Effect Of Chromatin Structure Research Articles

Abstract LB-024: The effects of chromatin structure on variation in fragment size of cell free DNA

Abstract Liquid biopsy using cell-free DNA (cfDNA) has established its clinical utilities in multiple clinical cohorts. However, the exact mechanism by which cell-free DNA (cfDNA) is generated remains undetermined. The fragment size of cfDNA sheds important insights on elucidating the process. Previous studies qualitatively concluded that tumor-derived cfDNA is shorter than cfDNA from healthy cells, but the exact size distribution of tumor-derived cfDNA remains unclear. To better understand the mechanism of cfDNA generation and directly study cfDNA of tumor origins, we analyzed the deep targeted sequencing results of cfDNA from 605 patients with various cancer types, and developed a method that separates tumor cfDNA through identification of somatic variant alleles-containing fragments. We show that tumor-derived cfDNA has an increased proportion of fragments less than 150bp compared to healthy controls, while the most common size is still 166 bp. It has been speculated that differences in nucleosome wrapping may contribute to differing fragment lengths of cfDNA.To better understand the relationship between the magnitude of chromatin packaging and cfDNA length, we quantitatively analyzed the size distribution of cfDNA collected from 5 cancer patients in genomic regions with the varying degree of chromatin openness. We discovered that the fragment size of cfDNA was closely associated with chromatin inaccessibility and chromosome condensation as measured by transcriptional start sites, DNase I hypersensitivity, histone modifications, and Giemsa banding, or as implied by chromosomal sub-nuclear localization. The shortening occurs at both 5' and 3' ends of cfDNA fragments without bias. Taken together, these results collectively established a positive correlation between cfDNA fragment size and chromatin inaccessibility. Citation Format: Hui Xia, Xingya Li, Hua Bao, Xiangyuan Ma, Xue Wu, Yang W. Shao, Feixiang Wu, Shucai Zhang. The effects of chromatin structure on variation in fragment size of cell free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-024.

Nuclear topology modulates the mutational landscapes of cancer genomes.

Nuclear organization of genomic DNA affects DNA damage and repair processes, and yet its impact on mutational landscapes in cancer genomes remains unclear. Here we analyzed genome-wide somatic mutations from 366 samples of 6 cancer types. We found that lamina-associated regions, which are typically localized at the nuclear periphery, displayed higher somatic mutation frequencies compared to the inter-lamina regions at the nuclear core. This effect remained even after adjusting for features such as GC%, chromatin, and replication timing. Furthermore, mutational signatures differed between the nuclear core and periphery, indicating differences in the patterns of DNA damage and/or DNA repair processes. For instance, smoking and UV-related signatures were more enriched in the nuclear periphery. Substitutions at certain motifs were also more common in the nuclear periphery. Taken together, we found that the nuclear architecture influences mutational landscapes in cancer genomes beyond the effects already captured by chromatin and replication timing.

Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin

Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Evidence of association between Nucleosome Occupancy and the Evolution of Transcription Factor Binding Sites in Yeast

BackgroundDivergence of transcription factor binding sites is considered to be an important source of regulatory evolution. The associations between transcription factor binding sites and phenotypic diversity have been investigated in many model organisms. However, the understanding of other factors that contribute to it is still limited. Recent studies have elucidated the effect of chromatin structure on molecular evolution of genomic DNA. Though the profound impact of nucleosome positions on gene regulation has been reported, their influence on transcriptional evolution is still less explored. With the availability of genome-wide nucleosome map in yeast species, it is thus desirable to investigate their impact on transcription factor binding site evolution. Here, we present a comprehensive analysis of the role of nucleosome positioning in the evolution of transcription factor binding sites.ResultsWe compared the transcription factor binding site frequency in nucleosome occupied regions and nucleosome depleted regions in promoters of old (orthologs among Saccharomycetaceae) and young (Saccharomyces specific) genes; and in duplicate gene pairs. We demonstrated that nucleosome occupied regions accommodate greater binding site variations than nucleosome depleted regions in young genes and in duplicate genes. This finding was confirmed by measuring the difference in evolutionary rates of binding sites in sensu stricto yeasts at nucleosome occupied regions and nucleosome depleted regions. The binding sites at nucleosome occupied regions exhibited a consistently higher evolution rate than those at nucleosome depleted regions, corroborating the difference in the selection constraints at the two regions. Finally, through site-directed mutagenesis experiment, we found that binding site gain or loss events at nucleosome depleted regions may cause more expression differences than those in nucleosome occupied regions.ConclusionsOur study indicates the existence of different selection constraint on binding sites at nucleosome occupied regions than at the nucleosome depleted regions. We found that the binding sites have a different rate of evolution at nucleosome occupied and depleted regions. Finally, using transcription factor binding site-directed mutagenesis experiment, we confirmed the difference in the impact of binding site changes on expression at these regions. Thus, our work demonstrates the importance of composite analysis of chromatin and transcriptional evolution.

H2B K123 ubiquitination and H3 K4 methylation play a positive role in DNA replication

DNA replication is initiated from sites in the genome known as origins of replication. In eukaryotic cells, control over the function of origins is essential to ensure complete genome replication, and its loss results in genome instability. Despite the importance of controlling origin function, little is known about the determinants of origin identity or the effects of chromatin structure on origin function. We hypothesize that unique elements of chromatin structure are responsible for defining functional origins and regulating replication initiation. Our objective is to characterize the effects of histone modifications on origin function using genetic and biochemical analyses. We have conducted a genetic screen to identify chromatin‐modifying enzymes that affect the licensing of origins. We discovered that loss of the Bre1 E3 ubiquitin ligase compromises the growth of a replication mutant. This enzyme, in conjunction with the E2 ubiquitin‐conjugating enzyme, Rad6, monoubiquitinates H2B K123, and this mark is a pre‐requisite for H3 K4 methylation. We are analyzing the mechanism by which these marks affect DNA replication. We will also determine whether the presence/absence of these marks affects recruitment of replication proteins, origin firing, or replication fork progression. These studies will provide a mechanistic view of how these and other histone modifications regulate DNA replication. (AHA 10PRE3740013, NIH 5R01GM083024‐02)

Abstract 4888: The effect of chromatin structure in regulation of telomere length in telomerase negative ALT cells

Abstract Telomeres are DNA-protein complexes at the ends of linear eukaryotic chromosomes and composed of tandem repeats of a TTAGGG sequence ending in a 3′ single-stranded overhang, or the G-strand overhang. And telomeres confer chromosomal stability protecting chromosome ends. Most immortalized human cell lines maintain and extend their telomere by activating telomerase. A small number of cells maintain and extend their telomere lengths without telomerase by homologous recombination of telomeres and this mechanism is called as alternative lengthening of telomere (ALT). There are many factors involved in telomere regulation. Recently, it has been known that chromatin structure is closely related to regulation of telomere length. Maintaining telomere length is important process in tumorigenesis and it has been suggested that telomeres are important target for cancer therapy. However, in ALT cells the effect of chromatin structure on regulation of telomere length is not understood. In this study, we induced change of chromatin structure by treating Trichostatin A (TSA), Histone deacetylase inhibitor, in ALT cells. Then, we measured APBs (ALT associated PML bodies) patterns, T-SCE (Telomere Sister Chromatid Exchange) frequency and telomere lengths. APBs patterns were analyzed by FISH (Fluorescence in situ hybridization) combined IF (Immunofluorescence staining). We compared the number of APBs in ALT cells with or without TSA. In GM 847 (ALT cell), APBs positive cells were found to be more frequent in GM 847 with TSA (63/100) compared to GM 847 without TSA (30/100) (p<0.001). A similar result was found in other ALT cell line, WI38-VA13. APB positive cells were more frequent in WI38-VA13 with TSA (38/100) compare to WI38-VA13 without TSA (18/100), (p<0.001). T-SCE frequency was determined by CO-FISH (Chromosome Orientation-FISH). Increse of T-SCE frequency was found in GM 847 with TSA (262/1026, 25.5%) compared to GM 847 without TSA (215/1026, 20.9%), (p<0.001). Telomere length was determined by telomere-FISH. Decrease of telomere length was found in GM 847 with TSA (telomere intensity mean 25.11%) compared to GM 847 without TSA (telomere intensity mean 29.8%), (p<0.001). This study demonstrated a close association between chromatin structure and telomere recombination in ALT cells and this result might provide insight into the mechanism of regulation of telomere length in telomerase-negative cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4888.

Accommodation and Repair of a UV Photoproduct in DNA at Different Rotational Settings on the Nucleosome Surface

Cyclobutane-thymine dimers (CTDs), the most common DNA lesion induced by UV radiation, cause 30 degrees bending and 9 degrees unwinding of the DNA helix. We prepared site-specific CTDs within a short sequence bracketed by strong nucleosome-positioning sequences. The rotational setting of CTDs over one turn of the helix near the dyad center on the histone surface was analyzed by hydroxyl radical footprinting. Surprisingly, the position of CTDs over one turn of the helix does not affect the rotational setting of DNA on the nucleosome surface. Gel-shift analysis indicates that one CTD destabilizes histone-DNA interactions by 0.6 or 1.1 kJ/mol when facing away or toward the histone surface, respectively. Thus, 0.5 kJ/mol energy penalty for a buried CTD is not enough to change the rotational setting of sequences with strong rotational preference. The effect of rotational setting on CTD removal by nucleotide excision repair (NER) was examined using Xenopus oocyte nuclear extracts. The NER rates are only 2-3 times lower in nucleosomes and change by only 1.5-fold when CTDs face away or toward the histone surface. Therefore, in Xenopus nuclear extracts, the rotational orientation of CTDs on nucleosomes has surprisingly little effect on rates of repair. These results indicate that nucleosome dynamics and/or chromatin remodeling may facilitate NER in gaining access to DNA damage in nucleosomes.

A Genetic and Molecular Characterization of Two Proximal Heterochromatic Genes on Chromosome 3 of Drosophila melanogaster

Heterochromatin comprises a transcriptionally repressive chromosome compartment in the eukaryotic nucleus; this is exemplified by the silencing effect it has on euchromatic genes that have been relocated nearby, a phenomenon known as position-effect variegation (PEV), first demonstrated in Drosophila melanogaster. However, the expression of essential heterochromatic genes within these apparently repressive regions of the genome presents a paradox, an understanding of which could provide key insights into the effects of chromatin structure on gene expression. To date, very few of these resident heterochromatic genes have been characterized to any extent, and their expression and regulation remain poorly understood. Here we report the cloning and characterization of two proximal heterochromatic genes in D. melanogaster, located deep within the centric heterochromatin of the left arm of chromosome 3. One of these genes, RpL15, is uncharacteristically small, is highly expressed, and encodes an essential ribosomal protein. Its expression appears to be compromised in a genetic background deficient for heterochromatin protein 1 (HP1), a protein associated with gene silencing in these regions. The second gene in this study, Dbp80, is very large and also appears to show a transcriptional dependence upon HP1; however, it does not correspond to any known lethal complementation group and is likely to be a nonessential gene.

Sperm chromatin integrity of bucks transgenic for the WAP bGH gene

The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13–18 months old. Semen was collected at 1-week intervals and 3–7 ejaculates from each rabbit were examined in total.Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes.The mean value of COMPαt was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.αt for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (−0.68, P<0.05) was found between S.D.αt of NTG males and percentages of pregnant does.The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. This suggests that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene.The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks.

The effect of chromatin structure on cisplatin damage in intact human cells.

The influence of chromatin structure on cisplatin DNA damage was investigated in intact human cells. The epsilon-globin gene promoter was utilised as the target DNA sequence and the terminal transferase-dependent PCR technique was employed to examine adduct formation at base pair resolution. It was found that cisplatin preferentially damaged at runs of consecutive guanine bases in intact cells. By comparing the relative intensity of adduct formation in intact cells and in purified genomic DNA, it was possible to assess the influence of chromatin proteins on the extent of cisplatin DNA damage. Enhanced damage in intact cells was found at the CACC site where a member of the Sp1 family of proteins is thought to bind. It is postulated that protein binding at this site bends the DNA double-helix so that enhanced cisplatin binding occurs. The altered DNA binding of cisplatin in the presence of chromatin proteins could be important in the properties of cisplatin as an anti-tumour drug.

Chromatin Domains and Prediction of MAR Sequences

Polynuceosomes are constrained into loops or domains and are insulated from the effects of chromatin structure and torsional strain from flanking domains by the cross-complexation of matrix-attached regions (MARs) and matrix proteins. MARs or SARs have an average size of 500 bp, are spaced about every 30 kb, and are control elements maintaining independent realms of gene activity. A fraction of MARs may cohabit with core origin replication (ORIs) and another fraction might cohabit with transcriptional enhancers. DNA replication, transcription, repair, splicing, and recombination seem to take place on the nuclear matrix. Classical AT-rich MARs have been proposed to anchor the core enhancers and core origins complexed with low abundancy transcription factors to the nuclear matrix via the cooperative binding to MARs of abundant classical matrix proteins (topoisomerase II, histone H1, lamins, SP120, ARBP, SATB1); this creates a unique nuclear microenvironment rich in regulatory proteins able to sustain transcription, replication, repair, and recombination. Theoretical searches and experimental data strongly support a model of activation of MARs and ORIs by transcription factors. A set of 21 characteristics are deduced or proposed for MAR/ORI sequences including their enrichment in inverted repeats, AT tracts, DNA unwinding elements, replication initiator protein sites, homooligonucleotide repeats (i.e., AAA, TTT, CCC), curved DNA, DNase I-hypersensitive sites, nucleosome-free stretches, polypurine stretches, and motifs with a potential for left-handed and triplex structures. We are establishing Banks of ORI and MAR sequences and have undertaken a large project of sequencing a large number of MARs in an effort to determine classes of DNA sequences in these regulatory elements and to understand their role at the origins of replication and transcriptional enhancers.

Mouse whey acidic protein gene is expressed ectopically in transgenic sheep

The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13–18 months old. Semen was collected at 1-week intervals and 3–7 ejaculates from each rabbit were examined in total.Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes.The mean value of COMPαt was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.αt for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (−0.68, P<0.05) was found between S.D.αt of NTG males and percentages of pregnant does.The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. This suggests that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene.The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks.

Irradiation Damage in Chromatin Isolated from V-79 Chinese Hamster Lung Fibroblasts

The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.

The binding of a carcinogen to the nucleosomal and non-nucleosomal regions of the simian virus 40 chromosome in vivo.

The effect of chromatin structure on the binding of a chemical carcinogen to the genomic DNA was studied. The binding in vivo of the ultimate carcinogen, benzo-pyrene 7,8,-diol,-9,10-epoxide, to various regions of the SV40 chromosome was revealed by an immunological method. Particular attention was given to restriction fragments which include the origin of replication which is "non-nucleosomal" in a significant fraction of the chromosomes. The distribution of (+/-) trans-7,8-dihydrobenzo[alpha]pyrene-7,8-diol-9,10-epoxide (BPDE) adducts was studied in 1) SV40 DNA modified in vitro to a level of 20 adducts/molecule, 2) DNA from SV40 chromosomes modified in vivo to a level of less than 1 adduct, and 3) DNA from only those chromosomes with an open origin of replication. In other experiments, the binding of BPDE to the origin region was compared to the binding to nucleosome core particle DNA from the viral chromosome. The origin region bound 1.7-fold more BPDE than core DNA, while linker DNA is 3-fold more modified than core DNA. However, the origin region was only about 20% more modified than any other region of the chromosome. We conclude that while the conformation of the DNA in chromatin has a slight effect on its accessibility to the carcinogen, the SV40 chromosome does not contain a particular "hot spot" which is preferentially modified by BPDE.