A major challenge in biomedical research is to understand the functional interplay between gene regulation, chromosome structure, chromatin modifications, and human disease. X-chromosome dosage compensation (DC) in the nematode C. elegans is exemplary for such analysis for the following reasons: DC is mediated by a condensin complex, a chromosome restructuring complex essential for chromosome segregation; DC is essential for proper gene expression and viability because it distinguishes X chromosome from autosomes to control hundreds of genes on X simultaneously; and X-chromosome-specific chromatin modifications are essential for DC. The dosage compensation complex (DCC) binds selectively to X chromosomes of XX embryos to reduce transcription by half, thereby equalizing X-chromosome transcription between XX and XO embryos. Our recent experiments indicate that the DCC imposes a unique spatial organization onto the X chromosomes of XX embryos by binding to its highest affinity recruitment sites on X (rex sites). Mutations that disrupt DCC binding disrupt that X-specific conformation. Proposed experiments extend this analysis and explore the relationship between chromosome structure and gene expression by using our highly efficient genome editing strategies to delete and insert rex sites on X and autosomes and then assess the consequent changes in chromosome conformation and gene expression. Our study is unique in providing a robust example of a major change in chromosome structure imposed by a specific complex, on a specific chromosome, through high- affinity binding to its targets. We have also shown recently that a DCC subunit has a demethylase activity that is responsible for the selective enrichment of H4K20me1 on X chromosomes of XX embryos upon DCC binding. Selective mutation of the catalytic residues abrogates H4K20me1 enrichment and disrupts dosage compensation. These highly specific mutations allow us to test, in an unusually precise way, the role of histone modifications in X-chromosome structure and gene expression. H4K20me1 is also enriched on the mammalian inactive X chromosome, but the role of this enrichment in transcriptional silencing is not known, nor is a selective reagent available to test its role. Changes in histone lysine methylation states are a common occurrence during tumor formation, and strong correlation now exists between an increase in activity of histone demethylases and tumor progression. Hence, our studies of a chromosome-specific demethylase enzyme may become directly relevant to human health. Lastly, we have gained an evolutionary perspective on the X-chromosome DNA sequences that recruit DCC complexes to X chromosomes of highly diverged nematode species. Surpisingly, while the DCC complexes are conserved, the cis-acting motifs are highly diverged. More typically, the target site specificity of conserved regulatory proteins that control multiple celluar processes by targeting hundreds of sites is far more evolutionarily constrained. Hence, the divergence in DCC binding specificity provides an unusual opportunity to understand the path for a concerted change in hundreds of target sites.

Public Health Relevance

Our research explores the relationship between chromosome structure, chromatin modifications, and long- distance regulation of gene expression by studying X-chromosome dosage compensation (DCC) in C. elegans and in highly diverged nematode species using our efficient genome editing strategies. We showed that the DCC, a condensin complex, imposes a unique conformation onto X chromososomes by binding to its high- affinity target sites on X and that a DCC subunit possesses a demethylase activity responsible for the selective enrichment on X of a specific mono-methylated histone, which is essential for proper X-chromosome expression. A strong correlation now exists between an increase in activity of histone demethylases and tumor progression, suggesting that our studies of a chromosome-specific demethylase may be directly relevant to human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030702-35
Application #
9185979
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
1982-09-01
Project End
2018-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
35
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Farboud, Behnom; Jarvis, Erin; Roth, Theodore L et al. (2018) Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms. J Vis Exp :
Yin, Da; Schwarz, Erich M; Thomas, Cristel G et al. (2018) Rapid genome shrinkage in a self-fertile nematode reveals sperm competition proteins. Science 359:55-61
Bian, Qian; Anderson, Erika C; Brejc, Katjuša et al. (2017) Dynamic Control of Chromosome Topology and Gene Expression by a Chromatin Modification. Cold Spring Harb Symp Quant Biol 82:279-291
Brejc, Katjuša; Bian, Qian; Uzawa, Satoru et al. (2017) Dynamic Control of X Chromosome Conformation and Repression by a Histone H4K20 Demethylase. Cell 171:85-102.e23
Tian, Ye; Garcia, Gilberto; Bian, Qian et al. (2016) Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPR(mt). Cell 165:1197-1208
Wheeler, Bayly S; Anderson, Erika; Frøkjær-Jensen, Christian et al. (2016) Chromosome-wide mechanisms to decouple gene expression from gene dose during sex-chromosome evolution. Elife 5:
Farboud, Behnom; Meyer, Barbara J (2015) Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design. Genetics 199:959-71
Crane, Emily; Bian, Qian; McCord, Rachel Patton et al. (2015) Condensin-driven remodelling of X chromosome topology during dosage compensation. Nature 523:240-4
Maxwell, Colin S; Kruesi, William S; Core, Leighton J et al. (2014) Pol II docking and pausing at growth and stress genes in C. elegans. Cell Rep 6:455-66
Farboud, Behnom; Nix, Paola; Jow, Margaret M et al. (2013) Molecular antagonism between X-chromosome and autosome signals determines nematode sex. Genes Dev 27:1159-78

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