# US 2025/0085285 A1 · Methods for Generating Biologically Specific Chromatin Condensates and Uses Thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0085285 A1 - Document ID: US20250085285A1 - Status: Published application - Kind: A1 - Application number: 18/728,827 - Filed: 2023-01-18 - Published: 2025-03-13 - Earliest claimed: 2022-01-18 - Assignees: The Regents of the University of California - Applicants: The Regents of the University of California - Inventors: Muryam Gourdet; Laura Hsieh; Tracy Lou; Geeta Narlikar; Daniel Elnatan - Web page: https://patentgrep.com/patent/US20250085285A1 ## CPC - G01N 33/5076: {involving cell organelles, e.g. Golgi complex, endoplasmic reticulum} - G01N 33/5758: {involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites} - C12N 15/85: for animal cells - G01N 33/582: {with fluorescent label} - G01N 33/6875: {Nucleoproteins} - C12N 2800/107: for mammalian - G01N 2500/20: cell-free systems - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Compositions and methods for phase-separation of chromatin in distinct states are provided. In particular, differentially decorated chromatin in different states can be isolated by phase separation according to the methods disclosed herein. In some cases, specific chromatin interacting proteins are added to chromatin to recreate specific, biologically relevant chromatin states in vitro, which can be phase-separated using the disclosed methods. In addition, methods of using phase-separated chromatin for in vitro screening of pharmacological agents that modulate chromatin states, including small molecule chemicals and biomolecules, are also provided. ## Claims ### Claim 1 A method of assessing a test agent for the ability to modulate a chromatin state of interest, the method comprising: contacting a phase-separated droplet comprising a nucleosome array condensate with the test agent; and assessing the nucleosome array condensate for a modulation of the chromatin state of interest by the test compound. ### Claim 2 (depends on 1) The method according to claim 1, wherein the test agent is a small molecule, a biomolecule, or a polypeptide. ### Claim 3 (depends on 1, 2) The method according to claim 1 or 2, wherein the chromatin state of interest mimics a naturally occurring chromatin state. ### Claim 4 (depends on 3) The method according to claim 3, wherein the naturally occurring chromatin state is a chromatin state of a normal cell. ### Claim 5 (depends on 3) The method according to claim 3, wherein the naturally occurring chromatin state is a chromatin state associated with a disease. ### Claim 6 (depends on 5) The method according to claim 5, wherein the disease is cancer, a neurodegenerative disease, or an autoimmune disease. ### Claim 7 (depends on 6) The method according to claim 6, wherein the chromatin state is specific to a type of cancer. ### Claim 8 (depends on 7) The method according to claim 7, wherein the cancer is diffuse intrinsic pontine glioma (DIPG). ### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The method according to any one of claims 1 to 8, wherein the nucleosome array condensate comprises histone modifications. ### Claim 10 (depends on 9) The method according to claim 9, wherein the histone modifications comprise one or more histone modifications selected from the group consisting of: H3K9me3, H4K20me3, H3K27me3, H3K4me3, H3K36me3, H3K79me3, H3K56ac, H3K9Ac, H4K16Ac, H4K14Ac, H4K18Ac, and any combination thereof. ### Claim 11 (depends on 9, 10) The method according to claim 9 or claim 10, wherein the one or more histone modifications have a nucleosome occupancy selected to mimic a naturally occurring chromatin state. ### Claim 12 (depends on 11) The method according to claim 11, wherein the nucleosome occupancy is 60% or greater. ### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) The method according to any one of claims 1 to 12, wherein the nucleosome array condensate comprises one or more histone variants. ### Claim 14 (depends on 13) The method according to claim 13, wherein the one or more histone variants comprise H2Az, macro H2A, H3.3, CENP-A, H2ABBD, or any combination thereof. ### Claim 15 (depends on 9, 10, 11, 12, 13, 14) The method according to any one of claims 9 to 14, wherein the nucleosome array condensate comprises one or more heterochromatin or euchromatin proteins bound to the one or more histone modifications. ### Claim 16 (depends on 15) The method according to claim 15, wherein the one or more heterochromatin or euchromatin proteins bind cooperatively to the nucleosome array condensate. ### Claim 17 (depends on 15, 16) The method according to claim 15 or 16, wherein the one or more heterochromatin or euchromatin proteins comprise chromobox protein homolog 5 (CBX5), chromobox protein homolog 3 (CBX3), chromobox protein homolog 1 (CBX1), polycomb repressive complex 1 (PRC1), polycomb repressive complex 2 (PRC2), or any combination thereof. ### Claim 18 (depends on 15, 16, 17) The method according to any one of claims 15 to 17, wherein the one or more heterochromatin or euchromatin proteins exhibit a specificity for histones of the nucleosome array of 2-fold or greater as compared to the specificity for histones of a corresponding nucleosome array in which the one or more histone modifications are absent. ### Claim 19 (depends on 15, 16, 17, 18) The method according to any one of claims 15 to 18, wherein the one or more heterochromatin or euchromatin proteins exhibit a dissociation constant (KD) for the nucleosome array of less than or equal to 5 μM. ### Claim 20 (depends on 15, 16, 17, 18, 19) The method according to any one of claims 15 to 19, wherein the one or more heterochromatin or euchromatin proteins are labeled, and wherein assessing the nucleosome array condensate for a modulation of the chromatin state of interest by the test compound comprises detecting the labeled heterochromatin or euchromatin proteins. ### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) The method according to any one of claims 1 to 20, wherein the nucleosome array condensate comprises one or more proteins that associate with chromatin or DNA. ### Claim 22 (depends on 21) The method according to claim 21, wherein the one or more proteins that associate with chromatin or DNA comprise a polycomb-group protein, a bromodomain-containing protein, a chromobox protein homolog, a mediator complex subunit, or an oncogenic fusion protein, or any combination thereof. ### Claim 23 (depends on 22) The method according to claim 22, wherein the polycomb-group protein is PRC1 or PRC2, the bromodomain-containing protein is BRD1, BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, or BRD9, the chromobox protein homolog is CBX1, CBX2, CBX3, CBX4, CBX5, CBX6, CBX7, or CBX8, and the oncogenic fusion protein is AML-ETO. ### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) The method according to any one of claims 1 to 23, wherein the nucleosome array condensate comprises labeled DNA, and wherein assessing the nucleosome array condensate for a modulation of the chromatin state of interest by the test compound comprises detecting the labeled DNA. ### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) The method according to any one of claims 1 to 24, wherein the DNA used to assemble the nucleosome array is custom-designed to position nucleosomes at specific locations. ### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) The method according to any one of claims 1 to 24, wherein the DNA used to assemble the nucleosome array comprises naturally occurring DNA sequences. ### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) The method according to any one of claims 1 to 26, wherein the method is used to screen for and identify test agents that affect cooperativity or binding of proteins known to associate with chromatin or DNA or phase separation properties of chromatin. ### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) The method according to any one of claims 1 to 27, further comprising counter-screening against other types of chromatin states to identify test agents that specifically modulate the chromatin state of interest. ### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) The method according to any one of claims 1 to 28, wherein the method is a cell-free method. ### Claim 30 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) The method according to any one of claims 1 to 29, wherein assessing the nucleosome array condensate for a modulation of the chromatin state of interest comprises assessing the nucleosome array condensate for a modulation of dissolution, shape change, size change, or any combination thereof. ### Claim 31 A method to distinguish nucleosome array condensates that have different chromatin states, the method comprising: g) performing fluorescence imaging of fluorescently labeled nucleosome array condensates to obtain fluorescence images; h) performing segmentation-free image quantification on the fluorescence images; i) computing size distributions of the nucleosome array condensates from the fluorescence images; j) reducing complexities of the size distributions into distinct features that discriminate between the fluorescence images to simplify quantification of the nucleosome array condensates; k) acquiring shape distributions for the nucleosome array condensates from the fluorescence images; and l) reducing complexities of the shape distributions into distinct features that discriminate between the fluorescence images to simplify quantification of the nucleosome array condensates. ### Claim 32 (depends on 31) The method according to claim 31, further comprising quantifying z-factors using the outputs from steps d) and f) to assess quality control and reproducibility. ### Claim 33 (depends on 31, 32) The method according to claim 31 or 32, further comprising comparing data from multiple screens to identify test agents that specifically modulate the chromatin state of interest. ### Claim 34 A computer implemented method for processing fluorescence images of fluorescently labeled nucleosome array condensates, the computer performing steps comprising: g) receiving the fluorescence images of the fluorescently labeled nucleosome array condensates; h) performing segmentation-free image quantification on the fluorescence images; i) computing size distributions of the nucleosome array condensates from the fluorescence images; j) reducing complexities of the size distributions into distinct features that discriminate between the fluorescence images to simplify quantification of the nucleosome array condensates; k) acquiring shape distributions for the nucleosome array condensates from the fluorescence images; and l) reducing complexities of the shape distributions into distinct features that discriminate between the fluorescence images to simplify quantification of the nucleosome array condensates. ### Claim 35 (depends on 34) The method according to claim 34, further comprising quantifying z-factors using the outputs from steps d) and f) to assess quality control and reproducibility. ### Claim 36 (depends on 34, 35) The method according to claim 34 or 35, further comprising comparing data from multiple screens to identify test agents that specifically modulate the chromatin state of interest. ### Claim 37 (depends on 34, 35, 36) A non-transitory computer-readable medium comprising program instructions that, when executed by a processor in a computer, causes the processor to perform the method of any one of claims 34 to 36. ### Claim 38 (depends on 34, 35, 36) A system for distinguishing nucleosome array condensates that have different chromatin states, the system comprising: a processor programmed to process fluorescence images of fluorescently labeled nucleosome array condensates according to the computer implemented method of any one of claims 34 to 36; and a display component for displaying information about the nucleosome array condensates regarding size distributions of the nucleosome array condensates, shape distributions of the nucleosome array condensates, quantification of the nucleosome array condensates, or histone modifications or DNA modifications, or any combination thereof. ### Claim 39 (depends on 38) The system according to claim 38, further comprising reagents for forming a phase-separated droplet comprising a fluorescently labeled nucleosome array condensate. ### Claim 40 (depends on 38, 39) The system according to claim 38 or 39, further comprising a test agent. ### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) A pharmaceutical composition comprising a test agent identified as modulating a chromatin state of interest by the method of any one of claims 1 to 28. ### Claim 42 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) A method comprising administering to an individual in need thereof an effective amount of a test agent identified as modulating a chromatin state of interest by the method of any one of claims 1 to 28. ### Claim 43 A method of producing a nucleosome array condensate that mimics a naturally occurring chromatin state, the method comprising: combining one or more heterochromatin or euchromatin proteins and a nucleosome array under conditions in which the nucleosome array forms a nucleosome array condensate that mimics a naturally occurring chromatin state, wherein the nucleosome array comprises histone modifications, histone variants, or both, selected to mimic the naturally occurring chromatin state. ### Claim 44 (depends on 43) The method according to claim 43, wherein the one or more heterochromatin or euchromatin proteins comprise CBX5, CBX3, CBX1, PRC1, PRC2, or any combination thereof. ### Claim 45 (depends on 43, 44) The method according to claim 43 or claim 44, wherein the one or more heterochromatin or euchromatin proteins exhibit a specificity for histones of the nucleosome array of 2-fold or greater as compared to the specificity for histones of a corresponding nucleosome array in which the one or more histone modifications and/or histone variants are absent. ### Claim 46 (depends on 43, 44, 45) The method according to any one of claims 43 to 45, wherein the one or more heterochromatin or euchromatin proteins exhibit a dissociation constant (KD) for the nucleosome array of less than or equal to 5 μM. ### Claim 47 (depends on 43, 44, 45, 46) The method according to any one of claims 43 to 46, wherein the one or more heterochromatin or euchromatin proteins are labeled. ### Claim 48 (depends on 43, 44, 45, 46, 47) The method according to any one of claims 43 to 47, wherein the nucleosome array comprises histone modifications comprising H3K9me3, H4K20me3, H3K27me3, H3K4me3, H3K36me3, H3K79me3, H3K56ac, H3K9Ac, H4K16Ac, H4K14Ac, H4K18Ac, or any combination thereof, selected to mimic the naturally occurring chromatin state. ### Claim 49 (depends on 43, 44, 45, 46, 47, 48) The method according to any one of claims 43 to 48, wherein the nucleosome array comprises histone variants comprising H2Az, macro H2A, H3.3, CENP-A, H2ABBD, or any combination thereof, selected to mimic the naturally occurring chromatin state. ### Claim 50 (depends on 43, 44, 45, 46, 47, 48, 49) The method according to any one of claims 43 to 49, further comprising adding one or more proteins that associate with chromatin or DNA to the nucleosome array condensate, wherein the one or more proteins that associate with chromatin or DNA comprise a polycomb-group protein, a bromodomain-containing protein, a chromobox protein homolog, a mediator complex subunit, or an oncogenic fusion protein, or any combination thereof, selected to mimic the naturally occurring chromatin state. ### Claim 51 (depends on 50) The method according to claim 50, wherein the polycomb-group protein is PRC1 or PRC2, the bromodomain-containing protein is BRD1, BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, or BRD9, the chromobox protein homolog is CBX1, CBX2, CBX3, CBX4, CBX5, CBX6, CBX7, or CBX8, and the oncogenic fusion protein is AML-ETO. ### Claim 52 (depends on 43, 44, 45, 46, 47, 48, 49, 50, 51) The method according to any one of claims 43 to 51, wherein the nucleosome array comprises labeled DNA. ### Claim 53 (depends on 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) The method according to any one of claims 43 to 52, wherein the method is a cell-free method.