Abstract
Haploinsufficiency for Nipbl, a cohesin loading protein, causes Cornelia de Lange Syndrome (CdLS), the most common “cohesinopathy”. It has been proposed that the effects of Nipbl-haploinsufficiency result from disruption of long-range communication between DNA elements. Here we use zebrafish and mouse models of CdLS to examine how transcriptional changes caused by Nipbl deficiency give rise to limb defects, a common condition in individuals with CdLS. In the zebrafish pectoral fin (forelimb), knockdown of Nipbl expression led to size reductions and patterning defects that were preceded by dysregulated expression of key early limb development genes, including fgfs, shha, hand2 and multiple hox genes. In limb buds of Nipbl-haploinsufficient mice, transcriptome analysis revealed many similar gene expression changes, as well as altered expression of additional classes of genes that play roles in limb development. In both species, the pattern of dysregulation of hox-gene expression depended on genomic location within the Hox clusters. In view of studies suggesting that Nipbl colocalizes with the mediator complex, which facilitates enhancer-promoter communication, we also examined zebrafish deficient for the Med12 Mediator subunit, and found they resembled Nipbl-deficient fish in both morphology and gene expression. Moreover, combined partial reduction of both Nipbl and Med12 had a strongly synergistic effect, consistent with both molecules acting in a common pathway. In addition, three-dimensional fluorescent in situ hybridization revealed that Nipbl and Med12 are required to bring regions containing long-range enhancers into close proximity with the zebrafish hoxda cluster. These data demonstrate a crucial role for Nipbl in limb development, and support the view that its actions on multiple gene pathways result from its influence, together with Mediator, on regulation of long-range chromosomal interactions.
Highlights
Cohesin, a ring-shaped, DNA-associated protein complex, is best known for its role in tethering sister chromatids together until mitosis [1,2]
Using 3-dimensional fluorescent in situ hybridization (3D-FISH) in zebrafish fin buds, we further show that Nipbls and Med12 are required for higher-order chromatin organization near the hoxda cluster
Given the gene expression changes we found in pectoral fin buds of Nipbl-deficient fish, we decided to investigate if Nipbl-deficient mouse limb buds show some of the same changes
Summary
A ring-shaped, DNA-associated protein complex, is best known for its role in tethering sister chromatids together until mitosis [1,2]. Growing evidence indicates that cohesin, and proteins such as Nipped-B-like (Nipbl) that regulate cohesin loading onto DNA, play critical roles in gene regulation [3,4,5,6,7,8,9,10,11,12,13]. It has been suggested that Nipbl and cohesin mediate interactions between promoters and distant enhancers, a process thought to involve the physical looping out of intervening DNA sequences [14,15,16]. The reported physical interaction between Mediator and Nipbl at active genes suggests that they function together in promoter-enhancer communication, but exactly how this occurs is unknown
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