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Principal Investigator: Edward E Luk
Organization: STATE UNIVERSITY NEW YORK STONY BROOK
Fiscal Year: 2024
Award: $316,309
Funding agency: National Institute of General Medical Sciences
7. PROJECT SUMMARY/ABSTRACT
The chromatin landscape of eukaryotic cells is decorated with landmarks characterized by covalent
modifications and variant nucleoprotein structures. Some of these chromatin marks contribute to productive
transcription, while others are involved in the establishment and maintenance of silenced chromatin regions.
The fact that more than half of human cancers have mutations in genes encoding histones and chromatin
regulators underscores the importance of understanding the fundamental mechanism of chromatin remodeling
in cells. The canonical nucleosome, which has an inner core consisted of two copies each of histone H2A,
H2B, H3, and H4 and an outer DNA coil of 147 bp in length, represents the predominant packaging unit of
cellular chromatin. But emerging evidence suggests that nucleoproteins with alternative histone stoichiometries
and DNA wrapping configurations are present. These variant packaging units can significantly alter the
biophysical and biochemical properties of chromatin, potentially affecting a wide spectrum of nuclear functions.
In the proposed studies, we focus on a nucleosome-like particle, called the R-octasome, in which its core
is made up of eight subunits of the arginine-rich histone H3 and H4 (without H2A and H2B). Although it has
been known for >40 years that R-octasomes can be assembled in vitro, the biological relevance is unknown.
Using new structural data of the R-octasome, we strategically placed cysteine probes in yeast H3 and showed
by site-directed crosslinking that structures specific to R-octasomes are present in yeast cells, providing the
first evidence that R-octasomes exist in vivo. To further study the nature of R-octasomes, in Aim 1, we propose
to develop a methodology to purify native R-octasomes from yeast that can be used for biochemical, genomic,
and structural studies. To study the biological roles of R-octasomes, in Aim 2, we will interrogate the role of R-
octasomes in telomeric gene silencing, as analysis of site-specific chemical mapping data suggest that R-
octasomes are linked to telomeres. In parallel, we will investigate how R-octasomes function as substrates of
ATP-dependent chromatin remodelers. Finally, in Aim 3, we will explore the role of R-octasomes in higher-
order chromatin organization, as our structural data suggest that R-octasomes can nucleate the concatenation
of additional H3 and H4 histones. Overall, the outcome of this research will give new insights into how
eukaryotes use the highly conserved H3 and H4 as multi-functional substrates to modulate genomic functions.
Terms: <30 nm Chromatin Fiber><30 nm Fiber><Affect><Affinity><Antibodies><Arginine><Baker's Yeast><Binding><Biochemical><Biological><Biological Function><Biological Process><Biology><Biophysics><Brewer's Yeast><Cancer Biology><Cancers><Cell Body><Cell Differentiation><Cell Differentiation process><Cell Function><Cell Growth in Number><Cell Multiplication><Cell Nucleus><Cell Physiology><Cell Process><Cell Proliferation><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular Proliferation><Chemicals><Chromatin><Chromatin Assembly><Chromatin Fiber><Chromatin Modeling><Chromatin Structure><Collaborations><Cryo-electron Microscopy><Cryoelectron Microscopy><Cysteine><DNA><Data><Data Set><Deoxyribonucleic Acid><Dimerization><Electron Cryomicroscopy><Electron Microscopy><Eukaryota><Eukaryote><Eukaryotic Cell><Gene Expression><Gene Inactivation><Gene Silencing><Gene Transcription><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genetics-Mutagenesis><Genome><Genomics><Grant><Half-Cystine><Heterochromatin><Histone H2A><Histone H3><Histone H4><Histones><Human><In Vitro><L-Arginine><L-Cysteine><Left><Length><Link><Maintenance><Malignant Neoplasms><Malignant Tumor><Maps><Methodology><Minor><Modern Man><Modification><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Mutagenesis><Mutagenesis Molecular Biology><Mutation><N-terminal><NH2-terminal><Nature><Negative Staining><Normal Cell><Nuclear><Nuclease Protection Assays><Nucleoproteins><Nucleosomes><Nucleus><Outcomes Research><Paper><Position><Positioning Attribute><Productivity><Property><Protein Dimerization><Publishing><RNA Expression><Recombinants><Resolution><Role><S cerevisiae><S. cerevisiae><Saccharomyces cerevisiae><Sedimentation process><Shapes><Site><Structure><Subcellular Process><Testing><Therapeutic Intervention><Transcription><Variant><Variation><Yeasts><adduct><biologic><biophysical foundation><biophysical principles><biophysical sciences><cancer progression><cellular differentiation><chromatin remodeling><conformation><conformational><conformational state><conformationally><conformations><crosslink><cryo-EM><cryoEM><cryogenic electron microscopy><dimer><gene function><genome mutation><in vivo><insight><intervention therapy><malignancy><mutant><neoplasm progression><neoplasm/cancer><neoplastic progression><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><particle><reconstitute><reconstitution><resolutions><sedimentation><social role><stoichiometry><telomere><transcriptional silencing><tumor><tumor progression>