Date

February 2003

UMMS Affiliation

Graduate School of Biomedical Sciences, Cell Biology

Document Type

Dissertation, Doctoral

Subjects

Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Genes, Tumor Suppressor; Mammals--growth & development; Mice, Transgenic; Tumor Suppressor Proteins; Academic Dissertations; Dissertations, UMMS

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

In vivo DNA is compacted tightly, via its association with histones and non-histone proteins, into higher-order chromatin structure. In this state, the DNA is refractory to the cellular factors that require access to DNA. The repressive nature of chromatin is alleviated in part by the action enzymes that modify chromatin structure. There are two major groups of chromatin modifying enzymes: those that post-translationally modify histones by the addition of small chemical moieties and those that utilize the energy derived from ATP hydrolysis to physically disrupt chromatin structure. The SWI/SNF enzyme belongs to this latter group.

The SWI/SNF complex was identified originally in yeast. Several of its subunits are required for the expression of a subset of inducible genes. The ATPase activity is provided by the SWI2/SNF2 protein. In mammals, there are two biochemically separable SWI/SNF complexes that contain either BRG1 or BRM, both homologs of yeast SWI2/SNF2. The yeast and mammalian SWI/SNF complexes are able to disrupt the Dnase I digestion pattern of in vitro assembled mononucleosomes and arrays, as well as facilitate the accessibility of restriction nucleases and transcription factors. The mechanism by which SWI/SNF functions has yet to be elucidated.

SNF5 is a component of the yeast SWI/SNF complex. It is required for sucrose fermentation and mating type switching. The mammalian homolog of Snf5 is SNF5/INI1. SNF5/INI1 was identified simultaneously by two groups as a protein that shares homology with Snf5 and via a yeast two hybrid assay as a protein that interacts with HIV integrase (INtegrase Interactor). INI1 is a component of all mammalian SWI/SNF complexes purified to date.

In humans, mutations and/or deletions in INI1 are associated with a variety of cancers, including malignant rhabdoid tumors, choroid plexus carcinomas, medullablastomas, primitive neuralectodermal tumors, and some cases of leukemia. Furthermore, constitutional mutations within INI1 in individuals presenting with these tumors support the role of INI1 as a tumor suppressor.

In this thesis, we show that Ini1 also functions as a tumor suppressor in mice. Approximately 20% of mice heterozygous for Ini1 present with tumors. Most of these tumors are undifferentiated or poorly differentiated sarcomas with variable rhabdoid features. All tumors examined to date show loss of heterozygosity at the Ini1 locus. We also show that Ini1 is essential for embryonic development. Mice homozygous-null for Ini1 die between days 4 and 5.5 post-fertilization due to an inability to adhere to their substratum, form trophectoderm, and expand their inner cell mass.

We further characterize the function of Ini1 in tumor suppression by generating mice heterozygous for both Ini1 and either Rb or p53. While heterozygosity at the Ini1 locus appears to have no effect on the rate of tumorigenesis in Rb-heterozygous mice, many of the tumors arising in compound heterozygous mice present with an altered morphology. This finding suggests that Ini1 may contribute to tumor progression due to loss of Rb. In contrast, mice compound heterozygous for Ini1 and p53 show a marked reduction in the rate of tumorigenesis compared to p53-heterozygous mice. Furthermore, the tumor spectrum is altered in these compound heterozygous mice. These findings suggest that Ini1 may function normally to repress p53 activity.

Lastly, we show that expression of the Ini1 tumor suppressor itself is regulated tightly. Tissues and cells heterozygous for Ini1 express roughly equivalent levels of Ini1 protein and mRNA as their wild-type counterparts. We further show that this compensation is mediated by an increase in the rate of transcription from the wild-type Ini1 allele. Moreover, when exogenous Ini1 is introduced into Ini1-heterozygous cells, expression from the Ini1 promoter is reduced. These data indicate that a compensatory mechanism exists to ensure that the steady-state levels of Ini1 are constant.

In summary, research detailed in this thesis has contributed to our understanding of the regulation of Ini1 as well as the role this protein plays in mammalian development and tumor suppression.

Comments

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