Affinity hierarchies and amphiphilic proteins underlie the co-assembly of nucleolar and heterochromatin condensates.

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.

Affinity hierarchies and amphiphilic proteins underlie the co-assembly of nucleolar and heterochromatin condensates.

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.

HP1-driven phase separation recapitulates the thermodynamics and kinetics of heterochromatin condensate formation.

The spatial segregation of pericentromeric heterochromatin (PCH) into distinct, membrane-less nuclear compartments involves the binding of Heterochromatin Protein 1 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid-liquid phase separation properties in vitro, its mechanistic impact on the structure and dynamics of PCH condensate formation in vivo remains largely unresolved. Here, using a minimal theoretical framework, we systematically investigate the mutual coupling between self-interacting HP1-like molecules and the chromatin polymer. We reveal that the specific affinity of HP1 for H3K9me2/3 loci facilitates coacervation in nucleo and promotes the formation of stable PCH condensates at HP1 levels far below the concentration required to observe phase separation in purified protein assays in vitro. These heterotypic HP1-chromatin interactions give rise to a strong dependence of the nucleoplasmic HP1 density on HP1-H3K9me2/3 stoichiometry, consistent with the thermodynamics of multicomponent phase separation. The dynamical cross talk between HP1 and the viscoelastic chromatin scaffold also leads to anomalously slow equilibration kinetics, which strongly depend on the genomic distribution of H3K9me2/3 domains and result in the coexistence of multiple long-lived, microphase-separated PCH compartments. The morphology of these complex coacervates is further found to be governed by the dynamic establishment of the underlying H3K9me2/3 landscape, which may drive their increasingly abnormal, aspherical shapes during cell development. These findings compare favorably to 4D microscopy measurements of HP1 condensate formation in live Drosophila embryos and suggest a general quantitative model of PCH formation based on the interplay between HP1-based phase separation and chromatin polymer mechanics.

Mouse genomic associations with in vitro sensitivity to simulated space radiation.

Exposure to ionizing radiation is considered by NASA to be a major health hazard for deep space exploration missions. Ionizing radiation sensitivity is modulated by both genomic and environmental factors. Understanding their contributions is crucial for designing experiments in model organisms, evaluating the risk of deep space (i.e. high-linear energy transfer, or LET, particle) radiation exposure in astronauts, and also selecting therapeutic irradiation regimes for cancer patients. We identified single nucleotide polymorphisms in 15 strains of mice, including 10 collaborative cross model strains and 5 founder strains, associated with spontaneous and ionizing radiation-induced in vitro DNA damage quantified based on immunofluorescent tumor protein p53 binding protein (53BP1) positive nuclear foci. Statistical analysis suggested an association with pathways primarily related to cellular signaling, metabolism, tumorigenesis and nervous system damage. We observed different genomic associations in early (4 and 8 h) responses to different LET radiation, while later (24 hour) DNA damage responses showed a stronger overlap across all LETs. Furthermore, a subset of pathways was associated with spontaneous DNA damage, suggesting 53BP1 positive foci as a potential biomarker for DNA integrity in mouse models. Our results suggest several mouse strains as new models to further study the impact of ionizing radiation and validate the identified genetic loci. We also highlight the importance of future human in vitro studies to refine the association of genes and pathways with the DNA damage response to ionizing radiation and identify targets for space travel countermeasures.

Complete genomic and epigenetic maps of human centromeres.

Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.9 megabases). Detailed maps of these regions revealed multimegabase structural rearrangements, including in active centromeric repeat arrays. Analysis of centromere-associated sequences uncovered a strong relationship between the position of the centromere and the evolution of the surrounding DNA through layered repeat expansions. Furthermore, comparisons of chromosome X centromeres across a diverse panel of individuals illuminated high degrees of structural, epigenetic, and sequence variation in these complex and rapidly evolving regions.

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.

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.

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.

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.

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. 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.

Heterochromatin: Guardian of the Genome.

Constitutive heterochromatin is a major component of the eukaryotic nucleus and is essential for the maintenance of genome stability. Highly concentrated at pericentromeric and telomeric domains, heterochromatin is riddled with repetitive sequences and has evolved specific ways to compartmentalize, silence, and repair repeats. The delicate balance between heterochromatin epigenetic maintenance and cellular processes such as mitosis and DNA repair and replication reveals a highly dynamic and plastic chromatin domain that can be perturbed by multiple mechanisms, with far-reaching consequences for genome integrity. Indeed, heterochromatin dysfunction provokes genetic turmoil by inducing aberrant repeat repair, chromosome segregation errors, transposon activation, and replication stress and is strongly implicated in aging and tumorigenesis. Here, we summarize the general principles of heterochromatin structure and function, discuss the importance of its maintenance for genome integrity, and propose that more comprehensive analyses of heterochromatin roles in tumorigenesis will be integral to future innovations in cancer treatment.

Exploring the role of CENP-A Ser18 phosphorylation in CIN and Tumorigenesis.

Chromosome instability (CIN) contributes to the development of many cancer. In this paper, we summarize our recent finding that a novel pathway by which FBW7 loss promotes Centromere Protein A (CENP-A) phosphorylation on Serine 18 through Cyclin E1/CDK2, therefore promoting CIN and tumorigenesis. Our finding demonstrates the importance of CENP-A post-translational modification on modulating centromere and mitotic functions in cancer.

FBW7 Loss Promotes Chromosomal Instability and Tumorigenesis via Cyclin E1/CDK2-Mediated Phosphorylation of CENP-A.

The centromere regulates proper chromosome segregation, and its dysfunction is implicated in chromosomal instability (CIN). However, relatively little is known about how centromere dysfunction occurs in cancer. Here, we define the consequences of phosphorylation by cyclin E1/CDK2 on a conserved Ser18 residue of centromere-associated protein CENP-A, an essential histone H3 variant that specifies centromere identity. Ser18 hyperphosphorylation in cells occurred upon loss of FBW7, a tumor suppressor whose inactivation leads to CIN. This event on CENP-A reduced its centromeric localization, increased CIN, and promoted anchorage-independent growth and xenograft tumor formation. Overall, our results revealed a pathway that cyclin E1/CDK2 activation coupled with FBW7 loss promotes CIN and tumor progression via CENP-A-mediated centromere dysfunction. Cancer Res; 77(18); 4881-93. ©2017 AACR.

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.

Drosophila Histone Demethylase KDM4A Has Enzymatic and Non-enzymatic Roles in Controlling Heterochromatin Integrity.

Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. We demonstrate that dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. We conclude that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions.

Phase separation drives heterochromatin domain formation.

Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). A prevalent view of heterochromatic silencing is that these and associated factors lead to chromatin compaction, resulting in steric exclusion of regulatory proteins such as RNA polymerase from the underlying DNA. However, compaction alone does not account for the formation of distinct, multi-chromosomal, membrane-less heterochromatin domains within the nucleus, fast diffusion of proteins inside the domain, and other dynamic features of heterochromatin. Here we present data that support an alternative hypothesis: that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments. We show that Drosophila HP1a protein undergoes liquid-liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. We conclude that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. We propose that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.

Influence of early life exposure, host genetics and diet on the mouse gut microbiome and metabolome.

Although the gut microbiome plays important roles in host physiology, health and disease1, we lack understanding of the complex interplay between host genetics and early life environment on the microbial and metabolic composition of the gut. We used the genetically diverse Collaborative Cross mouse system2 to discover that early life history impacts the microbiome composition, whereas dietary changes have only a moderate effect. By contrast, the gut metabolome was shaped mostly by diet, with specific non-dietary metabolites explained by microbial metabolism. Quantitative trait analysis identified mouse genetic trait loci (QTL) that impact the abundances of specific microbes. Human orthologues of genes in the mouse QTL are implicated in gastrointestinal cancer. Additionally, genes located in mouse QTL for Lactobacillales abundance are implicated in arthritis, rheumatic disease and diabetes. Furthermore, Lactobacillales abundance was predictive of higher host T-helper cell counts, suggesting an important link between Lactobacillales and host adaptive immunity.

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.

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.

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.