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 #
5R01GM101958-03
Application #
8598915
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
2014-01-01
Budget End
2014-12-31
Support Year
3
Fiscal Year
2014
Total Cost
$303,629
Indirect Cost
$133,529
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Alekseyenko, Artyom A; Walsh, Erica M; Zee, Barry M et al. (2017) Ectopic protein interactions within BRD4-chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma. Proc Natl Acad Sci U S A 114:E4184-E4192
Kang, Hyuckjoon; Jung, Youngsook L; McElroy, Kyle A et al. (2017) Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila. Genes Dev 31:1988-2002
McElroy, Kyle A; Jung, Youngsook L; Zee, Barry M et al. (2017) upSET, the Drosophila homologue of SET3, Is Required for Viability and the Proper Balance of Active and Repressive Chromatin Marks. G3 (Bethesda) 7:625-635
Zee, Barry M; Alekseyenko, Artyom A; McElroy, Kyle A et al. (2016) Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry. Proc Natl Acad Sci U S A 113:1784-9
Zee, Barry M; Dibona, Amy B; Alekseyenko, Artyom A et al. (2016) The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns. PLoS One 11:e0163820
Kang, Hyuckjoon; McElroy, Kyle A; Jung, Youngsook Lucy et al. (2015) Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila. Genes Dev 29:1136-50
Alekseyenko, Artyom A; McElroy, Kyle A; Kang, Hyuckjoon et al. (2015) BioTAP-XL: Cross-linking/Tandem Affinity Purification to Study DNA Targets, RNA, and Protein Components of Chromatin-Associated Complexes. Curr Protoc Mol Biol 109:21.30.1-32
Jung, Youngsook L; Kang, Hyuckjoon; Park, Peter J et al. (2015) Correspondence of Drosophila polycomb group proteins with broad H3K27me3 silent domains. Fly (Austin) 9:178-82
Alekseyenko, Artyom A; Walsh, Erica M; Wang, Xin et al. (2015) The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains. Genes Dev 29:1507-23
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

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