The genomes of higher organisms are highly annotated by specific chromosomal proteins and histone modifications along active genes, regulatory elements, or silent regions. An ongoing challenge is to decipher the rules that establish and maintain chromatin organization. Classic epigenetic regulators such as the Polycomb group (PcG), the Trithorax group (TrxG), and Heterochromatin Protein 1 (HP1), have profound roles in developmental control of the genome in all higher organisms examined. However, one obstacle to understanding their function has been the trade-off between removing them from the DNA, to allow purification, and the resultant loss of weak or transient interactions with key partners. Our new approach allows us to affinity-purify fragmented chromatin with proteins and RNAs attached, using cross-linking to avoid disruption of weak interactions. Using our approach, the linked DNA, protein, histone peptides, and RNA fractions can be separately analyzed using comprehensive sequencing and mass spectrometry.
In Aim 1, a dual tag allows affinity purification from low amounts of relatively crude extracts, giving us the ability to look at early events in the establishment of these key chromatin-associated complexes.
In Aim 2, we probe their molecular mechanisms by measuring their impact on RNA polymerase distribution across the genome at high resolution. Success in our studies will be relevant to understanding the establishment of conserved genome organization and function, which is central to normal human development and physiology.

Public Health Relevance

Cellular genomes are organized into active and silent chromatin, characterized by specific DNA, RNA, and protein composition. Perturbation of this organization can lead to aberrant development and diseases such as cancer in humans. The goal of our studies is to understand the rules for establishment and maintenance of chromatin organization, and to document its precise functions in gene regulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM101958-02
Application #
8459396
Study Section
Special Emphasis Panel (ZRG1-GGG-R (03))
Program Officer
Carter, Anthony D
Project Start
2012-04-13
Project End
2015-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
2
Fiscal Year
2013
Total Cost
$325,557
Indirect Cost
$143,172
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
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Jung, Youngsook L; Luquette, Lovelace J; Ho, Joshua W K et al. (2014) Impact of sequencing depth in ChIP-seq experiments. Nucleic Acids Res 42:e74
McElroy, Kyle A; Kang, Hyuckjoon; Kuroda, Mitzi I (2014) Are we there yet? Initial targeting of the Male-Specific Lethal and Polycomb group chromatin complexes in Drosophila. Open Biol 4:140006
Alekseyenko, Artyom A; Gorchakov, Andrey A; Kharchenko, Peter V et al. (2014) Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification. Proc Natl Acad Sci U S A 111:2488-93
Wang, Charlotte I; Alekseyenko, Artyom A; LeRoy, Gary et al. (2013) Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila. Nat Struct Mol Biol 20:202-9