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1.
Mol Cell ; 83(9): 1393-1411.e7, 2023 05 04.
Article in English | MEDLINE | ID: mdl-37030288

ABSTRACT

Polycomb repressive complex 2 (PRC2) mediates H3K27me3 deposition, which is thought to recruit canonical PRC1 (cPRC1) via chromodomain-containing CBX proteins to promote stable repression of developmental genes. PRC2 forms two major subcomplexes, PRC2.1 and PRC2.2, but their specific roles remain unclear. Through genetic knockout (KO) and replacement of PRC2 subcomplex-specific subunits in naïve and primed pluripotent cells, we uncover distinct roles for PRC2.1 and PRC2.2 in mediating the recruitment of different forms of cPRC1. PRC2.1 catalyzes the majority of H3K27me3 at Polycomb target genes and is sufficient to promote recruitment of CBX2/4-cPRC1 but not CBX7-cPRC1. Conversely, while PRC2.2 is poor at catalyzing H3K27me3, we find that its accessory protein JARID2 is essential for recruitment of CBX7-cPRC1 and the consequent 3D chromatin interactions at Polycomb target genes. We therefore define distinct contributions of PRC2.1- and PRC2.2-specific accessory proteins to Polycomb-mediated repression and uncover a new mechanism for cPRC1 recruitment.


Subject(s)
Histones , Polycomb Repressive Complex 2 , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolism , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Histones/genetics , Histones/metabolism , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Chromatin/genetics
2.
Nat Biotechnol ; 41(12): 1801-1809, 2023 Dec.
Article in English | MEDLINE | ID: mdl-36973556

ABSTRACT

Transcription factor binding across the genome is regulated by DNA sequence and chromatin features. However, it is not yet possible to quantify the impact of chromatin context on transcription factor binding affinities. Here, we report a method called binding affinities to native chromatin by sequencing (BANC-seq) to determine absolute apparent binding affinities of transcription factors to native DNA across the genome. In BANC-seq, a concentration range of a tagged transcription factor is added to isolated nuclei. Concentration-dependent binding is then measured per sample to quantify apparent binding affinities across the genome. BANC-seq adds a quantitative dimension to transcription factor biology, which enables stratification of genomic targets based on transcription factor concentration and prediction of transcription factor binding sites under non-physiological conditions, such as disease-associated overexpression of (onco)genes. Notably, whereas consensus DNA binding motifs for transcription factors are important to establish high-affinity binding sites, these motifs are not always strictly required to generate nanomolar-affinity interactions in the genome.


Subject(s)
Chromatin , Transcription Factors , Chromatin/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Protein Binding , DNA/genetics , DNA/metabolism , Gene Expression Regulation , Binding Sites/genetics , Sequence Analysis, DNA
3.
Nucleic Acids Res ; 51(3): 1277-1296, 2023 02 22.
Article in English | MEDLINE | ID: mdl-36625255

ABSTRACT

Microfold (M) cells reside in the intestinal epithelium of Peyer's patches (PP). Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. By using single-cell RNA sequencing, we identified an enterocyte and M cell precursor population. We used our newly developed computational tool SCEPIA to link precursor cell-specific gene expression to transcription factor motif activity in cis-regulatory elements, uncovering high expression of and motif activity for the transcription factor ONECUT2. Subsequent in vitro and in vivo perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling axis to support enterocyte differentiation, thereby restricting M cell lineage specification. This study sheds new light on the mechanism regulating cell fate balance in the PP, and it provides a powerful blueprint for investigation of cell fate switches in the intestinal epithelium.


Subject(s)
Enterocytes , M Cells , Animals , Mice , Cell Differentiation , Intestinal Mucosa , Intestine, Small , Multiomics , Transcription Factors/metabolism
4.
Cell Rep Med ; 4(2): 100915, 2023 02 21.
Article in English | MEDLINE | ID: mdl-36657447

ABSTRACT

More than half of patients with malignant mesothelioma show alterations in the BAP1 tumor-suppressor gene. Being a member of the Polycomb repressive deubiquitinating (PR-DUB) complex, BAP1 loss results in an altered epigenome, which may create new vulnerabilities that remain largely unknown. Here, we performed a CRISPR-Cas9 kinome screen in mesothelioma cells that identified two kinases in the mevalonate/cholesterol biosynthesis pathway. Furthermore, our analysis of chromatin, expression, and genetic perturbation data in mesothelioma cells suggests a dependency on PR complex 2 (PRC2)-mediated silencing. Pharmacological inhibition of PRC2 elevates the expression of cholesterol biosynthesis genes only in BAP1-deficient mesothelioma, thereby sensitizing these cells to the combined targeting of PRC2 and the mevalonate pathway. Finally, by subjecting autochthonous Bap1-deficient mesothelioma mice or xenografts to mevalonate pathway inhibition (zoledronic acid) and PRC2 inhibition (tazemetostat), we demonstrate a potent anti-tumor effect, suggesting a targeted combination therapy for Bap1-deficient mesothelioma.


Subject(s)
Lung Neoplasms , Mesothelioma, Malignant , Mesothelioma , Humans , Animals , Mice , Mevalonic Acid , Lung Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Mesothelioma/genetics , Mesothelioma/pathology , Cholesterol , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism
5.
J Proteome Res ; 22(1): 138-151, 2023 01 06.
Article in English | MEDLINE | ID: mdl-36450103

ABSTRACT

The development of metastasis severely reduces the life expectancy of patients with colorectal cancer (CRC). Although loss of SMAD4 is a key event in CRC progression, the resulting changes in biological processes in advanced disease and metastasis are not fully understood. Here, we applied a multiomics approach to a CRC organoid model that faithfully reflects the metastasis-supporting effects of SMAD4 inactivation. We show that loss of SMAD4 results in decreased differentiation and activation of pro-migratory and cell proliferation processes, which is accompanied by the disruption of several key oncogenic pathways, including the TGFß, WNT, and VEGF pathways. In addition, SMAD4 inactivation leads to increased secretion of proteins that are known to be involved in a variety of pro-metastatic processes. Finally, we show that one of the factors that is specifically secreted by SMAD4-mutant organoids─DKK3─reduces the antitumor effects of natural killer cells (NK cells). Altogether, our data provide new insights into the role of SMAD4 perturbation in advanced CRC.


Subject(s)
Colorectal Neoplasms , Multiomics , Humans , Cell Line, Tumor , Colorectal Neoplasms/pathology , Transforming Growth Factor beta/metabolism , Cell Proliferation/genetics , Smad4 Protein/genetics
6.
iScience ; 24(12): 103444, 2021 Dec 17.
Article in English | MEDLINE | ID: mdl-34877501

ABSTRACT

Retinoic acid (RA) signaling is an important and conserved pathway that regulates cellular proliferation and differentiation. Furthermore, perturbed RA signaling is implicated in cancer initiation and progression. However, the mechanisms by which RA signaling contributes to homeostasis, malignant transformation, and disease progression in the intestine remain incompletely understood. Here, we report, in agreement with previous findings, that activation of the Retinoic Acid Receptor and the Retinoid X Receptor results in enhanced transcription of enterocyte-specific genes in mouse small intestinal organoids. Conversely, inhibition of this pathway results in reduced expression of genes associated with the absorptive lineage. Strikingly, this latter effect is conserved in a human organoid model for colorectal cancer (CRC) progression. We further show that RXR motif accessibility depends on progression state of CRC organoids. Finally, we show that reduced RXR target gene expression correlates with worse CRC prognosis, implying RA signaling as a putative therapeutic target in CRC.

7.
Nat Genet ; 53(8): 1221-1232, 2021 08.
Article in English | MEDLINE | ID: mdl-34294917

ABSTRACT

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.


Subject(s)
Enhancer Elements, Genetic , Histones/metabolism , Neural Stem Cells/physiology , Polycomb Repressive Complex 2/genetics , Rhombencephalon/cytology , Animals , Brain Neoplasms/genetics , Cell Differentiation/genetics , Cell Line , Enhancer of Zeste Homolog 2 Protein/genetics , Epigenome , Gene Expression Regulation, Developmental , Glioma/genetics , Histones/genetics , Humans , Lysine/metabolism , Male , Mice, Inbred Strains , Mutation , Neural Stem Cells/transplantation , Oncogenes , Polycomb Repressive Complex 2/antagonists & inhibitors , Polycomb Repressive Complex 2/metabolism , Promoter Regions, Genetic , Rhombencephalon/physiology
8.
Mol Cell ; 76(3): 437-452.e6, 2019 11 07.
Article in English | MEDLINE | ID: mdl-31521505

ABSTRACT

Polycomb repressive complex 2 (PRC2) is composed of EED, SUZ12, and EZH1/2 and mediates mono-, di-, and trimethylation of histone H3 at lysine 27. At least two independent subcomplexes exist, defined by their specific accessory proteins: PRC2.1 (PCL1-3, EPOP, and PALI1/2) and PRC2.2 (AEBP2 and JARID2). We show that PRC2.1 and PRC2.2 share the majority of target genes in mouse embryonic stem cells. The loss of PCL1-3 is sufficient to evict PRC2.1 from Polycomb target genes but only leads to a partial reduction of PRC2.2 and H3K27me3. Conversely, disruption of PRC2.2 function through the loss of either JARID2 or RING1A/B is insufficient to completely disrupt targeting of SUZ12 by PCLs. Instead, the combined loss of both PRC2.1 and PRC2.2 is required, leading to the global mislocalization of SUZ12. This supports a model in which the specific accessory proteins within PRC2.1 and PRC2.2 cooperate to direct H3K27me3 via both synergistic and independent mechanisms.


Subject(s)
Chromatin/metabolism , Histones/metabolism , Mouse Embryonic Stem Cells/metabolism , Polycomb Repressive Complex 2/metabolism , Protein Processing, Post-Translational , Animals , Binding Sites , Cell Line, Tumor , Chromatin/genetics , Humans , Methylation , Mice , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 2/genetics , Protein Binding , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
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