Nipbl and Mediator Cooperatively Regulate Gene Expression to Control Limb Development

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.

The Mediator complex: a central integrator of transcription

“…In mammalian cells, long-range looping interactions between enhancer and promoter sequences appear to be important for driving high-level and cell type-specific gene expression 125-127 . Precisely how these loops are formed and stabilized remains uncertain, but Mediator, in conjunction with other factors, appears to play important roles 102,128-130 . Whereas yeast genes are not regulated via long-distance enhancer-promoter interactions 122 , it is notable that Sin4 mutants (the yeast ortholog of Med16) were identified in S. cerevisiae that enabled upstream activating sequences to activate transcription at longer distances from the promoter than normally observed in yeast (normally only 100 – 200 base pairs from the promoter TATA sequence) 131 .…”

Structure of the cohesin loader Scc2

“…Cohesin interacts with Nipbl Scc2 and the mediator complex to connect enhancers and promoters of actively transcribing genes15. Chromosome conformation capture assays show that cohesin helps organize key cell identity genes into insulated neighbourhoods by the formation of chromosomal loop structures16, while in zebrafish and mice, Nipbl Scc2 and mediator cooperatively regulate gene expression during limb development17. The intimate relation between cohesin and Nipbl Scc2 manifests its importance in Cornelia de Lange syndrome (CdLS), one of a family of genetic disorders known as cohesinopathies181920.…”

mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

“…In order to better understand the pathogenesis of the syndrome, it is necessary to study the functions of the NIPBL protein in greater depth, implicated not only in cohesin loading, but also in the regulation of gene expression during development [17,18]. This variety of functions could be achieved through the generation of alternative transcripts by splicing, one of the most efficient mechanisms of protein diversification [19].…”