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

Journal name:
Nature Genetics
Volume:
46,
Pages:
753–758
Year published:
DOI:
doi:10.1038/ng.2971
Received
Accepted
Published online

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.

At a glance

Figures

  1. Functional characterization of the 8q24 CL/P regulatory landscape.
    Figure 1: Functional characterization of the 8q24 CL/P regulatory landscape.

    (a) The human 8q24 interval associated with CL/P risk. Genes are shown as plain arrows (black, protein-coding genes; gray, annotated noncoding transcripts). The CL/P interval3 and the most significantly associated SNP (rs987525) are indicated in blue. (b) Syntenic organization of the mouse locus, depicting transposon insertions (blue triangles) and deletions (red bars) used in this study. An expanded list of insertions and alleles is given in Supplementary Table 1. The expression patterns of adjacent insertions (shown in c) define a broad 'medionasal regulatory domain' indicated by a blue bar whose width represents relative LacZ expression levels. (c) LacZ staining of E11.5 embryos with various insertions, with insertion number indicated below. Arrowheads indicate expression in the medionasal process (blue) and in the nasal epithelium (orange). Insertions located in the Gsdmc gene cluster did not result in any expression, whereas the more telomeric ones around Fam49b resulted in a different expression pattern. (d,e) Magnified view (d) and sections (e) of E11.5 embryos with strong LacZ expression in the medionasal process (MNP; blue) and nasal epithelium (NP; orange). MX, maxillary process; MD, mandibulary process; LNP, lateral nasal process. Most insertions in the 7a–17a interval also showed LacZ expression in somites and in the limb mesenchyme of E11.5 embryos but not in heart or liver9. (f) LacZ staining in the faces of E11.5 embryos carrying the different deletions. The medionasal process and nasal epithelium expression domains can be separated by different deletions, highlighting two distinct regulatory regions, the MNE and NEE regions, respectively. See also the description of additional lines and sections in Supplementary Figures 2 and 3. Precise genomic positions are given in Supplementary Table 2. (g) Enrichment for enhancer-associated marks (blue, H3K4me1; turquoise, H3K27ac) profiled by ChIP sequencing (ChIP-seq) of E11.5 facial tissues highlighted several candidate face-specific regulatory regions (dashed boxes; positions and evolutionary conservation are given in Supplementary Table 3). One representative profile of two biological replicates is shown for each track. The blue boxed peak corresponds to Vista element hs1877, which showed enhancer activity in a number of regions, including the facial mesenchyme14.

  2. Gene expression changes upon deletion of the MNE region.
    Figure 2: Gene expression changes upon deletion of the MNE region.

    (a) Gene expression in the face of wild-type (WT) and del(8–17) homozygous E11.5 embryos (normalized to Gusb levels and displayed with the wild-type expression level set as 1). Fam84b, A1bg, Myc, Pvt1, Gsdmc, Fam49b and Asap1 (Ddef1) are in the chr. 15: 60,000,000–64,000,000 region, and Gusb and Pdhb are on chromosomes 5 and 14, respectively. The primers used for Gsdmc cannot distinguish the different isoforms of the Gsdmc genes, which are duplicated in tandem. (b) Myc expression in different tissues from E11.5 embryos. Values are expressed after normalization to Gusb levels (set as 1 in each tissue). (c) Loss of Myc expression detected by whole-mount in situ hybridization in the medionasal process (MNP) but not in the liver (L) of del(8–17) homozygous embryos relative to wild-type E11.5 embryos. Staining in control embryos is highlighted by dashed circles. (d) Myc expression in the face of E11.5 embryos heterozygous for different deletions. Values are expressed after normalization to Gusb levels (set as 1 in each tissue). (e) Allele-specific analysis of Myc expression in the faces of E11.5 embryos showed that the MNE region acts in cis. The reference allele Myctm1Slek carries an insertion of GFP in the second exon of Myc15, and its expression is detected with GFP-specific primers. Expression levels of the other alleles (wild type or del(8–17)) were determined by subtracting GFP expression levels from the overall Myc expression levels (the Myc-specific primers used amplified both alleles). Myc expression levels are significantly lower in del(8–17) than in wild-type mice (Student's t test, P < 5.6 × 10−5), whereas GFP expression is instead higher. In all charts, error bars represent s.d. ***P < 0.005, Student's t test; NS, not statistically significant (P > 0.05).

  3. Facial dysmorphologies upon deletion of the CL/P-associated 8q24 region.
    Figure 3: Facial dysmorphologies upon deletion of the CL/P-associated 8q24 region.

    (a) Comparison of the skulls of del(8–17) homozygous mice to those of their wild-type littermates. Dorsal views of representative skulls from 3-week-old wild-type and mutant (del(8–17)) mice, stained with Alcian blue (cartilage) and Alizarin red (bone). IOD, interorbital distance; NBL, nasal bone length. Scale bars, 5 mm. (b) Enlarged views of the frontonasal regions showing the abnormal suture in del(8–17) mice (highlighted by dashed lines). (c) Comparison of different bone lengths and skull measures (FBL, frontal bone length; PBL, parietal bone length) in 3-week-old (del(8–17), n = 5; wild type, n = 7) mice. Del(8–17) mice showed reduced nasal and frontal bone lengths (Student's t test, P = 0.0033 and 0.0028, respectively). Box plots show medians and first and third quartiles. Whiskers indicate minimum and maximum values. (d) Comparison of the widths of the lateral and medial parts of the developing face in wild-type (n = 3) and del(8–17) (n = 4) E11.5 embryos. The landmarks (arrows) used to compare the lateral and medial widths of the developing faces of E11.5 embryos are shown on the left. NS = not significant. (e) Quantification of proliferation in the faces of wild-type (n = 5) and (del(8–17) homozygous (n = 7) E11.5 embryos. Values represent the proportion of mitotic cells, determined by staining for phosphorylated histone H3, averaged from 10–30 serial sections per embryo. In d and e, box plots show medians and first and third quartiles. Whiskers indicate 1.5 times the interquartile ranges of the first and third quartiles. *P = 0.011 (Student's t test). (f) Most E14.5 del(8–17) embryos have normal face morphology (top), but a minority have CL/P (arrow) with other craniofacial malformations (bottom). In all panels, statistical significance was determined with Student's t tests. ***P < 0.005, *P < 0.05.

  4. Alterations in gene expression upon deletion of the CL/P-associated 8q24 region.
    Figure 4: Alterations in gene expression upon deletion of the CL/P-associated 8q24 region.

    (a) Changes in expression measured by RNA sequencing in the medial faces of del(8–17) homozygous embryos (four replicate libraries from four different embryos) compared to wild-type controls (four replicate libraries from littermates). Genes with significant (FDR < 0.05) changes in expression are shown in red. See Supplementary Table 4 for a list of the misregulated genes and Supplementary Figure 7 for heat-map representations of the data. RNA sequencing analysis also showed downregulation of several blood-specific genes; their presence arose from the small blood vessels in the dissected tissues and suggested an additional role of the 8–17 interval in hematopoiesis, in which Myc has an important role49. Notably, the genomic region involved appears to be distinct from the MNE region, as the downregulation of Apoe and Csf1 was observed in del(14–17) but not in del(8–14) embryos (Supplementary Fig. 8). (b) Validation by qPCR of expression changes for some of the genes identified by RNA sequencing. The levels of Rplp1 and Rps20 are significantly (***P < 0.005, **P < 0.01, Student's t test) decreased to similar extents in the medial faces of del(8–17) (n = 4 to 7) and del(8–14) (n = 3) homozygote E11.5 embryos but not in del(14–17) (n = 3) homozygote E11.5 embryos. Nr2f1 levels also appeared lower (*P < 0.05, Student's t test). Error bars, s.d.

  5. Conserved organization of the 8q24 region in mice and humans.
    Supplementary Fig. 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.

  6. Deletion series to delineate the MNE region.
    Supplementary Fig. 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).

  7. Duplication series to delineate the MNE region.
    Supplementary Fig. 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.

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

  9. Transposon insertion does not induce expression changes.
    Supplementary Fig. 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.

  10. Morphological and cellular differences between del(8-17) and wild-type mice.
    Supplementary Fig. 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

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

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

  13. Genetic and functional organization of the CL/P interval on 8q24.
    Supplementary Fig. 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.

  14. Molecular nature of the 8q24 CL/P risk factor.
    Supplementary Fig. 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.

  15. RNA quality control and primer efficiency.
    Supplementary Fig. 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 Ct values from qRT-PCR amplification using the different primer pairs.

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Author information

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

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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Conserved organization of the 8q24 region in mice and humans. (281 KB)

    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.

  2. Supplementary Figure 2: Deletion series to delineate the MNE region. (298 KB)

    (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).

  3. Supplementary Figure 3: Duplication series to delineate the MNE region. (133 KB)

    (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.

  4. Supplementary Figure 4: Expression levels of the genes flanking the CL/P region in the face of E11.5 embryos. (70 KB)

    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.

  5. Supplementary Figure 5: Transposon insertion does not induce expression changes. (68 KB)

    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.

  6. Supplementary Figure 6: Morphological and cellular differences between del(8–17) and wild-type mice. (72 KB)

    (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

  7. Supplementary Figure 7: Expression changes in the face of del(8–17) embryos compared to wild-type controls. (153 KB)

    (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).

  8. Supplementary Figure 8: Reduced expression of blood-related genes in del(14–17) but not del(8–14) mice. (96 KB)

    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.

  9. Supplementary Figure 9: Genetic and functional organization of the CL/P interval on 8q24. (501 KB)

    (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.

  10. Supplementary Figure 10: Molecular nature of the 8q24 CL/P risk factor. (194 KB)

    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.

  11. Supplementary Figure 11: RNA quality control and primer efficiency. (174 KB)

    (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 Ct values from qRT-PCR amplification using the different primer pairs.

PDF files

  1. Supplementary Text and Figures (6,952 KB)

    Supplementary Figures 1–11 and Supplementary Tables 1, 2 and 6–10

Excel files

  1. Supplementary Table 3 (33 KB)

    Regions enriched for H3K27ac and H3K4me1.

  2. Supplementary Table 4 (619 KB)

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

  3. Supplementary Table 5 (39 KB)

    RNA-seq data for the genes surrounding the MNE.

Additional data