Mouse genomic associations with in vitro sensitivity to simulated space radiation
Not Available
60 APPLIED LIFE SCIENCES↗
Engineering topics
Karpen, Gary H.
Publications and source records attributed to Karpen, Gary H..
Innovation of heterochromatin functions drives rapid evolution of essential ZAD-ZNF genes in Drosophila
Contrary to dogma, evolutionarily young and dynamic genes can encode essential functions. We find that evolutionarily dynamic ZAD-ZNF genes, which encode the most abundant class of insect transcription factors, are more likely to encode essential functions in Drosophila melanogaster than ancient, conserved ZAD-ZNF genes. We focus on the Nicknack ZAD-ZNF gene, which is evolutionarily young, poorly retained in Drosophila species, and evolves under strong positive selection. Yet we find that it is necessary for larval development in D. melanogaster. We show that Nicknack encodes a heterochromatin-localizing protein like its paralog Oddjob, also an evolutionarily dynamic yet essential ZAD-ZNF gene. We find that the divergent D. simulans Nicknack protein can still localize to D. melanogaster heterochromatin and rescue viability of female but not male Nicknack-null D. melanogaster. Our findings suggest that innovation for rapidly changing heterochromatin functions might generally explain the essentiality of many evolutionarily dynamic ZAD-ZNF genes in insects.
59 BASIC BIOLOGICAL SCIENCES↗
Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin
Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct “territories.” Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.
Genetics & Heredity↗
RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster
Tandemly-repeated DNAs, or satellites, are enriched in heterochromatic regions of eukaryotic genomes and contribute to nuclear structure and function. Some satellites are transcribed, but we lack direct evidence that specific satellite RNAs are required for normal organismal functions. Here, we show satellite RNAs derived from AAGAG tandem repeats are transcribed in many cells throughout Drosophila melanogaster development, enriched in neurons and testes, often localized within heterochromatic regions, and important for viability. Strikingly, we find AAGAG transcripts are necessary for male fertility, and that AAGAG RNA depletion results in defective histone-protamine exchange, sperm maturation and chromatin organization. Since these events happen late in spermatogenesis when the transcripts are not detected, we speculate that AAGAG RNA in primary spermatocytes ‘primes’ post-meiosis steps for sperm maturation. In addition to demonstrating essential functions for AAGAG RNAs, comparisons between closely related Drosophila species suggest that satellites and their transcription evolve quickly to generate new functions.
59 BASIC BIOLOGICAL SCIENCES↗
Haplotypes spanning centromeric regions reveal persistence of large blocks of archaic DNA
Despite critical roles in chromosome segregation and disease, the repetitive structure and vast size of centromeres and their surrounding heterochromatic regions impede studies of genomic variation. Here we report the identification of large-scale haplotypes (cenhaps) in humans that span the centromere-proximal regions of all metacentric chromosomes, including the arrays of highly repeated α-satellites on which centromeres form. Cenhaps reveal deep diversity, including entire introgressed Neanderthal centromeres and equally ancient lineages among Africans. These centromere-spanning haplotypes contain variants, including large differences in α-satellite DNA content, which may influence the fidelity and bias of chromosome transmission. The discovery of cenhaps creates new opportunities to investigate their contribution to phenotypic variation, especially in meiosis and mitosis, as well as to more incisively model the unexpectedly rich evolution of these challenging genomic regions.
1000 genomes↗
Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A
Repair of DNA double-strand breaks (DSBs) must be orchestrated properly within diverse chromatin domains in order to maintain genetic stability. Euchromatin and heterochromatin domains display major differences in histone modifications, biophysical properties, and spatiotemporal dynamics of DSB repair. However, it is unclear whether differential histone-modifying activities are required for DSB repair in these distinct domains. Here, we showed previously that the Drosophila melanogaster KDM4A (dKDM4A) histone demethylase is required for heterochromatic DSB mobility. Here we used locus-specific DSB induction in Drosophila animal tissues and cultured cells to more deeply interrogate the impact of dKDM4A on chromatin changes, temporal progression, and pathway utilization during DSB repair. We found that dKDM4A promotes the demethylation of heterochromatin-associated histone marks at DSBs in heterochromatin but not euchromatin. Most importantly, we demonstrate that dKDM4A is required to complete DSB repair in a timely manner and regulate the relative utilization of homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways but exclusively for heterochromatic DSBs. We conclude that the temporal kinetics and pathway utilization during heterochromatic DSB repair depend on dKDM4A-dependent demethylation of heterochromatic histone marks. Thus, distinct pre-existing chromatin states require specialized epigenetic alterations to ensure proper DSB repair.
59 BASIC BIOLOGICAL SCIENCES↗
Pervasive epigenetic effects of Drosophila euchromatic transposable elements impact their evolution
Transposable elements (TEs) are widespread genomic parasites, and their evolution has remained a critical question in evolutionary genomics. Here, we study the relatively unexplored epigenetic impacts of TEs and provide the first genome-wide quantification of such effects in D. melanogaster and D. simulans. Surprisingly, the spread of repressive epigenetic marks (histone H3K9me2) to nearby DNA occurs at >50% of euchromatic TEs, and can extend up to 20 kb. This results in differential epigenetic states of genic alleles and, in turn, selection against TEs. Interestingly, the lower TE content in D. simulans compared to D. melanogaster correlates with stronger epigenetic effects of TEs and higher levels of host genetic factors known to promote epigenetic silencing. Our study demonstrates that the epigenetic effects of euchromatic TEs, and host genetic factors modulating such effects, play a critical role in the evolution of TEs both within and between species.
Life Sciences & Biomedicine - Other Topics↗
Drosophila Histone Demethylase KDM4A Has Enzymatic and Non-enzymatic Roles in Controlling Heterochromatin Integrity
Not Available
Colmenares, Serafin U.↗
Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy
Chromosomal instability (CIN) is a hallmark of cancer that contributes to tumour heterogeneity and other malignant properties. Aberrant centromere and kinetochore function causes CIN through chromosome missegregation, leading to aneuploidy, rearrangements and micronucleus formation. Here we develop a Centromere and kinetochore gene Expression Score (CES) signature that quantifies the centromere and kinetochore gene misexpression in cancers. High CES values correlate with increased levels of genomic instability and several specific adverse tumour properties, and prognosticate poor patient survival for breast and lung cancers, especially early-stage tumours. They also signify high levels of genomic instability that sensitize cancer cells to additional genotoxicity. Thus, the CES signature forecasts patient response to adjuvant chemotherapy or radiotherapy. Our results demonstrate the prognostic and predictive power of the CES, suggest a role for centromere misregulation in cancer progression, and support the idea that tumours with extremely high CIN are less tolerant to specific genotoxic therapies.
Science & Technology - Other Topics↗
The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic
Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.
59 BASIC BIOLOGICAL SCIENCES↗
The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic
Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.
59 BASIC BIOLOGICAL SCIENCES↗
A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin
Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.
59 BASIC BIOLOGICAL SCIENCES↗
Identification of genetic factors that modify motor performance and body weight using Collaborative Cross mice
Evidence has emerged that suggests a link between motor deficits, obesity and many neurological disorders. However, the contributing genetic risk factors are poorly understood. Here we used the Collaborative Cross (CC), a large panel of newly inbred mice that captures 90% of the known variation among laboratory mice, to identify the genetic loci controlling rotarod performance and its relationship with body weight in a cohort of 365 mice across 16 CC strains. Body weight and rotarod performance varied widely across CC strains and were significantly negatively correlated. Genetic linkage analysis identified 14 loci that were associated with body weight. However, 45 loci affected rotarod performance, seven of which were also associated with body weight, suggesting a strong link at the genetic level. As a result, we show that genes identified in this study overlap significantly with those related to neurological disorders and obesity found in human GWA studies. In conclusion, our results provide a genetic framework for studies of the connection between body weight, the central nervous system and behavior.
59 BASIC BIOLOGICAL SCIENCES↗
Identification of genetic loci that control mammary tumor susceptibility through the host microenvironment
The interplay between host genetics, tumor microenvironment and environmental exposure in cancer susceptibility remains poorly understood. Here we assessed the genetic control of stromal mediation of mammary tumor susceptibility to low dose ionizing radiation (LDIR) using backcrossed F1 into BALB/c (F1Bx) between cancer susceptible (BALB/c) and resistant (SPRET/EiJ) mouse strains. Tumor formation was evaluated after transplantation of non-irradiated Trp53-/- BALB/c mammary gland fragments into cleared fat pads of F1Bx hosts. Genome-wide linkage analysis revealed 2 genetic loci that constitute the baseline susceptibility via host microenvironment. However, once challenged with LDIR, we discovered 13 additional loci that were enriched for genes involved in cytokines, including TGFβ1 signaling. Surprisingly, LDIR-treated F1Bx cohort significantly reduced incidence of mammary tumors from Trp53-/- fragments as well as prolonged tumor latency, compared to sham-treated controls. We demonstrated further that plasma levels of specific cytokines were significantly correlated with tumor latency. Using an ex vivo 3-D assay, we confirmed TGFβ1 as a strong candidate for reduced mammary invasion in SPRET/EiJ, which could explain resistance of this strain to mammary cancer risk following LDIR. Our results open possible new avenues to understand mechanisms of genes operating via the stroma that affect cancer risk from external environmental exposures.
59 BASIC BIOLOGICAL SCIENCES↗
The Release 6 reference sequence of the Drosophila melanogaster genome
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
59 BASIC BIOLOGICAL SCIENCES↗
Nucleosomes Shape DNA Polymorphism and Divergence
An estimated 80% of genomic DNA in eukaryotes is packaged as nucleosomes, which, together with the remaining interstitial linker regions, generate higher order chromatin structures. Nucleosome sequences isolated from diverse organisms exhibit ,10 bp periodic variations in AA, TT and GC dinucleotide frequencies. These sequence elements generate intrinsically curved DNA and help establish the histone-DNA interface. We investigated an important unanswered question concerning the interplay between chromatin organization and genome evolution: do the DNA sequence preferences inherent to the highly conserved histone core exert detectable natural selection on genomic divergence and polymorphism? To address this hypothesis, we isolated nucleosomal DNA sequences from Drosophila melanogaster embryos and examined the underlying genomic variation within and between species. We found that divergence along the D. melanogaster lineage is periodic across nucleosome regions with base changes following preferred nucleotides, providing new evidence for systematic evolutionary forces in the generation and maintenance of nucleosome-associated dinucleotide periodicities. Further, Single Nucleotide Polymorphism (SNP) frequency spectra show striking periodicities across nucleosomal regions, paralleling divergence patterns. Preferred alleles occur at higher frequencies in natural populations, consistent with a central role for natural selection. These patterns are stronger for nucleosomes in introns than in intergenic regions, suggesting selection is stronger in transcribed regions where nucleosomes undergo more displacement, remodeling and functional modification. In addition, we observe a large-scale (~180 bp) periodic enrichment of AA/TT dinucleotides associated with nucleosome occupancy, while GC dinucleotide frequency peaks in linker regions. Divergence and polymorphism data also support a role for natural selection in the generation and maintenance of these super-nucleosomal patterns. Our results demonstrate that nucleosome-associated sequence periodicities are under selective pressure, implying that structural interactions between nucleosomes and DNA sequence shape sequence evolution, particularly in introns.
59 BASIC BIOLOGICAL SCIENCES↗
An interferon signature identified by RNA-sequencing of mammary tissues varies across the estrous cycle and is predictive of metastasis-free survival
The concept that a breast cancer patient's menstrual stage at the time of tumor surgery influences risk of metastases remains controversial. The scarcity of comprehensive molecular studies of menstrual stage-dependent fluctuations in the breast provides little insight. To gain a deeper understanding of the biological changes in mammary tissue and blood during the menstrual cycle and to determine the influence of environmental exposures, such as low-dose ionizing radiation (LDIR), we used the mouse to characterize estrous-cycle variations in mammary gene transcripts by RNA-sequencing, peripheral white blood cell (WBC) counts and plasma cytokine levels. We identified an estrous-variable and hormone-dependent gene cluster enriched for Type-1 interferon genes. Cox regression identified a 117-gene signature of interferon-associated genes, which correlated with lower frequencies of metastasis in breast cancer patients. LDIR (10cGy) exposure had no detectable effect on mammary transcripts. However, peripheral WBC counts varied across the estrous cycle and LDIR exposure reduced lymphocyte counts and cytokine levels in tumor-susceptible mice. Our finding of variations in mammary Type-1 interferon and immune functions across the estrous cycle provides a mechanism by which timing of breast tumor surgery during the menstrual cycle may have clinical relevance to a patient's risk for distant metastases.
38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗
Impact of sequencing depth in ChIP-seq experiments
In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40–50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data.
59 BASIC BIOLOGICAL SCIENCES↗