Can DNA enhance your look?

The shape of the face is one of the features that most distinguishes individual humans from one another. Differences in facial morphology have substantial implications in many areas, including social interaction, psychology, forensics, and clinical genetics (1–3). The resemblance of facial shapes within families in general, and between monozygotic twins in particular, suggests a major contribution of genetic factors to craniofacial morphology (4–6). Many protein-coding genes whose disruption causes major aberrations of craniofacial morphology are known. This includes pathological dysmorphologies of the face itself, such as clefts of the lip or palate, as well as distinctive facial features associated with genetic syndromes that are indicative of associated pathologies in other organ systems (7–14). In contrast to these disease-related genes, only a small number of candidate genes have been implicated in normal variation of craniofacial shape through genome-wide association studies, and collectively they explain only a minute fraction of the morphological variation observed in human populations (15–17). We are interested to understand how complex traits such as the individualized shape of the face can be modulated in subtle ways while avoiding the often severe consequences associated with protein-coding mutations (18).

Recent observations of large numbers of distant-acting transcriptional enhancers in mammalian genomes (19–21) raise the possibility that these sequences regulate development of structures such as the craniofacial complex. Enhancers can be located hundreds of kilobases away from their target genes and typically have highly restricted in vivo activity patterns. They often control the expression of their target genes in a modular fashion, where different enhancers activate the expression of the same gene in different cell types, anatomical regions, or at different developmental time points. In principle, such complex arrays of enhancers acting on individual genes may provide a general mechanism for the independent fine-tuning of distinct aspects of gene expression in different developmental processes, which in turn may affect specific phenotypic traits, including facial shape (22). This model is consistent with the extensive studies of the genes and gene regulatory networks involved in the development of the neural crest, a cell population contributing to multiple tissues, including facial bone and cartilage (23). In-depth studies of individual genes involved in neural crest development [e.g., (24–26)], as well as genome-wide studies of regulatory sequences active in human neural crest cells (27), support that many genes involved in craniofacial development are associated with complex regulatory architecture. We used chromatin immunoprecipitation on whole face tissue to explore the genome-wide landscape of craniofacial enhancers in mice, and we studied enhancer involvement in defining mouse craniofacial morphology using transgenic reporter assays and enhancer knockout studies.

Identification of in Vivo Craniofacial Enhancers

To identify craniofacial developmental enhancers on a genome-wide scale, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) analysis on mouse embryonic-day 11.5 (e11.5) facial tissue with the enhancer-associated p300 protein (Fig. 1) (21). At this developmental time point, key events of craniofacial development are in progress, including growth and morphogenetic processes affecting the size, shape, and structure of all major craniofacial prominences (28, 29). All major facial subregions were included in this tissue preparation (30), building on the previously described efficiency of this inclusive approach to identify enhancers with both broad and tightly confined patterns in subregions of developing embryonic structures (31,32).

Enrichment analysis identified 4399 distal candidate enhancers genome-wide, defined as regions that showed significant p300 binding in craniofacial tissue and were at least 2.5 kb from known transcription start sites (Fig. 2and tables S1 and S2). Candidate enhancers were located up to 1.4 Mb (median distance, 44 kb) from the nearest known transcript start site, with 38.4% in introns of genes and 54.7% located in noncoding regions outside of genes (intergenic). The majority of candidate enhancers also showed evidence of evolutionary constraint (87.5%) (table S1) and had unique orthologous sequences in the human genome (96.7%). Unbiased ontology analysis (33) revealed that candidate craniofacial enhancers are enriched near genes that are known to cause craniofacial phenotypes when deleted in mouse models or mutated in humans (table 1). Candidate craniofacial enhancers were also enriched at loci implicated in human craniofacial traits and birth defects through genome-wide association studies (fig. S1). These observations are consistent with a role of the identified enhancer candidate sequences in the regulation of genes with known roles in craniofacial development. Taken together, these results suggest that thousands of distant-acting enhancers are involved in orchestrating the genome-wide gene expression landscape during craniofacial development.

Large-Scale Transgenic Analysis of Craniofacial Enhancers

ChIP-Seq performed directly on craniofacial tissues provided a genome-wide catalog of sequences that are likely to be active in vivo enhancers during craniofacial development at e11.5. However, this approach does not provide direct insight into the exact activity patterns of individual candidate enhancer sequences. To examine craniofacial enhancer activity patterns in detail, we used transgenic enhancer reporter assays in mice, coupled to high-resolution three-dimensional (3D) mapping of LacZ reporter activities by means of optical projection tomography (OPT) (Fig. 1) (30, 34, 35). Because many, but not all, in vivo enhancers can be identified through p300 binding (36), we also considered sequence conservation (34) and proximity to genes or loci with a known role in craniofacial development as additional criteria in the selection of candidate sequences. In total, we tested 205 candidate sequences in transgenic mice, with the majority (123, or 60%) located within or near regions associated with craniofacial development through experimental, genetic, or genome-wide association studies (properties of all tested candidate sequences are provided in table S3). Each candidate enhancer sequence was coupled to a minimal promoter and used to generate multiple transgenic embryos by means of pronuclear injection (30). Only patterns that were independently observed in at least three different embryos were considered reproducible. In total, 121 of 205 tested sequences showed reproducible reporter gene expression in at least one craniofacial structure. We further extended the set of in vivo–characterized craniofacial enhancers by reexamining data from previously described large-scale enhancer screens not specifically targeted at craniofacial enhancer discovery (21, 31, 32, 34, 37–39), providing an additional 75 craniofacial enhancers (table S3). Transgenic results for the 196 craniofacial enhancers identified or reexamined in this study are available through the Vista Enhancer Browser (http://enhancer.lbl.gov) or the National Institute of Dental and Craniofacial Research (NIDCR) FaceBase consortium web site (http://facebase.org) (40).

To gain higher-resolution insight into the 3D activity patterns of craniofacial enhancers in the context of developing embryos, we used optical projection tomography (OPT). In total, representative embryos for 55 craniofacial enhancers, including 48 from this study, were analyzed with OPT. Selected examples of 3D views are provided as supplementary movies (movies S1 to S11). More comprehensive OPT data collections can be interactively explored through a dedicated viewer at the NIDCR FaceBase database (fig. S2) (40). Examination of this large set of in vivo–validated and –characterized craniofacial enhancers highlights several salient features and resulting potential applications of these data sets, which we will describe using selected examples. Specifically, this collection of enhancers (i) highlights the diversity of enhancer activity patterns and the regulatory complexity of the genetic code, (ii) enables the dissection of the regulatory landscapes of individual genes known to be involved in craniofacial development, and (iii) provides a starting point for the mechanistic exploration of genomic intervals implicated in craniofacial development through genome-wide association studies.

Diversity of Patterns

To illustrate the reproducibility and diversity of craniofacial activity patterns identified in transgenic embryos, selected examples of enhancers identified in this study are shown in Fig. 3A. For all craniofacial prominences (medial nasal, lateral nasal, maxillary, and mandibular), structure-specific active enhancers were identified (Fig. 3A; a schematic view of the e11.5 mouse face is provided in fig. S4A). In depth analysis of craniofacial activity patterns through the combined use of whole-mount LacZ staining and OPT imaging revealed that in many cases only subregions of these structures were reproducibly targeted by an enhancer. For example, enhancer mm387 drives expression in the anterior part of the maxillary prominence, whereas enhancer mm458 is restricted to a posterior ventral region (Fig. 3B, top). Similar region-specific activities are observed in other facial substructures—such as the nose, where enhancer mm933 is active in the medial nasal prominence, whereas the activity of enhancer mm426 is confined to the lateral nasal prominence (Fig. 3B, top). OPT scans of whole-mount embryos provide additional spatial information about enhancer activity pattern by capturing the activity signal in internal embryonic structures (Fig. 3B, bottom). These data highlight the complexity, diversity, and spatially highly restricted activity patterns of distant-acting enhancer sequences active during craniofacial development.

Regulatory Landscap?es of Craniofacial Genes

Systematic screening of individual genomic loci via ChIP-Seq followed by transgenic characterization enables functional dissection of the distant-acting enhancer landscapes of individual genes with known roles in craniofacial development. As an example, mouse Msx1 and human MSX1 have been studied for their role in craniofacial development (supplementary text) (41). Msx1 is surrounded by several hundred kilobases of noncoding DNA, which renders the search for distant-acting enhancers challenging. Transgenic testing of seven candidate sequences identified with ChIP-Seq and located up to 235 kb away from the Msx1 transcription start site resulted in the identification of five distinct craniofacial enhancers potentially regulating its expression (Fig. 4A). At e11.5, each of these enhancers drove patterns that partially recapitulated the endogenous Msx1 RNA expression. For instance, Msx1 activity in the second branchial arch and in the maxillary process of the e11.5 embryo is recapitulated by the combined activity of two separate enhancers located at 1 and 235 kb upstream of the promoter (mm426 and hs746) (Fig. 4A). These observations support the notion that complex spatial expression patterns of key developmental genes are driven by modular arrays of distant-acting enhancers (42) and highlights the potential of enhancers to provide a mechanism for fine tuning of in vivo gene expression patterns.

Craniofacial Enhancers Within Disease-Associated Intervals

To illustrate the utility of these enhancer data sets in the follow-up of genome-wide association, population-scale sequencing, and candidate locus studies, 50 candidate enhancers mapping to intervals implicated in craniofacial morphology or orofacial birth defects through human genetic studies were included in the transgenic assays (table S3). Trait-associated variants that map to noncoding genome regions or are not linked to any protein-altering variants are a common challenge in the interpretation of such genetic studies. A prototypical example is a region of human chromosome 8q24 that is devoid of protein-coding genes. A 640-kb stretch located within this region is a major susceptibility locus for cleft palate, with a calculated population attributable risk of 41% (43–45). Variants at this locus are also significantly linked to normal variation in several facial morphology traits (16). We identified four craniofacial enhancer candidate sequences in the mouse genome region orthologous to the human risk interval, two of which drive reproducible craniofacial reporter activity at e11.5 in transgenic mice (Fig. 4B). As a second example, we examined the 1p22 locus. In this interval, markers located near and within the ABCA4 gene are associated with an increased risk for cleft palate in humans, but it remains unclear whether these variants are linked to deleterious protein-coding mutations of ABCA4 (46, 47). On the basis of RNA expression data, the neighboring gene ARHGAP29, rather than ABCA4 itself, has been proposed to be causatively involved in craniofacial development (48). However, ARHGAP29 falls outside the genomic boundaries of the risk-associated linkage block. By scanning the region comprising these two genes for possible associated enhancers, we identified a human-mouse conserved sequence in the first intron of Abca4 that drove highly-reproducible reporter activity in the facial midline, a pattern reminiscent of Arhgap29 RNA expression, suggesting that this enhancer may drive expression of Arhgap29 during craniofacial development (Fig. 4C and movie S10) (49). A causative effect of sequence or copy number variants in these particular enhancers on craniofacial morphology remains to be demonstrated; furthermore, we cannot exclude the existence of additional enhancer sequences at these loci that were not captured in the present screen. These possible limitations notwithstanding, our results illustrate the utility of collections of validated enhancers as starting points for the mechanistic interpretation of human genetic studies by linking functional genomic and human genetic data sets.

Targeted Deletions of Craniofacial Enhancers

The existence of large numbers of distant-acting enhancers with precise tissue-specific activities during craniofacial development raises the question of their functional impact on craniofacial morphology through the regulation of their respective target genes. To examine such contributions in more detail, we selected three enhancers with highly reproducible craniofacial activity patterns and explored their functions through targeted deletions in mice (Fig. 1). The three enhancers—termed hs1431 (near Snai2), hs746 (near Msx1), and hs586 (near Isl1)—were chosen on the basis of their association with known craniofacial genes (supplementary text) (7,50, 51), the robustness of their activity patterns, and the absence of additional known enhancers with overlapping activity near the same gene. Furthermore, the in vivo activity patterns driven by these enhancers partially recapitulate the known expression patterns of their presumptive target genes (Fig. 4A and fig. S3). The enhancers were intentionally chosen from different, functionally unrelated loci in order to provide a representative sample of the genome-wide enhancer data set, rather than an in-depth exploration of a single gene or pathway. All selected enhancers are located at a very long distance from their respective target genes (350, 235, and 190 kb, respectively) and are active in the craniofacial complex through multiple stages of embryonic development (Figs. 4A and 5, fig. S3, and movies S1 to S9).

To test whether these enhancers are important in modulating craniofacial morphology, we created three separate mouse lines carrying deletion alleles for each of the three enhancers using a standard homologous recombination strategy in embryonic stem cells (30). Mice homozygous for any of the three enhancer deletions do not display gross craniofacial malformations or other obvious deficiencies. To evaluate the effect of each enhancer deletion on the expression of the presumptive target genes (Snai2, Msx1, and Isl1), we used quantitative reverse transcription polymerase chain reaction to measure transcript levels in different craniofacial structures of individual wild-type and enhancer deletion embryos (littermates) at e11.5 and e13.5 (Fig. 6 and fig. S4). Depending on time-point and substructure, we observed up to 3.9-fold down-regulation (P = 4 x 10^–5) of Snai2 in homozygous Δhs1431 embryos, 1.5-fold down-regulation (P = 0.015) of Msx1 in Δhs746, and 1.3-fold down-regulation (P = 0.04) of Isl1 in Δhs586 (Fig. 6, C and D, and fig. S4E). In all cases, the changes in transcript levels of the respective target gene were confined to subregions in which the enhancer was active. However, not all subregions with enhancer reporter activity showed significant down-regulation of the target gene. These observations raise the possibility of partial functional redundancy between the enhancers studied here and overlapping regulatory activities from gene promoters or additional distant-acting enhancers that were not captured in our genome-wide screen. Regardless of the presence of possible additional regulatory sequences in these genome intervals, these results provide evidence for the requirement of enhancers for normal gene expression during craniofacial development.

To examine whether the deletion of these enhancers altered craniofacial morphology, we compared mouse skulls from wild-type and enhancer deletion mice at 8 weeks of age. Because it is challenging to quantify possible differences in craniofacial morphology with visual observation alone, we used micro-computed tomography (micro-CT) to obtain accurate 3D measurements of the skulls. Three cohorts, each consisting of at least 30 mice homozygous for a deletion of one of the three enhancers, were compared with a cohort of 44 wild-type littermates. Micro-CT reconstructions of each mouse head were measured by using 54 standardized skeletal landmarks (fig. S5). The cohorts of wild-type and enhancer deletion mice were compared by using canonical variate analysis (CVA) to identify possible changes in craniofacial morphology resulting from the enhancer deletions (Fig. 7). Procrustes analysis of variance (ANOVA) (F = 12.0, P < 0.0001) and multivariate ANOVA (Pillau’s Trace 2.5, P < 0.0001) tests both showed that enhancer deletion genotypes were significantly associated with alterations of craniofacial shape. All individual pair-wise permutation tests (Procrustes distances) between wild-type and enhancer deletion lines revealed significant differences (table S4), with the most pronounced differences observed for Δhs1431 and Δhs746 (both P < 0.0001 compared with wild-type). Differences between wild-type, Δhs1431, and Δhs746 mice were also significant after Bonferroni adjustment for the six pairwise comparisons between groups. The largest magnitude of effect on shape was observed for Δhs1431, followed by an intermediate quantitative effect for Δhs746 (Fig. 7B), whereas possible changes in Δhs586 were not statistically significant after correction for multiple hypothesis testing. These results mirror the magnitude of expression phenotypes, which were most pronounced in Δhs1431, followed by intermediate changes in Δhs746 and only a limited expression phenotype observed in Δhs586 (Fig. 6 and fig. S4). These results show that deletion of enhancers can affect craniofacial morphology.

Each enhancer deletion causes a distinct set of differences as compared with wild-type morphology. This is evident from the CVA, in which the first three canonical variates (CV1 to CV3) most clearly separate wild-type mice from Δhs1431, Δhs746, and Δhs586, respectively (Fig. 7). Each enhancer deletion produces phenotypic effects that are not confined to a single feature but involve multiple regions of the skull (Fig. 7C and movies S12 to S20). For example, deletion of hs1431 results in an increase in facial length, a relative increase in the width of the anterior neurocranium, and a shortening of the anterior cranial base. In contrast, Δhs746 results in a shortening of the face, a widening of the posterior neurocranium, a narrowing of the palate, and shortening of the cranial base. Although both Δhs1431 and Δhs746 have significant effects on facial morphology in structures derived from regions with enhancer activity at e11.5 and e13.5 (Fig. 6), there are also changes in other parts of the skull. These correlated patterns of change are consistent with numerous studies demonstrating that cranium development is a highly integrated process and that variation of the skull is structured by complex interactions between the growing chondrocranium, neurocranium, and other nearby tissues (52, 53). Regardless of the precise molecular pathways and developmental mechanisms that underlie the morphological changes observed upon deletion of these enhancers, these results demonstrate that distant-acting enhancers contribute to the development of craniofacial shape in mammals. The observation of significant but nonpathological alterations of craniofacial morphology as a result of enhancer deletions supports the notion that enhancers contribute to normal variation in facial shape.

Conclusions

The general shape of the human face and skull, the differences in facial shape between individuals, and the high heritability of facial shape are subjects of broad interest because they have far-reaching implications well beyond basic scientific and biomedical considerations. In this study, we examined the possible impact of distant-acting regulatory sequences on craniofacial morphology. Throughout the genome, we identified several thousand sequences that are likely to be distant-acting enhancers active in vivo during mammalian craniofacial development. Although this epigenomic analysis was performed in the mouse, the vast majority of these enhancer candidate sequences are conserved between mouse and human. Large-scale characterization of more than 200 candidate sequences in transgenic mice showed the versatility of enhancers in orchestrating gene expression during craniofacial development. These observations are consistent with genome-wide analyses of enhancers active in human neural crest cells, as well as studies of regulatory sequences associated with individual members of the neural crest gene regulatory network (23–27). We also demonstrated that deletion of craniofacial enhancers results in nonpathological but measurable changes in craniofacial morphology in mice. Taken together, these data support that enhancers are involved in determining craniofacial shape. Systematic genome-wide studies of normal morphological variation in human populations are beginning to emerge (15–17) and will offer the opportunity to compare in vivo–derived genome-wide maps of craniofacial enhancers identified in this study with variation data in order to gain further mechanistic insight into the molecular underpinnings of human facial shape and variation therein.

Beyond the spectrum of normal morphological variation in craniofacial shape, these results also provide a functional genomic framework for the analysis of craniofacial birth defects. We showed that deletion of craniofacial enhancers results in noticeable but nonpathological changes in morphology. Even for Δhs1431, the enhancer deletion resulting in the most severe reduction in craniofacial gene expression, the morphological phenotype was overall much less severe than the pathological changes observed upon deletion of the Snai2 gene itself (54). This milder phenotype is not surprising, considering that remaining baseline activity of the gene was observed in all craniofacial structures examined (Fig. 6A and fig. S4C). Although some enhancer deletions may lead to more severe phenotypes (26), these observations highlight the potential of enhancers to modulate craniofacial morphology in quantitatively subtle ways, without the pathological consequences potentially associated with deleterious protein-coding mutations. These results raise the possibility that sequence or copy number variation affecting more than one enhancer of the same gene may cumulatively result in more severe and potentially pathological phenotypes. Isolated examples of sequence variants in distant-acting enhancers associated with malformations such as clefts of the lip or palate have been described (49), and there is circumstantial evidence that noncoding sequences, including enhancers, contribute substantially to these processes (43). There is partial overlap between loci involved in normal facial shape variation and in craniofacial birth defects, supporting the possibility that some dysmorphologies represent the extreme ends of the normal spectrum of variation (15, 16). The improved genome-wide functional annotation of craniofacial in vivo enhancers obtained through this study is expected to aid not only in the functional exploration of isolated studies of craniofacial dysmorphologies but may also facilitate an understanding of the links between normal and pathological variation in craniofacial shape.

Supplementary Materials

www.sciencemag.org/content/342/6157/1241006/suppl/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S6

Tables S1 to S6

References (62–91)

Movies S1 to S20

References and Notes 

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