Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Dec;1832(12):2097-102.
doi: 10.1016/j.bbadis.2013.07.020. Epub 2013 Aug 3.

Reduction of Nipbl Impairs Cohesin Loading Locally and Affects Transcription but Not Cohesion-Dependent Functions in a Mouse Model of Cornelia De Lange Syndrome

Affiliations
Free PMC article

Reduction of Nipbl Impairs Cohesin Loading Locally and Affects Transcription but Not Cohesion-Dependent Functions in a Mouse Model of Cornelia De Lange Syndrome

Silvia Remeseiro et al. Biochim Biophys Acta. .
Free PMC article

Abstract

Cornelia de Lange Syndrome (CdLS) is a genetic disorder linked to mutations in cohesin and its regulators. To date, it is unclear which function of cohesin is more relevant to the pathology of the syndrome. A mouse heterozygous for the gene encoding the cohesin loader Nipbl recapitulates many features of CdLS. We have carefully examined Nipbl deficient cells and here report that they have robust cohesion all along the chromosome. DNA replication, DNA repair and chromosome segregation are carried out efficiently in these cells. While bulk cohesin loading is unperturbed, binding to certain promoters such as the Protocadherin genes in brain is notably affected and alters gene expression. These results provide further support for the idea that developmental defects in CdLS are caused by deregulated transcription and not by malfunction of cohesion-related processes.

Keywords: Cohesin; Cornelia de Lange Syndrome; Mouse model; Nibpl; Transcription.

Figures

Figure 1
Figure 1. Reduced Nipbl levels do not affect cohesion and progression through the cell cycle
(A) Metaphase spreads from wild-type and Nipbl heterozygous MEFs showing robust centromere cohesion. (B) Telomere fragility measured in two clones each of wild-type and Nipbl heterozygous MEFs untreated or treated with 0.5 μM aphidicolin for 24 h. White arrows on the images indicate the aberrant telomeres displaying two instead of a single dot. The number of chromosomes examined is indicated above each bar. (C) Quantification of breaks along the chromosome arms (white arrows) in cells treated as in (A). (D) Frequency of normal anaphases and aberrant anaphases showing lagging chromosomes or bridges in wild-type and Nipbl heterozygous MEFs (n ≥ 50 cells per clone from two independent clones per genotype). (E) Growth curves of wild-type and Nipbl heterozygous MEFs (n=2 clones per genotype).
Figure 2
Figure 2. Nipbl deficiency does not increase DNA damage sensitivity in MEFs
(A) Survival of wild-type and Nipbl heterozygous cells after exposure to the DNA damaging agents aphidicolin, hydroxyurea, mitomycin C and gamma irradiation at the indicated doses (n=2 clones per genotype). (B) Left: 53BP1 staining of G2 irradiated (8Gy) cells, identified as pH3(S10) positive, was categorized in three different classes: homogenous nuclear staining (class I), few foci with diffuse staining (class II) and many strong foci without diffuse staining (class III). Right: Quantification of these phenotypes before, immediately after and 3 hours after irradiation of wild-type and Nipbl heterozygous MEFs. (n ≥ 50 G2-cells per condition and per clone from two independent clones per genotype).
Figure 3
Figure 3. Nipbl deficiency does not affect bulk cohesin loading
Asynchronous cultures of two clones per genotype were subjected to chromatin fractionation followed by immunoblotting with the indicated antibodies. Mek2 cytoplasmatic kinase and histone H3 are used as control for the fractionation procedure. WCE whole-cell extract; Cyt, cytoplasm; Chr, chromatin-enriched fraction. The soluble nucleoplasmic fraction was not loaded in this gel. In the immunoblot of Wapl, the lower band indicated by the asterisk correspond to SA2, since the membrane was reused.
Figure 4
Figure 4. Nipbl deficiency alters cohesin binding at certain promoters and affects gene expression
(A) mRNA levels of the indicated genes in the brains from E17.5 Nipbl +/− (grey bars) and SA1 +/− embryos (white bars). Three embryos per genotype were tested and three independent qPCR reactions per condition were performed. Values are represented as log2 of FC versus its respective wild-type controls. * P <0.05, ** P <0.01, n.s = not significant. (B) In vivo ChIP-qPCR of SA1 and SMC1 binding at the promoter of the indicated genes in E17.5 brains from Nipbl +/− (n=5) and their wild-type littermates (n=5). (C) Same analysis by ChIP-qPCR of SA1 and SMC1 binding in E17.5 brains from SA1 +/− (n=6) and their wild-type littermates (n=4).

Similar articles

See all similar articles

Cited by 17 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback