The objective of this proposal is to examine the interplay between the ATP-dependent remodeler INO80 and the histone variant H2A.Z in order to better understand their roles in activating transcription and preventing bidirectional transcription from promoter regions. It has been confusing as to the role of H2A.Z in transcription given the contradictory information. Incorporation of H2A.Z in vitro into nucleosomes and nucleosomal arrays stabilizes the nucleosome more than canonical H2A and promotes higher-order folding into a chromatin fiber, all of which suggests H2A.Z should be refractory to transcription. However, H2A.Z is a consistent marker of active genes in higher eukaryotes and has been thought to poise genes for transcription. Early in transcription H2A.Z is rapidly replaced by H2A from promoter regions and H2A.Z nucleosomes are more rapidly turned over and lost than canonical nucleosomes leading to the suggestion that they can open the chromatin structure. Although we know INO80 exchanges H2A.Z for H2A during remodeling, we know little about how this happens and if the reaction intermediates involved predispose nucleosomes to become more accessible and prone to disassemble. In addressing whether INO80 remodeling of H2A.Z nucleosomes can explain the discrepancy between in vitro and in vivo observations, the resolution of this dilemma will not only answer basic question in transcription but also potentially in DNA repair, genomic stability linked to the formation of centromeres and telomeres, and replication fork stability; all of which involve INO80 and H2A.Z. The binding interface between INO80 and nucleosomes will be mapped by determining the INO80 domains associated with specific sites in core histones and nucleosomal and extranucleosomal DNA when INO80 is bound. The functional relevance of the crosslinked regions in INO80 for binding and remodeling will be examined by deletion or mutagenesis of conserved residues within these domains. The structural dynamics of histone exchange will be examined using ensemble and single molecule techniques to determine the rate and order of events such as disruption of the contacts of H2A.Z-H2B with DNA and the H3-H4 tetramer, and its eventual loss from nucleosomes. We will determine the parts of INO80 facilitating dimer ejection and the entry of the incoming H2A-H2B dimer using the described mutants. The effects of acetylation of lysine 56 of histone H3 (H3K56ac) on the contacts of INO80 with nucleosomes will be examined by histone and DNA crosslinking. Alterations in the reaction pathways of H2A.Z and H2A exchange when nucleosomes contain H3K56ac versus unacetylated H3 will be examined using the same ensemble and single molecule techniques to understand how acetylation enhances the exchange reaction.

Public Health Relevance

The proposed research is relevant to public health because chromatin remodelers regulate gene expression, DNA replication, and chromosome integrity; and alterations of these complexes are found to be the cause of different human diseases such as cancer and have important roles in cell development. This project is relevant to NIH's mission because it focuses on understanding the mechanism whereby INO80, a chromatin remodeler, changes the composition of chromosomes by replacement of the histone variant H2A.Z and will determine the molecular basis of its specificity and the impact of its intermediates on nucleosome accessibility and stability. Aberrant incorporation of H2A.Z has been shown to promote metastasis and correlates with decreased patient survival as well as be associated with MYC-induced B cell transformation and lymphoma- genesis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Carter, Anthony D
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University of Texas MD Anderson Cancer Center
Internal Medicine/Medicine
Schools of Medicine
United States
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Brahma, Sandipan; Ngubo, Mzwanele; Paul, Somnath et al. (2018) The Arp8 and Arp4 module acts as a DNA sensor controlling INO80 chromatin remodeling. Nat Commun 9:3309
Brahma, Sandipan; Udugama, Maheshi I; Kim, Jongseong et al. (2017) INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers. Nat Commun 8:15616
Lafon, Anne; Taranum, Surayya; Pietrocola, Federico et al. (2015) INO80 Chromatin Remodeler Facilitates Release of RNA Polymerase II from Chromatin for Ubiquitin-Mediated Proteasomal Degradation. Mol Cell 60:784-796