| Genome Instability and Chromatin-Remodeling Section |
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Weidong Wang, Ph.D., Chief
Senior Investigator |
| Overview: Recently, multiprotein complexes have been implicated in the regulation or modulation of many cellular processes. Often, one protein can be discovered in several complexes, with each complex performing its unique function. Thus, the biological functions of a given protein can be understood only when the consequences of its association in complexes are defined. The Genome Instability and Chromatin-Remodeling Section studies selected nuclear regulatory complexes. |
| In the eucaryotic nucleus, the chromatin structures that allow efficient storage of genetic information also tend to render the DNA inaccessible to metabolizing enzymes. The repressive chromatin structure must be remodeled to allow transcription and other metabolic reactions to occur. Chromatin-remodeling multiprotein complexes are critically involved in processes that include transcription, replication, chromatin assembly, and chromosome condensation. Furthermore, multiple human diseases, including several types of cancer, are caused by mutations in remodeling complexes; and aging in several lower species (and in several human disorders with features of premature aging) can be modulated by alterations in remodeling enzymes. Our Section aims to discover novel chromatin-remodeling molecules and investigate their composition and mechanism of action. We have taken a biochemical approach to defining targeted complexes, starting with the development of a highly efficient immunopurification protocol to isolate the endogenous complexes from mammalian nuclear extracts in highly purified form. We have focused on studies of two families of multiprotein complexes involved in DNA expression and genome stability, in two corresponding projects: |
| Project I. Chromatin-remodeling Complexes that Participate in Gene Regulation |
| 1. Mammalian SWI/SNF-Related Chromatin-Remodeling Complexes: The SWI/SNF complex, originally identified in yeast, functions as a chromatin remodeling machine in signaling pathways that lead to activation of gene expression. In mammals, the SWI/SNF-related complexes are involved not only in gene regulation, but also in targeting of HIV integration, cell cycle regulation, and in tumor suppression by interacting with Rb protein. Mutation of the hSNF5 subunit has been shown to be a cause for pediatric rhabdomyosarcoma. We have completely purified several distinct mammalian SWI/SNF-related complexes. By microsequencing, we have cloned all subunits from two major complexes of human KB cells, BAF and PBAF. We have recently isolated a novel complex containing a chromosomal translocation fusion partner for mixed lineage leukemia protein (MLL). We are continuing characterization of these complexes and investigate their mechanism of action. |
| 2. Chromatin Remodeling in ATRX Syndrome: ATRX syndrome represents a combination of a-thalassemia, mental retardation, and multiple associated developmental abnormalities. The gene defective in ATRX has been localized to the X chromosome and recently cloned. The ATRX gene encodes a gene product containing a SWI2/SNF2-type DNA-dependent ATPase domain. Thus, it has been hypothesized that ATRX could function in an ATP-dependent chromatin-remodeling complex and participate in regulation of gene expression. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. We also demonstrate that this complex has ATP-dependent chromatin remodeling activity. Our study suggests that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATR-X syndrome may result from inappropriate expression of genes controlled by this complex. |
| Project II. RecQ DNA Helicase Complexes Involved in Genome Instability Syndromes |
| 1. Purification of a Complex Containing WRN, the Helicase Involved in Werner's Premature Aging Disease: Many human helicases discovered to date are related to DNA repair diseases, including Werner Syndrome (WRN), Cockayne's Syndrome (ERCC6), Xermaderma pigmentosum, and Bloom's Syndrome. Many of the gene products have only been identified recently and their mechanisms of action are not known. We recently found that the gene product encoded by WRN is present in a high molecular weight complex in HeLa cells. We have now purified this complex and identified all of its subunits by microsequencing. We are now studying the functions of the WRN complex. Hopefully, this will lead to better understanding of the human aging process. |
| 2. Purification of a Complex Containing BLM, the Helicase Involved in Bloom Syndrome: This disease resembles Werner syndrome in genomic instability and cancer predisposition; but the patients do not display premature aging conditions. The gene defective in this disease belongs to the same family of RecQ helicase as WRN. We have purified three distinct BLM-containing complexes from HeLa cells. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (see below). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIa and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from a FA cell line have a lower molecular mass. Our study suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex consistent with a function for FA proteins in DNA repair. Currently, we are investigating the role of a component of the BRAFT complex, BLAP75, in BLM function. |
| 3. Identify New Fanconi Anemia Genes and Understand the Disease Mechanism: Fanconi anemia (FA) is a genome instability disease and the patients have higher risks to develop cancer. Genetic studies have identified 8 complementation groups for the disease. Among them, 6 genes have been cloned. However, these gene products show no sequence homology to known proteins in the database and they have not been reported to have any biochemical activity. We have isolated an FA core complex to a significant level of purity. We found that this complex has five known FA proteins and four new components (they are named FAAPs for FA-Associated Proteins). We identified all these components by mass spectrometry. One new component, PHF9, was found to contain a ubiquitin ligase motif as well as the corresponding activity. By three different approaches-small interfering RNA (siRNA) knockdown, knockout mice, and identification of an FA patient carrying a PHF9 mutation-we show that PHF9 represents a new FA complementation group and is required for FANCD2 monoubiquitylation in vivo. Our data suggest that PHF9 plays a crucial role in the FA/BRCA pathway as the catalytic subunit required for FANCD2 monoubiquitylation. We are continuing to investigate whether other components of the FA core complex are novel FA genes. Several of these new components were found to have DNA-interacting domains. We are investigating whether the FA core complex may have corresponding activities, which should help to understand how the FA core complex is involved in the pathophysiology of this disease. |
| 4. Purification of a Complex Involved in Rothmund-Thompson Syndrome: This disease also is characterized by genome instability and higher risk of cancer. The gene mutated in the disease belongs to the same RecQ helicase family as WRN and BLM. We have now purified the RecQ4 complex from HeLa cells and have identified all its components. The components in this complex are completely different from those in WRN or BLM complexes, and the functional studies are now underway. |
| Position Available: Postdoctoral position available to work on purification of multiprotein complexes and analysis of their structures and functions. Projects include studies of chromatin-remodeling mechanisms (Proc. Natl. Acad. Sci. USA. 100:10635-40), DNA damage response, and human genomic instability diseases (Nat. Genet. 35:165-170). |
