GSBS Dissertations and Theses

Title

The Role of CHD2 in Mammalian Development and Disease: a Dissertation

Approval Date

March 2007

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences

Subjects

Chromatin Assembly and Disassembly; Chromatin; DNA-Binding Proteins; DNA Helicases; Embryonic Development; Academic Dissertations

Abstract

Chromatin structure is intricately involved in the mechanisms of eukaryotic gene regulation. In general, the compact nature of chromatin blocks DNA accessibility such that components of the transcriptional machinery are unable to access regulatory sequences and gene activation is repressed. These repressive effects can be overcome or augmented by the actions of chromatin remodeling enzymes. Numerous studies highlight two classes of these enzymes: those that covalently modify nucleosomal histones and those that utilize energy derived from ATP hydrolysis to destabilize the histone-DNA contacts within the nucleosome (13, 14, 92). Members of each of these groups of chromatin remodeling enzymes play pivotal roles in modulating chromatin structure and in facilitating or blocking the binding of transcription factors. Mutations in genes encoding these enzymes can result in transcriptional deregulation and improper protein expression. Therefore, the regulation of chromatin structure is critical for precise regulation of almost all aspects of gene expression. Consequently, enzymes regulating chromatin structure are important modulators of cellular processes such as cell viability, growth, and differentiation. There remain many uncharacterized members of the ATP-dependent class of remodeling enzymes; characterization of these proteins will further elucidate the cellular functions these enzymes control.

Here, we focus primarily on the ATP-dependent remodeling complexes, specifically the chromodomain helicase DNA-binding (CHD) family. The CHD proteins are distinguished from other ATP-dependent complexes by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. These proteins also contain a SNF2-like ATPase motif and are further classified based on the presence or absence of additional domains. Genetic, biochemical, and structural studies demonstrate that CHD proteins are important regulators of transcription and play critical roles during developmental processes. Numerous CHD proteins have also been implicated in human disease.

The first CHD family member, mChd1, was identified in 1993 in a search for DNA-binding proteins with an affinity for immunoglobin promoters. Since then, additional CHD genes have been identified based on sequence and structural homology to mChd1. Despite an increase in the number of studies relating to CHD proteins, the function of most remains unknown or poorly characterized. Using embryonic stem (ES) cells containing an insertional mutation in the murine Chd2 locus, we generated a Chd2-mutant mouse model to address the biological effects of Chd2 in development and disease. The targeted Chd2 allele resulted in a stable Chd2-βgeo fusion protein that contained the tandem chromodomains, the SNF2-like ATPase motif, but lacked the C-terminal portion of the DNA-binding domain.

We demonstrated that the mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis as well as perinatal lethality. Similarly, heterozygous mice showed a decreased neonatal viability. Moreover, the surviving heterozygous mice showed a general growth delay during the neonatal period and increased susceptibility to non-neoplastic lesions affecting multiple organs, most notably the kidneys.

We further examined the connection between Chd2 and kidney disease in this murine model. Our findings revealed that the kidney phenotype observed in Chd2 mutant mice led to the development of membranous glomerulopathy, proteinuria, and ultimately to impaired kidney function. Additionally, serum analysis revealed decreased hematocrit levels in the Chd2-mutant mice, suggesting that the membranous glomerulopathy observed in these mice is associated with anemia.

Lastly, we investigated whether the type of anemia observed in the Chd2-mutant mice. Red blood cell (RBC) indices and morphological examination of the RBCs indicated that the anemia seen in the Chd2-mutant mice can be classified as normocytic and normochromic. Further analyses have been initiated to determine if the anemia is due to an intrinsic effect in erythropoiesis or a secondary consequence of the glomerular disease.

In summary, our findings have contributed to our understanding of the putative chromatin remodeling enzyme Chd2. Although much remains to be studied, these findings demonstrate a role for Chd2 in mammalian development and have revealed a link between Chd2 and disease.

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