Phase separation in genome organization across evolution

Abstract

Phase separation is emerging as a paradigm to explain the self-assembly and organization of membraneless bodies in the cell. Recent advances show that this principle also extends to nucleoprotein complexes, including DNA-based structures. We discuss here recent observations on the role of phase separation in genome organization across the evolutionary spectrum from bacteria to mammals. These findings suggest that molecular interactions amongst DNA-binding proteins evolved to form a variety of biomolecular condensates with distinct material properties that affect genome organization and function. We suggest that phase separation contributes to genome organization across evolution and that the resulting phase behavior of genomes may underlie regulatory mechanisms and disease.

Keywords: biomolecular condensates; evolution; genome organization; phase separation; transcription.

Figure 1. Molecular driving forces of phase separation of the mammalian nuclear genome

(A) Phase diagram for a simple, two-component system where an interaction parameter, such as temperature, salt concentration, pH, etc., is plotted as a function of concentration of one of the components. The dark solid line represents the boundary between a single, homogenous phase and the two-phase coexistence region. Insets show representative examples of the solution in different regions of the phase diagram. (B) Schematic of a typical phase separating protein. Many phase separating proteins contain substrate-binding (i.e. DNA-binding, protein-interaction) domains and unstructured, disordered domains that can both be arranged in (n) multiplicities, giving rise to multivalent interactions. (C) Representative arrangement of DNA-binding phase separating proteins to form condensates in association with DNA. (D) The nucleus as an emulsion of DNA condensates. (E-H) Levels of phase separation in the nucleus include interactions between histone tails as part of nucleosomal arrays (E), transcriptional condensates involving the interaction of enhancer elements and transcription factors (F), partial miscibility or immiscibility between neighboring chromatin domains (G), and large gene-silenced regions compacted in heterochromatin (H). Bar represents typical length scale of interactions.

Figure 2. Conservation of phase separation in organization of nucleoids

(A,B) Phase separation interactions drive the organization of the mitochondrial genome in a mitochondrion (A) into nucleoprotein complexes called mitochondrial nucleoids (B). (B) Mitochondrial Transcription Factor A (TFAM): primary nucleoid architectural protein containing two HMG domains and two IDRs (linker and C-tail); mt-nucleoid associated proteins (mt-NAPs): core proteins associated with mtDNA involved in transcription (TFB2M, POLRMT, mTERF) and replication (mtSSB, POLG1/2, TOP1MT), many of which have DNA-binding domains and intrinsically disordered regions; mtDNA: mitochondrial genome consisting of ∼16 kB circular DNA existing as ∼1–2 copies per nucleoid; mtRNA: mitochondrial RNA transcribed directly from the mitochondrial genome inside the condensate but later associates into separate RNP condensates. (C,D) The larger bacterial genome is analogously organized as a phase-separated nucleoid (D) in a bacterium (C). HU: one of the major architectural, histone-like proteins that exists as a dimer with flexible β-sheet arms that package DNA into a dense core surrounded by less dense phase of DNA and associated proteins; Transcriptional foci: dynamic condensates comprised of RNA polymerase (RNAP) and other transcription factors, many of which contain IDRs; SSB compartments: single-stranded DNA binding protein (SSB) forms a tetramer around single-stranded DNA with protruding intrinsically disordered linkers that are capped by conserved C-terminal peptide motif.

Figure 3. Phylogenetic tree comparing the evolution of TFAM’s modular domains

FASTA sequences were obtained for fifty representative organisms for TFAM and/or the homologue Abf2 (S. cerevisiae). Organisms were grouped using NCBI Taxonomy Common Tree algorithm, and the generated tree was visualized using the EMBL Interactive Tree of Life (iTOL) tool. Sequences were aligned using the NCBI Cobalt algorithm using default settings, and color coding was assigned based on frequency-based differences, where red indicates highly variable regions with high frequency of mutations and grey indicates highly conserved regions with low frequency of mutations. Sequence gaps are indicated by solid black lines.

Figure 4. Regulation of phase separation in genome organization and function

(A) Phase diagram describing phase separation of a DNA condensate within the cell nucleus. There is an interplay between organization of the chromatin and droplet coarsening. (B) Droplets nucleate around specific genomic loci, which are brought together as droplets grow and coarsen, while other genomic loci are excluded from the droplet. (C) Individual proteins and small condensates can diffuse and rearrange in the nucleus, while larger condensates are immobilized within the chromatin network. (D) Droplets concentrate DNA which may lead to transcriptional repression. (E) Droplets concentrate proteins, such as transcription factors, which can alter reaction kinetics, leading to changes in gene activity. (F) In DNA repair, droplets nucleate scaffold proteins such as 53BP1 that later recruit client proteins which partition into the droplet to augments DNA repair. (G) The viscous physicochemical environment within the droplet stabilizes stabilize the chromatin fiber from thermal fluctuations and excludes immiscible molecules from entering, such as potentially damaging reactive oxygen species (ROS). (H-J) DNA condensates exhibit a spectrum of material properties: highly dynamic, liquid-like behavior (H), intermediary, viscoelastic behavior (I), and solid-like, crystalline behavior (J). These material properties correlate with the level of biological activity, such as transcriptional bursting, controlled processing, or genome preservation, respectively.