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Title: Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

Abstract

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations,more » display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in euchromatin. Remarkably, human euchromatin and fly heterochromatin share similar features; such as repeated DNA content, intron lengths and open reading frame sizes. Human cells likely stabilize their DNA content via mechanisms and factors similar to those in Drosophila heterochromatin. Furthermore, my thesis work raises implications for H3K9me and chromatin functions in complex-DNA genome stability, repeated DNA homogenization by molecular drive, and in genome reorganization through evolution.« less

Authors:
 [1]
  1. Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE; National Institutes of Health (NIH)
OSTI Identifier:
913167
Report Number(s):
LBNL-62693
R&D Project: L0042; TRN: US200802%%568
DOE Contract Number:
AC02-05CH11231; NIHR01GM061169
Resource Type:
Thesis/Dissertation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ANIMAL CELLS; CENTROMERES; CHROMATIN; CHROMOSOMES; DNA; DNA DAMAGES; DNA REPAIR; DNA REPLICATION; DROSOPHILA; GENES; GENETICS; HETEROCHROMATIN; NUCLEOSOMES; PROTEINS; STABILITY; TELOMERES

Citation Formats

Peng, Jamy C. Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity. United States: N. p., 2007. Web. doi:10.2172/913167.
Peng, Jamy C. Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity. United States. doi:10.2172/913167.
Peng, Jamy C. Mon . "Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity". United States. doi:10.2172/913167. https://www.osti.gov/servlets/purl/913167.
@article{osti_913167,
title = {Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity},
author = {Peng, Jamy C.},
abstractNote = {Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in euchromatin. Remarkably, human euchromatin and fly heterochromatin share similar features; such as repeated DNA content, intron lengths and open reading frame sizes. Human cells likely stabilize their DNA content via mechanisms and factors similar to those in Drosophila heterochromatin. Furthermore, my thesis work raises implications for H3K9me and chromatin functions in complex-DNA genome stability, repeated DNA homogenization by molecular drive, and in genome reorganization through evolution.},
doi = {10.2172/913167},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}

Thesis/Dissertation:
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  • This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that evenmore » in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' ..-->.. 5' exonuclease activity of T4 DNA polymerase.« less
  • In Physarum polycephalum, the nucleoli are extra chromosomal structures containing 200 to 400 copies of a linear 60 kilobase palindromic rDNA molecule. These rDNA molecules are organized into minichromosomes which apparently are held within a nucleolar protein matrix. To obtained evidence for attachment of the rDNA to such a matrix, both intact and lithium diiodosalicylate/NaCl-extracted nucleoli were digested for various lengths of time with micrococcal nuclease, so that portions of the rDNA molecules not attached within the nucleolar structure would be released. Nucleolar DNA-binding proteins were determined by blotting electrophoretically separated proteins from SDS-polyacrylamide gels onto nitrocellulose paper and probingmore » them with radiolabeled DNA. In addition to the histones and lexosome proteins, eight DNA-binding proteins were identified having molecular weights of 25, 38, 47, 53, 55, 67, and 70 kD, with the 47, 53, 67, and 70 kD proteins requiring Ca{sup 2+} for binding.« less
  • DNA damage and repair in response to ultraviolet light (UV) or furocoumarin photoadducts were studied in cultured primate cells. First, the sensitivity to digestion by the enzyme staphylococcal nuclease (SN) of repair patches synthesized in response to damage by UV or the angular furocoumarin angelicin was examined in human cells. Second, excision repair was examined in the 172 base pair highly repeated alpha DNA sequence in African green monkey cells. Excision repair after treatment of cells with UV was the same in alpha DNA as in the bulk of the genome. However repair of furocoumarin photoadducts was only about 30%more » as great in alpha DNA as in the bulk of the genome, even through the initial amount of damage, the time course of excision repair, and the excision repair patch size were all indistinguishable for the two DNA species.« less
  • Micrococcal nuclease, which preferentially cleaves linker DNA in chromatin, was immobilized by covalent attachment to CNBr-activated agarose beads and used to study the accessibility of linker DNA in chromatin fibers prepared from chicken erythrocyte nuclei. This immobilized nuclease was able to cleave chromatin fibers into the typical pattern of fragments corresponding to multiples of mononucleosomes. Cleavage from only the ends of the fibers was ruled out by examining the products of cleavage of fibers end-labelled with /sup 35/P. Comparison of the rate of digestion by immobilized and soluble micrococcal nuclease indicated that the fiber structure does not significantly affect accessmore » to linker DNA. The absence of an effect of reducing temperatures on the rate of digestion of fibers, as compared to short oligonucleosomes, indicated that breathing motions to allow access to the fiber interior were not required for cleavage of linker DNA.« less
  • Aerosols are an ever-present part of our daily environment and have extensive effects on both human and environmental health. Particles in the inhalable range (1-10 μm diameter) are of particular concern because their deposition in the lung can lead to a variety of illnesses including allergic reactions, viral or bacterial infections, and cancer. Understanding the transport of inhalable aerosols across both short and long distances is necessary to predict human exposures to aerosols. To assess the transport of hazardous aerosols, surrogate tracer particles are required to measure their transport through occupied spaces. These tracer particles must not only possess similarmore » transport characteristics to those of interest but also be easily distinguished from the background at low levels and survive the environmental conditions of the testing environment. A previously-developed DNA-tagged particle (DNATrax), composed of food-grade sugar and a DNA oligonucleotide as a “barcode” label, shows promise as a new aerosol tracer. Herein, the use of DNATrax material is validated for use in both indoor and outdoor environments. Utilizing passive samplers made of materials commonly found in indoor environments followed by quantitative polymerase chain reaction (qPCR) assay for endpoint particle detection, particles detection was achieved up to 90 m from the aerosolization location and across shorter distances with high spatial resolution. The unique DNA label and PCR assay specificity were leveraged to perform multiple simultaneous experiments. This allowed the assessment of experimental reproducibility, a rare occurrence among aerosol field tests. To transition to outdoor testing, the solid material provides some protection of the DNA label when exposed to ultraviolet (UV) radiation, with 60% of the DNA remaining intact after 60 minutes under a germicidal lamp and the rate of degradation declining with irradiation time. Additionally, exposure of the DNATrax material using formulations of two different food-grade sugars (maltodextrin and erythritol) to humidity as high as 66% had no significant effect on the DNA label’s degradation or the particle’s aerodynamic diameter, confirming particle stability under such conditions. In summary, confirmation of the DNATrax particles’ size and label integrity under variable conditions combined with experiment multiplexing and high resolution sampling provides a powerful experimental design for modeling aerosol transport through occupied indoor and outdoor locations.« less