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. 2012 Dec 11;109(50):20204-11.
doi: 10.1073/pnas.1217659109. Epub 2012 Nov 7.

Impact of copy number variations (CNVs) on long-range gene regulation at the HoxD locus

Thomas Montavon  1 , Laurie Thevenet, Denis Duboule
Affiliations

Impact of copy number variations (CNVs) on long-range gene regulation at the HoxD locus

Thomas Montavon et al. Proc Natl Acad Sci U S A. .

Abstract

Copy number variations are genomic structural variants that are frequently associated with human diseases. Among these copy number variations, duplications of DNA segments are often assumed to lead to dosage effects by increasing the copy number of either genes or their regulatory elements. We produced a series of large targeted duplications within a conserved gene desert upstream of the murine HoxD locus. This DNA region, syntenic to human 2q31-32, contains a range of regulatory elements required for Hoxd gene transcription, and it is often disrupted and/or reorganized in human genetic conditions collectively known as the 2q31 syndrome. Unexpectedly, one such duplication led to a transcriptional down-regulation in developing digits by impairing physical interactions between the target genes and their upstream regulatory elements, thus phenocopying the effect obtained when these enhancer sequences are deleted. These results illustrate the detrimental consequences of interrupting highly conserved regulatory landscapes and reveal a mechanism where genomic duplications lead to partial loss of function of nearby located genes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Long-range transcriptional control of Hoxd genes during limb development. (A) The mammalian HOXD cluster is flanked by two conserved gene deserts on its centromeric (CEN) and telomeric (TEL) sides (Upper). In humans, multiple structural variants are found within this interval and are associated with various limb malformations. Some of them are shown with blue arrowheads indicating breakpoints for either translocations (t) or an inversion (inv), which modified this interval. (Lower) Examples of haplo-insufficient deletions (orange lines) and duplications (double green lines) are depicted. (B) Enlargement of the mouse syntenic region, including the HoxD cluster and the centromeric gene desert. In developing digits (schematized limb bud; blue territory), the coordinated expression of the Hoxd13 to Hoxd10 genes as well as of Lnp and Evx2 is under the control of a regulatory archipelago, which consists of multiple regulatory islands located either within the gene desert (ovals I–V and GCR) or between Lnp and Hoxd13 (Prox). (C) Chromatin looping brings these various elements to the vicinity of their target gene promoters, thus forming a transcriptionally active conformation (19).
Fig. 2.
Fig. 2.
A set of nested inversions disrupts the regulatory archipelago controlling Hoxd gene transcription in digits. (A) Map of the centromeric gene desert along with the positions of the various LoxP sites located within the HoxD regulatory archipelago (red triangles) used for the inversions, deletions, and duplications shown in this study. (B) The WT genomic context of the HoxD cluster is shown in Left, with the location of a remote loxP site within the Itga6 gene used for the set of nested inversions described in C–F. Gray rectangles represent genes, and the HoxD cluster is in white. (Right) The expression of Hoxd13 in an E12.5 limb bud is depicted as well as a WT hand skeleton at birth. (C) A 3-Mb large inversion separates the HoxD cluster from its regulatory elements and thus, abrogates all Hoxd13 expression in developing digits. The resulting phenotype is identical to a full deletion of the HoxD cluster (24). (D) A smaller inversion separating the gene desert from the HoxD cluster maintains a limited Hoxd13 expression to a faint posterior domain. Only the GCR and Prox elements keep their vicinity to Hoxd13. (E) An inversion separating the distal half of the gene desert leads to a decreased anterior expression of Hoxd13 and a shortening of digit II at birth. In this inversion, islands I and II have been removed from the archipelago. (F) An inversion leaving the gene desert uninterrupted had no detectable impact on either Hoxd13 expression or limb morphology. All specimens are homozygous for the various indicated inversions.
Fig. 3.
Fig. 3.
Morphological effects of inducing duplications within the regulatory archipelago. (A) The various duplications were produced by using the same LoxP sites as for Fig. 2 (red arrowheads), and they are depicted by double thick black lines. Below the set of duplications, the position of the del(SB-Atf2) is indicated by a dashed gray line. (B–G) Schematics of the locus after the various duplications (or deletions) were produced are shown in Left, with a hand skeleton at birth shown in Right. In vivo, each configuration was balanced by a chromosome carrying a deletion of Hoxd13 to Hoxd8 [the Del(8–13) allele, indicated as ∆]. For the sake of simplicity, the three segments of the regulatory archipelago, as defined by the positions of LoxP sites, are highlighted using different colors (control in B). In B–G, these colors are used to identify the parts of the archipelago that are duplicated (C–F) or deleted (G). In all cases, LacZ reporter genes were associated with the various configurations. They are indicated on the schemes by a blue rectangle along with the presence of the associated LoxP site (red arrowheads). In E, two such LacZ reporters are present (in the text). (B) WT configuration. (C) Duplication of the full archipelago from Evx2 to Atf2. (D) Duplication of islands I–V. (E) A duplication extending from Evx2 until the proximal half of the gene desert is associated with a shortening of digit II at birth (arrowhead). (F) Duplication of the Prox-GCR segment. (G) Deletion of the distal half of the gene desert complementary to the duplication in E. Note the similar shortening of digit II (arrowhead).
Fig. 4.
Fig. 4.
Duplications induce a partial loss of Hoxd gene expression. (A) Schemes of the genetic configurations are as for Fig. 3. (B–G) Hoxd13 expression at E12.5 in the various mutant configurations. Each allele is balanced with a Del(8–13) chromosome (∆). Dup(Nsi-Atf2) (C) and Dup(Rel5-Atf2) (D) do not affect the Hoxd13 expression domain. In contrast, Hoxd13 expression is lost in the anterior part of Dup(Nsi-SB) distal limbs (E) and significantly decreased in Dup(Rel1-Rel5) (F). Similar reductions are observed in embryos carrying a deletion of the distal gene desert (G). (H) RT-qPCR analysis of Hoxd gene expression levels in E12.5 developing digits of embryos homozygous for the various genetic rearrangements. Dup(Nsi-SB) and Del(SB-Atf2) elicit a similar down-regulation of Hoxd13 to Hoxd10. Milder decreases are observed in Dup(Rel1-Rel5). Dup(Nsi-Atf2) and Dup(Rel5-Atf2) are not associated with significant changes in Hoxd gene expression levels. A supernumerary copy of Hoxd11 is associated with Dup(Nsi-Atf2) and Dup(Nsi-SB) as an Hoxd11LacZ reporter gene, and Hoxd11 levels were, thus, not assessed in these configurations (N.A.). The WT levels are set to one for each gene. Error bars indicate SD (n = 4).
Fig. 5.
Fig. 5.
Expression levels of the two duplicated genes Evx2 and Lnp. (A) Schemes of the various genetic configurations. (B and C) RT-qPCR analysis of Lnp and Evx2 expression levels in E12.5 developing digits dissected from embryos homozygous for the various rearrangements. For each allele, the number of copies of these two genes is indicated above the graphs. Both genes are clearly expressed at higher levels in the Dup(Nsi-Atf2) allele but not other configurations, where they are present in two copies, indicating an influence of genomic topology rather than a mere consequence of copy number. Lnp and Evx2 mRNAs levels do not correlate with the impact of these modified configurations on Hoxd gene expression. The WT levels are set to one for each gene. Error bars indicate SD (n = 4).
Fig. 6.
Fig. 6.
Reporter gene expression in the modified configurations. (A–D) Scheme of the genetic configurations (Left) along with the expression profiles of the associated LacZ reporter genes (Right). The parental alleles used to produce the duplications are associated with different LacZ insertions within the regulatory landscape (A and B). Although the expression of these reporter transgenes slightly varies with insertion sites, with specific domains in the posterior mesoderm, the proximal limb (presumptive forearm), and the CNS, they all display the same expression pattern in developing digits. (C and D) The Dup(Nsi-SB) and Dup(Nsi-Atf2) rearrangements are associated with a distinct pattern in the CNS, but they do not display an altered digit expression compared with parental configurations. (E) RT-qPCR analysis of LacZ expression levels in E12.5 developing digits of embryos heterozygous for the duplicated configurations indicated similar RNA levels in both duplications, although only the Dup(Nsi-SB) elicited a phenotype. Error bars indicate SD (n = 4).
Fig. 7.
Fig. 7.
The duplication interferes with regulatory interactions. (A) 4C analysis with a viewpoint in Hoxd13 (orange bar) showing long-range interactions over the centromeric gene desert in developing digits of E12.5 WT (green) or Dup(Nsi-SB) (blue) embryos. The red profile displays the ratio (log2 scale) of Dup/WT intensities. The duplicated interval is highlighted in light blue over the profiles. Hoxd13 interactions are increased with sequences within the duplicated segment (gray arrowhead) and decreased with sequences located farther centromeric (black arrowheads). The x axis shows chromosomal coordinates (mm8, 2006 University of California, Santa Cruz assembly) in megabases and the y axes are the ratio of 4C-amplified/genomic DNA intensities. (B) Similar analysis with a viewpoint taken within island I. (C) Enlargement of the HoxD cluster from B. The interactions of island I with the 5′ HoxD cluster are reduced (arrowheads). The light gray bar highlights the sequence corresponding to the Hoxd11LacZ reporter gene present at two positions on this genetic configuration (Fig. 3). (D and E) Distinct conformations in WT or Dup(Nsi-SB) digits, with schematic representation of the transcriptional output in developing digits. (D) In the WT situation, the locus adopts an active conformation, bringing the various islands in the vicinity of the HoxD cluster. (E) The duplication impairs the association of the distal elements (orange circles) with the cluster. The scheme represents one of the possible conformations of the locus, because the interactions experienced by each of the duplicated copies of the islands (purple and green circles) cannot be discriminated with our approach.
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