Biology subjects

Karpen, G.

Publications and source records attributed to Karpen, G..

7 recordsLinked to original sources

bioRxiv2026

ORC binding to Heterochromatin Protein 1 through intrinsically disordered regions is required for heterochromatin structure and function

The Origin Recognition Complex (ORC) is known for initiating DNA replication in eukaryotic cells, but increasing evidence suggests that ORC has multiple functions. Here we show that the organization and function of two major nuclear components, heterochromatin and the nucleolus, depend on multivalent interactions between Orc1 and Heterochromatin Protein 1a (HP1a) in Drosophila melanogaster. Specifically, binding requires two short motifs (R1 and R2) in the intrinsically disordered region (IDR) of Orc1, and two motifs (HGM and CTE) located in the N- and C-terminal regions of HP1a. Pairing of these four motifs promotes ORC-HP1 interactions, which also requires HP1 dimerization. Disrupting ORC-HP1a interactions by mutating the R1/R2 motifs causes defects in heterochromatin functions, specifically suppression of Position-Effect Variegation (PEV), ribosomal DNA (rDNA) decondensation, increased rRNA transcription, and nucleolar expansion, without global loss of H3K9me2 epigenetic modifications. Our findings indicate that ORC acts as a structural regulator that organizes heterochromatic regions and promotes proper genome regulation through multivalent interactions with HP1 and chromatin.

molecular biology↗

bioRxiv2024

Expanding the HP1a-binding consensus and molecular grammar for heterochromatin assembly

The recruitment of Heterochromatin Protein 1 (HP1) partners is essential for heterochromatin assembly and function, yet our knowledge regarding their organization in heterochromatin remains limited. Here we show that interactors engage the Drosophila HP1 (HP1a) dimer through a degenerate and expanded form of the previously identified PxVxL motif, which we now term HP1a Access Codes (HACs). These HACs reside in disordered regions, possess high conservation among Drosophila homologs, and contain alternating hydrophobic residues nested in a cluster of positively charged amino acids. These findings and molecular dynamics simulations identify key electrostatic interactions that modulate HP1a-binding strength and provide a dramatically improved HP1a-binding consensus motif that can reveal protein partners and the molecular grammar involved in heterochromatin assembly. We propose HP1a acts as a scaffold for other heterochromatin components containing HAC motifs, which in turn may regulate the function and higher order structure of the heterochromatin compartment.

molecular biology↗

bioRxiv2024

HP1a promotes chromatin liquidity and drives spontaneous heterochromatin compartmentalization

Compartmentalization of the nucleus into heterochromatin and euchromatin is highly conserved across eukaryotes. Constitutive heterochromatin (C-Het) constitutes a liquid-like condensate that packages the repetitive regions of the genome through the enrichment of histone modification H3K9me3 and recruitment of its cognate reader protein Heterochromatin Protein-1 (HP1a). The ability for well-ordered nucleosome arrays and HP1a to independently form biomolecular condensates suggests that the emergent material properties of C-Het compartments may contribute to its functions such as force-buffering, dosage-dependent gene silencing, and selective permeability. Using an in vitro reconstitution system we directly assess the contributions of H3K9me3 and HP1a on the biophysical properties of C-Het. In the presence of HP1a, H3K9me3 (Me-) and unmodified (U-) chromatin form co-condensates composed of distinct, immiscible domains. These chromatin domains form spontaneously and are reversible. Independently of HP1a, H3K9me3 modifications are sufficient to increase linker-DNA length within chromatin arrays and slow chromatin condensate growth. HP1a increases the liquidity of chromatin condensates while dramatically differentiating the viscoelastic properties of Me-chromatin versus U-chromatin. Mutating key residues in HP1a show that HP1a interactions with itself and chromatin determine the relative interfacial tension between chromatin compartments, however the formation of condensates is driven by the underlying chromatin. These direct measurements map the energetic landscape that determines C-Het compartmentalization, demonstrating that nuclear compartmentalization is a spontaneous and energetically favorable process in which HP1a plays a critical role in establishing a hierarchy of affinities between H3K9me3-chromatin and unmodified-chromatin. Highlights HP1a is necessary and sufficient for heterochromatin compartmentalization. Heterochromatin compartmentalization is reversible and proceeds through microphase-separation. H3K9me3 is sufficient to change nucleosome-array dynamics and mesoscale material properties. HP1a increases chromatin liquidity. HP1a-chromatin interaction modes tune the interfacial tensions and morphologies of heterochromatin compartments.

biophysics↗

bioRxiv2023

DNA Double-Strand Break Movement in Heterochromatin Depends on the Drosophila Histone Acetyltransferase Gcn5.

Cells employ diverse strategies to repair double-strand breaks (DSBs), a dangerous form of DNA damage that threatens genome integrity. Eukaryotic nuclei consist of different chromatin environments, each displaying distinct molecular and biophysical properties that can significantly influence the DSB repair process. Specifically, DSBs arising in the compact and silenced heterochromatin domains have been found to move to the heterochromatin periphery in mouse and Drosophila to prevent aberrant recombination events. However, it is poorly understood how chromatin components, such as histone post-translational modifications, contribute to these DSB movements within heterochromatin. Using locus-specific DSB induction in Drosophila tissues and cultured cells, we identify that histone H3 lysine 9 acetylation (H3K9ac) is enriched at DSBs in heterochromatin but not euchromatin. We find that this enrichment is mediated by the histone acetyltransferase dGcn5, which rapidly localizes to heterochromatic DSBs. Moreover, we demonstrate that in the absence of dGcn5, heterochromatic DSBs display impaired recruitment of the SUMO E3 ligase Nse2/Qjt and fail to relocate to the heterochromatin periphery to complete repair. In summary, our results reveal a previously unidentified role for dGcn5 and H3K9ac in heterochromatin DSB repair and underscore the importance of differential chromatin responses at heterochromatic and euchromatic DSBs to promote safe repair.

cell biology↗

bioRxiv2022

Liquid-liquid phase separation recapitulates the thermodynamics and kinetics of heterochromatin 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 biophysical modeling, 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 crosstalk 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 condensates that we perform 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. SIGNIFICANCE STATEMENTThe compartmentalization of pericentromeric heterochromatin (PCH), the highly-repetitive part of the genome, into membrane-less organelles enriched in HP1 proteins, is critical to both genetic stability and cell fate determination. While HP1 can self-organize into liquid-like condensates in vitro, the roles of HP1 and the polymer chromatin in forming 3D PCH domains in vivo are still unclear. Using molecular simulations, we show that key kinetic and thermodynamic features of PCH condensates are consistent with a phase-separation mode of organization driven by the genomic distribution of methylated domains and HP1 self-attraction and affinity for heterochromatin. Our predictions are corroborated by live-microscopy performed during early fly embryogenesis, suggesting that a strong crosstalk between HP1-based phase separation and chromosome mechanics drive PCH condensate formation.

biophysics↗

bioRxiv2022

Mapping protein-DNA interactions with DiMeLo-seq

We recently developed Directed Methylation with Long-read sequencing (DiMeLo-seq) to map protein-DNA interactions genome wide. DiMeLo-seq is capable of mapping multiple interaction sites on single DNA molecules, profiling protein binding in the context of endogenous DNA methylation, identifying haplotype specific protein-DNA interactions, and mapping protein-DNA interactions in repetitive regions of the genome that are difficult to study with short-read methods. With DiMeLo-seq, adenines in the vicinity of a protein of interest are methylated in situ by tethering the Hia5 methyltransferase to an antibody using protein A. Protein-DNA interactions are then detected by direct readout of adenine methylation with long-read, single-molecule, DNA sequencing platforms such as Nanopore sequencing. Here, we present a detailed protocol and practical guidance for performing DiMeLo-seq. This protocol can be run on nuclei from fresh, lightly fixed, or frozen cells. The protocol requires 1-2 days for performing in situ targeted methylation, 1-5 days for library preparation depending on desired fragment length, and 1-3 days for Nanopore sequencing depending on desired sequencing depth. The protocol requires basic molecular biology skills and equipment, as well as access to a Nanopore sequencer. We also provide a Python package, dimelo, for analysis of DiMeLo-seq data. Key papersAltemose, N., Maslan, A., Smith, O.K., Sundararajan, K., Brown, R.R., Mishra, R., Detweiler, A.M., Neff, N., Miga, K.H., Straight, A.F. and Streets, A., 2022. DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide. Nature Methods, pp.1-13. (https://doi.org/10.1038/s41592-022-01475-6)

genomics↗

bioRxiv2022

Mouse Genomic Associations with Ex Vivo 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 ex vivo DNA damage quantified based on immunofluorescent 53BP1+ 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 hour) 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+ foci as a potential biomarker for DNA integrity in mouse models. Based on our results, we 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 ex vivo studies to refine the association of genes and pathways with the DNA damage response to ionizing radiation and identify targets for space travel countermeasures.

genomics↗