Genome-Wide Mapping of Chromosomal Proteins in Drosophilia
| Gary Karpen (PI) | Lawrence Berkeley National Laboratory | |
|---|---|---|
| Sarah Elgin | Washington University | |
| Mitzi Kuroda | Brigham and Women's Hospital | |
| Peter Park | Brigham and Women's Hospital | |
| Vincent Pirrotta | Rutgers University | |
The packaging of DNA into chromatin and chromosomes has major implications for understanding how genome sequences function in eukaryotic cells. We propose to participate in the ENCODE project by determining the locations of 125 chromosomal proteins and histone modifications across the Drosophila melanogaster genome. The proteins and modifications under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) with antibodies obtained commercially and generated and validated by this project, isolate and label the precipitated DNA, and apply the probes to genomic tiling arrays. Data generated by scanning the hybridized arrays will be analyzed by statistical methods, and the array data will be validated by independent analyses in cells and animals. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages. We will perform a variety of bioinformatic comparisons between protein "landscape" data sets, including analyses of combinatorial patterns of modifications and chromosomal proteins, tissue-specific differences, and interactions among proteins involved in the same epigenetic pathways. All validated data and analyses will be made available to members of the ENCODE project and the scientific community. Successful completion of this project will provide basic information about the distributions of chromatin components across the Drosophila genome, which will serve as a foundation for future functional studies. The data and analysis are very likely to provide information critical to understanding the roles of chromatin packaging in human cells, and the mispackaging that can be associated with disease.
Experimental Approaches
Our aims are:
Aim 1: Determine the genomic distributions of histone modifications and chromosomal proteins. The main approach will be to perform ChIP for ~50 histone modifications and variants, and ~50 chromosomal proteins, and hybridize the IP'd DNA to Affymetrix Genome TIling arrays. Histone antibodies will be obtained from commercial sources, and we will generate and validate antibodies to a subset of chromosomal proteins for which antibodies are not currently available.
Aim 2: Perform bioinformatic and statistical analyses of ChIP-array data. We will examine the distributions of histone modifications/variants and chromosomal proteins in the euchromatin and heterochromatin, and will examine the data for combinatorial patterns of localization.
Aim 3: Generate technical and biological validations of protein distributions. We will validate the array results with a combination of molecular and cytological approaches, and by performing arrays on chromatin derived from cells depleted for the protein or proteins that establish histone modifications.
Information Resources to be Generated
We will generate maps of the distributions of individual proteins and nucleosome components relative to the genome sequence, as well as data on how the patterns change after protein depletion. We intend to integrate these results with annotations present in FlyBase and other genome databases in order to analyze correlations with genes and other elements.
Reagents to be Generated
Antibodies to ~50 chromosomal proteins.
Contact Information
1. PI: Gary Karpen, karpen@fruitfly.org
2. co-PI: Sarah Elgin, selgin@biology.wustl.edu
3. co-PI: Mitzi Kuroda, mkuroda@genetics.med.harvard.edu
4. co-PI: Vince Pirrotta, pirrotta@biology.rutgers.edu
5. co-PI/Informatics: Peter Park, peter_park@harvard.edu