buffalo-State University 4. Gisselbrecht, S.S. et al. (2019) Transcriptional silencers in of New York, Buffalo, NY, USA Drosophila serve a dual role as transcriptional enhancers in al- regulatory roles in RNA polymerase II 2NY State Center of Excellence in Bioinformatics and Life ternate cellular contexts. Mol. Cell. Published online November recruitment, release of paused RNA poly- Sciences, Buffalo, NY, USA 5, 2019. https://doi.org/10.1016/j.molcel.2019.10.004 3Department of Biological Sciences, University at Buffalo-State 5. Halfon, M.S. (2019) Studying transcriptional enhancers: merase II, or targeting of genes to nuclear University of New York, Buffalo, NY, USA the founder fallacy, validation creep, and other biases. 4 subregions [5,6]. Not only may there be Department of Biomedical Informatics, University at Buffalo- Trends Genet. 35, 93–103 State University of New York, Buffalo, NY, USA 6. Catarino, R.R. and Stark, A. (2018) Assessing sufficiency distinct subclasses of enhancers and 5Department of Molecular and Cellular Biology and Program in and necessity of enhancer activities for gene expression silencers, but it has also been suggested Cancer Genetics, Roswell Park Comprehensive Cancer Center, and the mechanisms of transcription activation. Genes Buffalo, NY, USA Dev. 32, 202–223 that there is significant functional overlap 7. Lewis, J.J. et al. (2019) Parallel evolution of ancient, pleiotropic enhancers underlies butterfly wing pattern mimicry. Proc. between enhancers and promoters [9], *Correspondence: Natl. Acad. Sci. U. S. A. 116, 24174–24183 [email protected] (M.S. Halfon). promoters and insulators [10], enhancers 8. Sabaris, G. et al. (2019) Actors with multiple roles: pleiotropic and PREs [2], and enhancers and si- https://doi.org/10.1016/j.tig.2019.12.005 enhancers and the paradigm of enhancer modularity. Trends Genet. 35, 423–433 lencers [4]. © 2019 Elsevier Ltd. All rights reserved. 9. Andersson, R. and Sandelin, A. (2019) Determinants of enhancer and promoter activities of regulatory elements. Nat. Rev. Genet. Published online October 11, 2019. Our understanding of the regulatory ge- References https://doi.org/10.1038/s41576-019-0173-8 nome is still in its infancy, and we should ex- 1. Payankaulam, S. et al. (2010) Transcriptional repression: 10. Raab, J.R. and Kamakaka, R.T. (2010) Insulators and conserved and evolved features. Curr. Biol. 20, promoters: closer than we think. Nat. Rev. Genet. 11, pect to see many ‘settled’ ideas upended in R764–R771 439–446

Figure 1. Transcriptional Silencers as Bifunctional Regulatory Elements. (A) The screen for silencers used by Gisselbrecht et al. [4]. Transgenic flies are constructed bearing the sequences to be tested for silencer activity placed upstream of a ubiquitously active enhancer driving GFP expression (‘Screen’). The embryos are dissociated and mesodermal cells assayed by fluorescence activated cell sorting (FACS) based on a red-fluorescent mesodermal cell marker (magenta outline). Cells with reduced GFP expression are indicative of silencing and are sequenced to recover the identity of the silencer sequence. The identified putative silencers are then validated by placing them upstream of a mesoderm-specific enhancer driving GFP in stabile, individual transgenic flylines(‘Validate’). Silencer activity leads to a reduction in mesodermal GFP expression relative to negative controls (light green, arrow). Enhancer function of the same sequences manifests as GFP expression in non-mesodermal tissues (dark green, arrowhead). (B,C) Two possibilities for silencer/enhancer bifunctionality. In (B), the bifunctional regulatory element regulates a single gene, silencing ‘gene 1’ in tissue A but activating ‘gene 1’ in tissue B. In (C), the bifunctional element is a silencer with respect to ‘gene 1’ in tissue A, but an enhancer with respect to ‘gene 2’ in tissue B. The current data are insufficient to clearly support either model, although the evidence slightly favors the case shown in (B). (D,E) Integrated bifunctional elements versus adjacent silencers and enhancers. In (D), silencer functions (blue) and enhancer functions (yellow) are mixed within a single bifunctional sequence. An alternative is shown in (E), where silencer and enhancer functions reside in separable but adjacent sequence elements.

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