Biology subjects

Kingston, R. E.

Publications and source records attributed to Kingston, R. E..

12 recordsLinked to original sources

bioRxiv2025

Mutual Antagonism Between PRC1 Condensates and SWI/SNF in Chromatin Regulation

Opposing activities of conserved chromatin regulatory complexes, such as the Polycomb Repressive Complex 1 (PRC1) and the activating chromatin remodeler SWI/SNF play critical roles in regulating gene expression during development and differentiation. The mechanisms by which these complexes compete to regulate chromatin states remain poorly understood. We combine single-molecule analysis and genomic approaches in cultured cells to demonstrate that the condensate-forming properties of PRC1 play an important role in excluding SWI/SNF from chromatin. Consistently, PRC1 compositions with a higher propensity for condensate formation are more effective in preventing SWI/SNF binding. Conversely, SWI/SNF-bound chromatin significantly reduces PRC1 binding and subsequent condensate formation. Notably, SWI/SNF can suppress PRC1 condensate formation in an ATP-hydrolysis independent manner. We propose that the condensate properties of different PRC1 compositions drive mutual PRC1-SWI/SNF antagonism to properly balance these competing regulatory activities during development.

biophysics↗

bioRxiv2025

Fbxl10/Kdm2b is required for Kmt2b/Mll2 binding across thegenome and regulates H3K4 methylation on bivalent promoters

The presence of histone modifications associated with both transcriptional repression (H3K27me3) and activation (H3K4me3) on key developmental promoters in embryonic stem cells results from the co-localization of repressive Polycomb group (PcG) and activating Trithorax group (TrxG) protein complexes. Functional interactions between PcG and TrxG on these promoters are not fully understood. Here we focus on the relationships between Fbxl10/Kdm2b, a component of a PcG complex PRC1, and Kmt2b/Mll2, an essential component of TrxG at bivalent promoters. Computational analysis of previously published data revealed genome-wide correlation between chromatin occupancies of these two proteins, suggesting potential crosstalk between Kdm2b and Mll2 at both active and repressed promoters. We tested this hypothesis experimentally and found that loss of Kdm2b resulted in depletion of Mll2 at promoters genome-wide, suggesting that Kdm2b is required for Mll2 occupancy at both bivalent and active promoters. Loss of Kdm2b or the core PRC1 component Ring1b also resulted in the reduction of H3K4me3 specifically at bivalent promoters. These findings provide a direct pathway for cooperation between PcG and TrxG at bivalent promoters, suggesting an unexpected modification to the current model of bivalency. In addition, these findings reveal genome-wide role of Kdm2b independent of the full PRC1 complex.

molecular biology↗

bioRxiv2024

Leukemia aggressiveness is driven by chromatin remodeling and expression changes of core regulators

Mechanisms driving the increase of cell growth in developing leukemia are not fully understood. We focused on epigenomic regulation of this process by analyzing the changes of chromatin marks and gene expression in leukemic cell clones as they progressed towards increased proliferation in a mouse model of acute myeloid leukemia (AML). This progression was characterized by gradual modulation of chromatin states and gene expression across the genome, with a surprising preferential trend of reversing the prior changes associated with the origins of leukemia. Our analyses of this modulation in independently developing clones predicted a small set of potential growth regulators whose transcriptomic and epigenomic progression was consistent between clones and maintained both in vivo and ex vivo. We selected three of these genes as candidates (Irx5 and Plag1 as growth suppressors and Smad1 as a driver) and successfully validated their causal growth effects by overexpression in leukemic cells. Public patient data confirmed expression levels of IRX5 and SMAD1 as markers of AML status and survival, suggesting that multiomic analysis of evolving clones in a mouse model is a valuable predictive approach relevant to human AML.

molecular biology↗

bioRxiv2023

Modularity of PRC1 Composition and Chromatin Interaction define Condensate Properties

Polycomb repressive complexes (PRC) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells and the ability of PRC1 to form condensates has been proposed to contribute to maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. By reconstituting and imaging PRC1 with various subunit compositions, we find that the exact combination of PHC and CBX subunits determine the initiation, morphology, stability, and dynamics of condensates. In particular, the polymerization activity of PHC2 strongly influences condensate dynamics to promote formation of structures with distinct domains that adhere to each other but do not coalesce. Using live cell imaging, we confirmed that CBX properties are critical for condensate initiation and that PHC polymerization is important to maintain stable condensates. Together, we propose that PRC1 can fine-tune the degree and type of condensation by altering its composition which might offer important flexibility of regulatory function during different stages of development.

molecular biology↗

bioRxiv2022

Nuclear autophagy interactome unveils WSTF as a constitutive nuclear inhibitor of inflammation

Macroautophagy (hereafter referred to as autophagy) degrades a variety of cellular components. A poorly understood area is autophagic degradation of nuclear substrates, or "nuclear autophagy". It remains unclear what can be degraded by autophagy from the mammalian nuclei. We began our study by investigating the nuclear binding partners of ATG8 family proteins that play important roles in recognizing autophagy substrates. We systematically evaluated the ATG8 nuclear interactome in primary human cells and in mouse brain, identifying hundreds of novel interactions. We continued our study by evaluating the nuclear proteomes of cellular senescence, a stable form of cell cycle arrest program associated with inflammation, in which nuclear autophagy is involved. Combined with the ATG8 nuclear interactome data, we identified WSTF, a component of the ISWI chromatin remodeling complex, as a novel substrate of nuclear autophagy. The degradation of WSTF, mediated by a direct interaction with the GABARAP isoform of ATG8, promotes chromatin accessibility of inflammatory genes and induces senescence-associated inflammation. Furthermore, WSTF directly binds the p65 subunit of NF-{kappa}B and inhibits its acetylation, thus blocking inflammatory gene expression in the setting of senescence, cancer, and pathogen infection. In addition, we show that loss of WSTF is required for the immuno-surveillance of oncogenic Ras in mouse liver; forced expression of WSTF inhibited tumor-suppressive inflammation and led to the development of liver tumors. Taken together, our study provides a global view of mammalian nuclear autophagy and reveals a novel nuclear inhibitor of inflammation implicated in diverse pathological contexts. Targeting WSTF may be broadly valuable as therapeutic intervention of inflammatory diseases.

cell biology↗

bioRxiv2022

Stimulation-responsive enhancers regulate inflammatory gene activation through retention and modification of H2A.Z-variant accessible nucleosomes

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies1,2. The mechanism of gene regulation in response to dmPGE2 is poorly understood. Here, we show that dmPGE2 regulates inflammatory gene induction by modulating the chromatin architecture and activity of enhancer elements in human HSPCs. We identified the specific genomic reorganization at stimuli-responsive enhancers that permits rapid transcriptional activation. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes that are permissive to binding of the transcription factor CREB. CREB binding to enhancer nucleosomes is concomitant with deposition of the histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone variant nucleosomes retained within inducible enhancers facilitate transcription factor (TF) binding. Acetylation of histone variant nucleosomes by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism by which inflammatory mediators such as dmPGE2 lead to acute transcriptional changes and alter HSPC behavior.

developmental biology↗

bioRxiv2022

Cell type-specific role of CBX2-cPRC1 at the onset of spermatogonial differentiation

Polycomb group (PcG) proteins maintain the repressed state of lineage-inappropriate genes and are therefore essential for embryonic development and adult tissue homeostasis. One critical function of PcG complexes is modulating chromatin structure. Canonical Polycomb repressive complex 1 (cPRC1), particularly its component CBX2, can compact chromatin and phase separate in vitro. These activities are hypothesized to be critical for forming a repressed physical environment in cells. While much has been learned by studying these PcG activities in cell culture models, it is largely unexplored how cPRC1 regulates adult stem cells and their subsequent differentiation during tissue homeostasis in living animals. Here, we show that CBX2 is upregulated as spermatogonial stem cells differentiate and is required in the differentiating spermatogonia of the male germ line. CBX2 forms condensates, similar to previously described Polycomb bodies, that co-localize with repressed target genes in differentiating spermatogonia. Single cell analyses of mosaic Cbx2 mutant testes show CBX2 is specifically required to produce differentiating, A1 spermatogonia. Furthermore, the domain of CBX2 responsible for compaction and phase separation is needed for the long-term maintenance of male germ cells in the animal. These results emphasize that the regulation of chromatin structure by CBX2 at a specific stage of spermatogenesis is critical for the continued production of sperm and, ultimately, the transmission of genetic material to the next generation.

developmental biology↗

bioRxiv2020

H3K27me3 is dispensable for early differentiation but required to maintain differentiated cell identity

Polycomb repressive complex 2 (PRC2) catalyzes trimethylation of histone H3 on lysine 27 and is required for normal development of complex eukaryotes. The requirement for H3K27me3 in various aspects of mammalian differentiation is not clear. Though associated with repressed genes, the modification is not sufficient to induce gene repression, and in some instances is not required. To examine the role of the modification in mammalian differentiation, we blocked trimethylation of H3K27 with both a small molecule inhibitor, GSK343, and by introducing a point mutation into EZH2, the catalytic subunit of PRC2. We found that cells with substantively decreased H3K27 tri-methylation were able to differentiate, which contrasts with EZH2 null cells. Different PRC2 targets had varied requirements for H3K27me3 in repressive regulation with a subset that maintained normal levels of repression in the absence of methylation. The primary cellular phenotype when H3K27 tri-methylation was blocked was an inability of the altered cells to maintain a differentiated state when challenged. This phenotype was determined by H3K27me3 deposition both in embryonic stem cells and in the first four days of differentiation. H3K27 tri-methylation therefore was not necessary for formation of differentiated cell states but was required to maintain a stable differentiated state.

molecular biology↗

bioRxiv2019

Changing the Waddington landscape to control mesendoderm competence

As pluripotent human embryonic stem cells progress towards one germ layer fate, they lose the ability to adopt alternative fates. It is unknown how the cells competence for these alternative fates changes along their developmental trajectory or if this competence can be modulated. Here, we show that a differentiating stem cells probability of adopting a mesendodermal fate when given the appropriate signal falls sharply at a specific point along the ectodermal trajectory, and we further demonstrate that this point can be moved using genetic perturbations. Using a low-dimensional reaction coordinate to monitor progression towards ectoderm, we can determine the probability that individual cells at different points along this path can transition to the mesendodermal fate upon BMP4 and Activin A signal exposure. Knowing this probability allows us to prospectively isolate and profile differentiating cells based on their mesendoderm competence. Analysis and validation of these RNA-seq and ATAC-seq profiles identified transcription factors that can independently control the cells mesendoderm competence and its progression along the ectodermal developmental trajectory. In the classical picture of a Waddington landscape, these effects correspond to altering the barrier between fates and changing the cells location on the landscape, respectively. The ability of the underlying gene regulatory network to modulate these two aspects of the developmental landscape could allow separate control of the dynamics of differentiation and tissue size proportions.

developmental biology↗

bioRxiv2019

Dynamics of activating and repressive histone modifications in Drosophila neural stem cell lineages and brain tumors

During central nervous system (CNS) development, spatiotemporal gene expression programs mediate specific lineage decisions to generate neuronal and glial cell types from neural stem cells (NSCs). However, little is known about the epigenetic landscape underlying these highly complex developmental events. Here, we perform ChIP-seq on distinct subtypes of Drosophila FACS-purified neural stem cells (NSCs) and their differentiated progeny to dissect the epigenetic changes accompanying the major lineage decisions in vivo. By analyzing active and repressive histone modifications, we show that stem cell identity genes are silenced during differentiation by loss of their activating marks and not via repressive histone modifications. Our analysis also uncovers a new set of genes specifically required for altering lineage patterns in type II neuroblasts, one of the two main Drosophila NSC identities. Finally, we demonstrate that this subtype specification in NBs, unlike NSC differentiation, requires Polycomb-group (PcG)-mediated repression.\n\nSummary statementDynamic epigenetic landscape of Drosophila neural stem cell lineages.

developmental biology↗

bioRxiv2019

lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation

Gene programs that control differentiation are regulated through the interplay between DNA, RNA, and protein. Cooperation among these fundamental cellular components can occur through highly structured interactions connecting domains formed by specific sequences of nucleotides, ribonucleotides, and/or amino acids and also through the assembly of biomolecular condensates. Here, we show that endoderm differentiation is regulated through the interaction of the long noncoding (lnc) RNA DIGIT and the bromodomain and extra-terminal (BET) domain family protein BRD3. BRD3 forms phase-separated condensates that contain DIGIT, occupies enhancers of endoderm transcription factors, and is required for endoderm differentiation. Purified BRD3 binds to acetylated histone H3 lysine 18 (H3K18ac) in vitro and occupies regions of the genome enriched in H3K18ac during endoderm differentiation, including the key transcription factors that regulate endoderm differentiation. DIGIT is also enriched in regions of H3K18ac, and depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings support a model where cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest a broader role for protein-lncRNA phase-separated condensates as regulators of transcription in development.

molecular biology↗

bioRxiv2018

Phase separation and nucleosome compaction are governed by the same domain of Polycomb Repressive Complex 1

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb Repressive Complex 1 (PRC1) family complexes are central to maintaining this repression1. These include a set of canonical PRC1 complexes that each contain four core proteins, including one from the CBX family. These complexes have previously been shown to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus2,3. We show here that reconstituted PRC1 readily phase separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of CBX2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells, and have previously been shown to eliminate nucleosome compaction in vitro4 and to generate axial patterning defects in mice5. Thus, a single domain in CBX2 is required for phase separation and nucleosome compaction, a finding that relates these functions to each other and to proper development.

molecular biology↗