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Long-range enhancers regulating Myc expression are required for normal facial morphogenesis

Nature Genetics volume 46, pages 753–758 (2014)Cite this article

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Abstract

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital malformations observed in humans, with 1 occurrence in every 500–1,000 births1,2. A 640-kb noncoding interval at 8q24 has been associated with increased risk of non-syndromic CL/P in humans3,4,5, but the genes and pathways involved in this genetic susceptibility have remained elusive. Using a large series of rearrangements engineered over the syntenic mouse region, we show that this interval contains very remote cis-acting enhancers that control Myc expression in the developing face. Deletion of this interval leads to mild alteration of facial morphology in mice and, sporadically, to CL/P. At the molecular level, we identify misexpression of several downstream genes, highlighting combined impact on the craniofacial developmental network and the general metabolic capacity of cells contributing to the future upper lip. This dual molecular etiology may account for the prominent influence of variants in the 8q24 region on human facial dysmorphologies.

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Figure 1: Functional characterization of the 8q24 CL/P regulatory landscape.
Figure 2: Gene expression changes upon deletion of the MNE region.
Figure 3: Facial dysmorphologies upon deletion of the CL/P-associated 8q24 region.
Figure 4: Alterations in gene expression upon deletion of the CL/P-associated 8q24 region.

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Acknowledgements

We thank B. Sleckman (Washington University, St. Louis) and A. Trumpp (DKFZ, Heidelberg) for providing the Myctm1Slek strain. We thank members of the EMBL Laboratory Animal Resources Facility for animal welfare and husbandry; the EMBL Genomics Core Facility for advice and support in processing ChIP and RNA sequencing experiments; and Genome Biology Computational Support for help with the analyses. We thank members of the Spitz laboratory and colleagues at EMBL for sharing reagents and helpful comments. V.V.U. and M.P. were supported by PhD fellowships from the Jeff Schell Darwin Trust and the EMBL International PhD program, respectively. This work was supported by EMBL.

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Authors and Affiliations

  1. Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Veli Vural Uslu, Massimo Petretich, Sandra Ruf, Katja Langenfeld & François Spitz

  2. European Bioinformatics Institute–European Molecular Biology Laboratory (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

    Nuno A Fonseca & John C Marioni

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  1. Veli Vural Uslu
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Contributions

F.S. designed the experiments. V.V.U., M.P., S.R. and K.L. performed the experiments. N.A.F. and J.C.M. performed RNA-seq data, bioinformatics and statistical analyses. V.V.U., M.P. and F.S. analyzed the data. F.S. wrote the manuscript with V.V.U., M.P. and J.C.M.

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Correspondence to François Spitz.

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Integrated supplementary information

Supplementary Figure 1 Conserved organization of the 8q24 region in mice and humans.

Representation of the 8q24 interval (hg19, chr. 8: 127,200,000–131,500,000) from the UCSC Genome Browser64 with the 640-kb CL/P risk interval boxed3. ENCODE tracks summarizing regulatory and transcription activities (from seven cell lines) are shown65, as well as the score of evolutionary conservation of the sequence (GERP track66). The paucity of gene annotation, transcriptional activity (RNA-seq tracks) and promoter-associated chromatin marks (H3K4me3) highlights the 'gene desert' constituted by this region between PVT1 and GSDMC. The region comprises, however, many evolutionarily conserved elements (peaks in the GERP track) and potential tissue-specific enhancers (peaks in the H3K4me1 and H3K27ac tracks). The Mouse Net track shows the extensive syntenic chain linking mouse and human orthologous sequences, with extreme conservation in sequence and relative order between the two species.

Supplementary Figure 2 Deletion series to delineate the MNE region.

(a) Schematic representation of the different deletions (red bars) generated and analyzed along the interval, with the different regulatory regions identified (blue, medionasal enhancer (MNE); orange, nasal epithelial enhancer (NEE)) shown as ovals. (b–e) LacZ staining of E11.5 embryos with different deletions, highlighting the persistence or loss of the two expression domains (blue arrowhead, MNP; orange arrowhead, NC). Insets in c–e, 150-μm vibratome sections through the head of embryos, showing strong expression in the nasal epithelium of del(8–14) heterozygous embryos (c). This domain of staining is absent in del(14–15) embryos (d) and weak but present in del(15–17) embryos (e).

Supplementary Figure 3 Duplication series to delineate the MNE region.

(a) Schematic representation of the positions and LacZ expression patterns in E11.5 embryos for the 10a, 13a and 20a transposon insertions. Regulatory regions are indicated as before. The topological boundary found around the Gsdmc cluster67, which overlaps with the regulatory transition between the different landscapes, is shown with double red brackets. (b) Schematic representation of the trans-allelic Cre-mediated recombination51 used to produce the different duplications, as a reciprocal product of the deletions. (c) Representation of the different duplications and (d) associated LacZ expression in E11.5 embryos. Duplications encompassing the region (10–13) led to expression in the fronto- and medionasal processes, whereas a duplication of the region (13–20) conferred expression in the nasal epithelium only. Even though it is unclear whether topological boundaries are fully respected in the context of rearrangements68, the different expression of the LacZ sensor for the dup(10–20) and dup(13–20) alleles, which place it at the same distance from the centromeric CL/P region (blue oval), can be better explained by the contribution of enhancer elements lying in the duplicated telomeric regions.

Supplementary Figure 4 Expression levels of the genes flanking the CL/P region in the face of E11.5 embryos.

Expression levels were measured by qRT-PCR and are shown with the lowest expression levels (for Gsdmc) set as 1 (log10 scale). Error bars represent ±s.d. from four independent biological replicates. *, the primers used cannot distinguish the different tandemly duplicated Gsdmc genes.

Source data

Supplementary Figure 5 Transposon insertion does not induce expression changes.

Endogenous gene expression in the face of E11.5 embryos homozygous for expression showing the strongest LacZ expression is not different from wild-type control. Expression was determined by qRT-PCR (three biological replicates). Expression levels were normalized to Gusb levels between samples and, for each gene, represent with wild-type levels equal to 1. Error bars are ±s.d.

Source data

Supplementary Figure 6 Morphological and cellular differences between del(8–17) and wild-type mice.

(a) Comparison of different bone lengths and skull measures (IOD, interorbital distance; NBL, nasal bone length; FBL, frontal bone length; PBL, parietal bone length) in 5-week-old (n = 4 (del(8–17); n = 4 (wild-type)) mice. Del(8–17) mice showed reduced nasal and frontal bone lengths (Student's t test, P = 0.00398 and P = 0.00099, respectively). Boxplots show median, 1st and 3rd quartiles. Whiskers indicate min./max (b) Cell proliferation in the face of del(8–17) and wild-type E11.5 embryos. Mitotic cells were identified by staining for phosphorylated H3 and counted on serial sections. Each dot represents the normalized proportion of cells positive for phosphorylated H3 for a given section. Del(8–17) embryos showed slight but significant differences (Student's t test, P =1.77 ×10–6). Boxplots show median, 1st and 3rd quartiles. Whiskers indicate 1.5 IQR of the 1st and 3rd quartiles. *** indicates P < 0.005

Source data

Supplementary Figure 7 Expression changes in the face of del(8–17) embryos compared to wild-type controls.

(a) A heat map showing normalized expression values for all genes with a minimum expression of 100 reads (summed across all samples). Each row corresponds to 1 of the 13,586 genes under consideration, and the columns correspond to the different samples (black, wild type; gray, deletion). Colors show gene expression on the log2 scale (blue, low expression; yellow, high expression). (b) A heat map showing normalized expression values for differentially expressed genes. Each row corresponds to a differentially expressed gene, and columns correspond to the different samples (black, wild type; gray, deletion). Colors show gene expression on the log2 scale (blue, low expression; yellow, high expression).

Supplementary Figure 8 Reduced expression of blood-related genes in del(14–17) but not del(8–14) mice.

Several genes with restricted expression in blood cells had downregulated expression in del(8–17) versus wild-type face samples. Overall, their expression levels were low, consistent with the presence of a few small blood vessels in the dissected facial mesenchyme. qPCR analysis of expression changes for some of these genes shows that this misexpression is associated with another regulatory region, located in (14–17) and therefore distinct from the MNE. **P < 0.01, *P < 0.05, Student's t test. Error bars are ± s.d.

Source data

Supplementary Figure 9 Genetic and functional organization of the CL/P interval on 8q24.

(a) Schematic representation of the 8q24 region, from the UCSC browser. The interval showing strong association with CL/P identified by Birnbaum and colleagues3 is outlined in red, with the position of the SNP (rs987525) with the lowest P value indicated by a red bar. This interval consists of multiple LD blocks (HapMap Phased LOD track). Importantly, multiple SNPs along this broad interval showed association with CL/P, in part independently of rs987525 (refs. 3,5). The orthologous region to the (10–13) MNE is outlined in blue, with ovals showing candidate enhancer modules in the region, including the Vista hs1877 element14. (b) The critical MNE region contains two main LD blocks, as shown by Haploview, using HapMap CEU data (phase 2, r24)69.

Supplementary Figure 10 Molecular nature of the 8q24 CL/P risk factor.

The 8q24 CL/P risk interval is a remote regulatory region (MNE) that specifically controls the high levels of expression of MYC in the developing medionasal region. Genetic variation in the MNE may perturb the GRN controlling the fate of the neural crest–derived mesenchymal cells, possibly through NR2F1 and TFAP2A, and may alter the growth and metabolic potential of the medial nasal process. This imbalance may be exacerbated by environmental (or genetic) conditions, leading to defective fusions of the different facial processes.

Supplementary Figure 11 RNA quality control and primer efficiency.

(a) RNA quality measured by Bioanalyzer. RNA Integrity Number (RIN; value assigned from 0 to 10) was calculated with Agilent 2100 Bioanalyzer software. Example histograms for three samples are shown, and the minimum RIN value of the samples used for qRT-PCR was 9.10. (b) Primer efficiency was measured using four- to eightfold dilutions of the cDNA stock. Curves show log2 values for the dilution ratio plotted against Ctvalues from qRT-PCR amplification using the different primer pairs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 1, 2 and 6–10 (PDF 6789 kb)

Supplementary Table 3

Regions enriched for H3K27ac and H3K4me1. (XLS 33 kb)

Supplementary Table 4

Misexpressed genes in del(8–17) versus WT mice (P value < 0.05). (XLS 604 kb)

Supplementary Table 5

RNA-seq data for the genes surrounding the MNE. (XLS 39 kb)

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Uslu, V., Petretich, M., Ruf, S. et al. Long-range enhancers regulating Myc expression are required for normal facial morphogenesis. Nat Genet 46, 753–758 (2014). https://doi.org/10.1038/ng.2971

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