Characterization of the NIPBL protein associated with Cornelia de Lange syndrome

Daniel K. Keter

doi:10.17918/etd-3005

Cornelia de Lange Syndrome (CdLS) is a disorder that variably affects many physiologic features of the human body. These include but are not limited to deformed limbs, facial features and mental retardation. CdLS occurs in approximately 1 in 10,000 births. Of all CdLS cases, 50% have recently been associated with heterozygous mutation of the NIPBL gene. Four cases of CdLS have been associated with mutation of SMCL1 which encodes a cohesin subunit. NIPBL is a homolog of the Drosophila melanogaster gene Nipped-B which has been shown to have roles in chromosome cohesion and gene regulation. Because mutant NIPBL as characterized in CdLS affects many organs in the body, we and others speculate that NIPBL has important roles during early stages of development. It has been shown that NIPBL binds to another protein, HP1. HP1 is known to be involved in chromatin based regulation by controlling access of transcription factors to the DNA strand. The proposition therefore would be that, since HP1 is a gene regulatory protein and NIPBL binds to it, a mutation on NIPBL would lead to an abnormal gene expression that leads to dysfunctional cell behavior displayed by Cornelia de Lange Syndrome. The NIPBL protein has not yet been well characterized in human cells. The hypothesis would be that NIPBL has an important role in gene expression. When this role is disrupted, it leads to defects in gene expression such as those seen in CdLS. To test this hypothesis, we characterized the NIPBL protein properties. Using immunohistochemistry, we were able to show that NIPBL colocalizes with HP1 distinctly in the nucleus. Physical observation of [delta]NIPBL cell lines showed lower doubling times and had a higher incidence of the 'wrinkling' phenomenon compared to wild type cell lines. At the protein level, only purified recombinant proteins were able to bedetected by mouse monoclonal and rabbit polyclonal antibodies. Only one [alpha]-NIPBL antibody, XScc2N was able to show a protein at 300kDa which corresponds to the size of NIPBL. It was also confirmed that a mutation of NIPBL leads to increased sister chromatid separation. The same did not hold true for mouse embryo fibroblasts. Characterization of the NIPBL protein would help better understand the resultant role of NIPBL in gene expression and cellular events that underlie the disease process in CdLS.

Characterization of the NIPBL protein associated with Cornelia de Lange syndrome

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Characterization of the NIPBL protein associated with Cornelia de Lange syndrome

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