COMMENTARY

Large genomic insertion at the Shh results in hammer toes through enhancer adoption COMMENTARY Christina Palioua and Guillaume Andreya,1

Enhancers are cis-regulatory elements which control through natural (DNA recombination, repair, or repli- the expression of in a defined spatiotemporal cation) or induced (X-irradiation) mutational mecha- pattern, enabling the normal morphogenesis of or- nisms. As this type of SVs changes the number of base gans and structures during embryogenesis. Enhancers pairs in the genome, they can be referred to as copy control their target genes independently of their ori- number or unbalanced variants. Large insertions can be entation or distance through chromosomal looping due to TE insertions or more complex rearrangements, and are thought to evolve through various mutational known as insertional translocations. TEs are DNA mechanisms (1). A critical biological process that leads sequences that have the ability to move themselves to the acquisition of new expression domains as to a different genomic location. They are divided into well as pathological outcomes is termed “enhancer two classes: the DNA transposons which mobilize in a adoption,” whereby a gene is regulated by an en- “cut-and-paste” mechanism and the retrotransposons hancer that is not normally its own (2). Enhancer adop- that sequentially transcribe and reverse-transcribe them- tion can derive from the insertion of transposable selves before “jumping” in another locus. Retrotranspo- elements (TEs) with regulatory capacities or genomic sons include, among others, LINEs (long interspersed structural variants (SVs). In particular, many studies nuclear elements), which comprise almost 20% of the have demonstrated the importance of SVs, including and can be “hotspots” of structural rear- deletions, inversions, duplications, and translocations, rangements (11–13). In contrast, insertional transloca- with regard to 3D genome organization, gene regula- tions are fragments of a , that can vary in tion, and disease (3–5). Recent advances in genome size, transferred into a nonhomologous genomic region engineering and in sequencing technologies have (13). In humans, studies have demonstrated that de novo contributed to a deeper understanding of the molec- insertions might originate from parental balanced trans- ular mechanisms behind genomic rearrangements locations (14, 15) or they can include multiple segments and their role in evolution and pathology. Therefore, in either orientation (16, 17). However, it remains a chal- researchers have seized the opportunity to reinterpret lenge to identify them and even more to reveal their old mouse alleles obtained by spontaneous mutations origin. Array CGH is commonly used to detect copy like X-irradiation-induced phocomelia, Hemimelic ex- number variations in patients but is not sufficient to find tra toes (Hx), Ulnaless, and so on, in the light of mod- and map the location of insertions. Complementary ap- ern molecular tools (6–8). In PNAS, Mouri et al. (10) set proaches, such as inverse PCR experiments, chromo- out to reanalyze a mouse mutant with syndactyly and some banding for larger rearrangements, and FISH interdigital webbing, known as Hammer toe (Hm), first are additionally used. More recently, long read se- described in 1964 (9). The authors found that a geno- quencing technologies and different computational ap- mic insertion of an interdigital regulatory region in the proaches provided another efficient way to uncover vicinity of the gene Sonic hedgehog (Shh) results in its insertions (18). In Mouri et al. (10), the authors charac- ectopic expression and the Hm phenotype. The au- terize a 150-kb de novo genomic insertion in a preex- thors use chromatin technologies and CRISPR/Cas9 isting LINE at the Shh locus at chromosome 5. Although genetic engineering to dissect the regulatory function the mechanism underlying its generation remains un- of the inserted region and to reverse the Hm pheno- known, the authors functionally analyze its effect on Shh type. Their results bring an important conceptual transcription and digit development. framework to the interpretation of diseases caused Despite the high impact of structural variations on by large genomic insertions (10). genome evolution and disease there have been a Insertions are SVs that occur when DNA chromo- limited number of functional studies focusing on their somal segments integrate into new genomic locations biological outcome. On the one hand, pathological

aRG Development & Disease, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany Author contributions: C.P. and G.A. wrote the paper. The authors declare no conflict of interest. Published under the PNAS license. See companion article on page 1021. 1To whom correspondence should be addressed. Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1721351115 PNAS | January 30, 2018 | vol. 115 | no. 5 | 839–841 Downloaded by guest on September 27, 2021 Limb E11.5 Limb E13.5 adult paws

Shh Lmbr1 Shh Shh chr 5 Wildtype Wildtype ZRS/MFCS1

chr 14 HIS Shh Shh Shh Lmbr1 chr 5 Hm/Hm Hm/Hm ZRS/MFCS1

Fig. 1. The pathomechanism of the Hm mouse mutation. The Hm mice are characterized by a genomic insertion (HIS), originating from chromosome 14, between Shh and its limb enhancer ZRS/MFCS1 (orange oval). HIS contains enhancer elements (green ovals) that can drive Shh expression ectopically at the interdigital space of the limb at embryonic stage E13.5 (green staining in the limb above the HIS region). Shh misexpression (purple staining in limbs at E13.5) leads to decreased interdigital cell death, soft tissue syndactyly, and interdigital webbing in adults. Note that the expression of Shh in the posterior part of the limb at embryonic stage E11.5 (purple staining in limbs), which is controlled by the ZRS/MFCS1 enhancer (orange staining in limb above the ZRS/MFCS1), remains unchanged between wild-type and Hm mice.

SVs can affect the coding part of the genome by altering the dosage efficient genome editing (22). The use of CRISPR/Cas9 technology of genes or by disrupting their normal sequences (19). On the other was in this study critical for the characterization of the Hm mice. hand, SVs can affect the noncoding part of the genome, which is Specifically, Mouri et al. (10) identified first the HIS subregions critical to ensure normal gene expression, for example by altering responsible for its regulatory activity using a chromatin accessibil- enhancer dosage (20). Alternatively, several studies have shown that ity assay and assessed their function in reporter experiments. SVs might lead to loss of insulation between chromatin domains, Next, to test if the Hm phenotype can be rescued they used the called topologically associating domains (TADs), which normally con- CRISPR/Cas9 technology and deleted the HIS from the Hm ge- strain the pairing of enhancers and promoters (21). Indeed, these SVs nome. As a result, the limb phenotype was reversed and the an- were shown to enable enhancers and promoters from neighboring imals were indistinguishable from wild-type animals. In parallel, TADs to interact, leading to gene misexpression and disease (3–5). the authors obtained a CRISPR/Cas9-mediated inversion of HIS, Therefore, genomic rearrangements have been shown to either affect which resulted in stronger Shh overexpression. The Hm-inv mice, the gene content, the cis-regulatory information around it, or the TAD as they were named, exhibited swollen soles and cleft palate lead- structure of loci. In the case of the Hm mice, the insertion was found in ing to neonatal lethality, in contrast to Hm mice, which are fully a gene desert within the Shh TAD. First, Mouri et al. (10) narrowed viable. The authors argue that the observed phenotype is likely down the size of the insertion using classical linkage analysis. After due to an orientation-dependent transcription factor. CTCF is a analyzing numerous recombinant mice they showed that the Hm factor known for facilitating enhancer–promoter communication inserted sequence (HIS) acts in cis on Shh. Next, by using inverse through DNA looping. Based on the current model, looping can PCR they were able to locate the origin of the fragment at chromo- be achieved between two convergent CTCF sites mediated by the some 14. Although the deletion of the original HIS fragment on chro- cohesin ring-like (23). However, there were no CTCF mo- mosome 14 does not result in obvious limb phenotype, the authors tifs found at this insertion site (10). Alternatively, the relative en- showed, using reporter assays, that it contains limb enhancer activity. hancer proximity to the insertion borders, which differs in both As a result of the HIS within the Shh regulatory landscape Shh becomes alleles, might influence the strength of chromatin interactions be- ectopically expressed in the interdigital space (Fig. 1). This ectopic tween the enhancers and Shh and thereby result in variable over- expression induces bone morphogenetic protein signaling and loss expression levels. Generally, this surprising result remains for of apoptosis in the interdigital space, ultimately leading to interdigital further future investigation. webbing and syndactyly phenotype. Moreover, the authors could Finally, the discovery of this insertion-based enhancer adop- demonstrate that this 150-kb integration did not perturb the early tion mechanism provides a new framework for the understanding regulatory activity of the endogenous ZRS/MFCS1 limb enhancer. of pathomechanisms underlying genomic insertions. It further CRISPR/Cas9 has revolutionized the field of functional geno- urges the need to develop efficient tools to detect and charac- mics by providing researchers with tools enabling fast and terize pathological insertions genome-wide in human patients.

1 Long HK, Prescott SL, Wysocka J (2016) Ever-changing landscapes: Transcriptional enhancers in development and evolution. Cell 167:1170–1187. 2 Lettice LA, et al. (2011) Enhancer-adoption as a mechanism of human developmental disease. Hum Mutat 32:1492–1499. 3 Lupia ´~nez DG, et al. (2015) Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell 161:1012–1025. 4 Franke M, et al. (2016) Formation of new chromatin domains determines pathogenicity of genomic duplications. Nature 538:265–269. 5 Weischenfeldt J, et al. (2017) Pan-cancer analysis of somatic copy-number alterations implicates IRS4 and IGF2 in enhancer hijacking. Nat Genet 49:65–74. 6 Galloway JL, Delgado I, Ros MA, Tabin CJ (2009) A reevaluation of X-irradiation-induced phocomelia and proximodistal limb patterning. Nature 460:400–404. 7 Lettice LA, et al. (2014) Development of five digits is controlled by a bipartite long-range cis-regulator. Development 141:1715–1725. 8 Lonfat N, Duboule D (2015) Structure, function and evolution of topologically associating domains (TADs) at HOX loci. FEBS Lett 589:2869–2876. 9 Green M (1964) New mutation. Mouse News Lett 31:27. 10 Mouri K, et al. (2018) Enhancer adoption caused by genomic insertion elicits interdigital Shh expression and syndactyly in mouse. Proc Natl Acad Sci USA 115:1021–1026. 11 Lander ES, et al.; International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409:860–921. 12 Startek M, et al. (2015) Genome-wide analyses of LINE-LINE-mediated nonallelic homologous recombination. Nucleic Acids Res 43:2188–2198.

840 | www.pnas.org/cgi/doi/10.1073/pnas.1721351115 Paliou and Andrey Downloaded by guest on September 27, 2021 13 Weckselblatt B, Rudd MK (2015) Human structural variation: Mechanisms of chromosome rearrangements. Trends Genet 31:587–599. 14 Nowakowska BA, et al. (2012) Parental insertional balanced translocations are an important cause of apparently de novo CNVs in patients with developmental anomalies. Eur J Hum Genet 20:166–170. 15 Kang SHL, et al. (2010) Insertional translocation detected using FISH confirmation of array-comparative genomic hybridization (aCGH) results. Am J Med Genet A 152A:1111–1126. 16 Chiang C, et al. (2012) Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration. Nat Genet 44:390–397, S1. 17 Weckselblatt B, Hermetz KE, Rudd MK (2015) Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis. Genome Res 25:937–947. 18 Guan P, Sung W-K (2016) Structural variation detection using next-generation sequencing data: A comparative technical review. Methods 102:36–49. 19 Spielmann M, Mundlos S (2013) Structural variations, the regulatory landscape of the genome and their alteration in human disease. BioEssays 35:533–543. 20 Will AJ, et al. (2017) Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog). Nat Genet 49:1539–1545. 21 de Laat W, Duboule D (2013) Topology of mammalian developmental enhancers and their regulatory landscapes. Nature 502:499–506. 22 Wang H, et al. (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918. 23 Dixon JR, Gorkin DU, Ren B (2016) Chromatin domains: The unit of chromosome organization. Mol Cell 62:668–680.

Paliou and Andrey PNAS | January 30, 2018 | vol. 115 | no. 5 | 841 Downloaded by guest on September 27, 2021