Project description:Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation, but their role in pluripotency remains elusive. Here we establish that PML is required for basal SUMO2/3 conjugation in mESCs and oxidative stress-driven sumoylation in mESCs or in vivo. PML NBs create an oxidationprotective environment for UBC9-driven SUMO2/3 conjugation of PML partners, often followed by their poly-ubiquitination and degradation. Differential in vivo proteomics identified several members of the KAP1 complex as PML NB-dependent SUMO2-targets. The latter drives functional activation of this key epigenetic repressor. Accordingly, Pml-/- mESCs reexpress transposable elements and display features of totipotent-like cells, a process further enforced by PML-controlled SUMO2-conjugation of DPPA2. Finally, PML is required for adaptive stress responses in mESCs. Collectively, PML orchestrates mESC fate through SUMO2-conjugation of key transcriptional or epigenetic regulators, raising new mechanistic hypotheses about PML roles in normal or cancer stem cells.
Project description:Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation, but their role in pluripotency remains elusive. Here we establish that PML is required for basal SUMO2/3 conjugation in mESCs and oxidative stress-driven sumoylation in mESCs or in vivo. PML NBs create an oxidationprotective environment for UBC9-driven SUMO2/3 conjugation of PML partners, often followed by their poly-ubiquitination and degradation. Differential in vivo proteomics identified several members of the KAP1 complex as PML NB-dependent SUMO2-targets. The latter drives functional activation of this key epigenetic repressor. Accordingly, Pml-/- mESCs reexpress transposable elements and display features of totipotent-like cells, a process further enforced by PML-controlled SUMO2-conjugation of DPPA2. Finally, PML is required for adaptive stress responses in mESCs. Collectively, PML orchestrates mESC fate through SUMO2-conjugation of key transcriptional or epigenetic regulators, raising new mechanistic hypotheses about PML roles in normal or cancer stem cells.
Project description:TRIM33 is a chromatin reader required for mesendoderm differentiation upon activation of Nodal signaling. But, its role in mESCs is still elusive. Here, we found that TRIM33 co-localizes with promyelocytic leukemia nuclear bodies (PML NBs) specifically in mESCs to mediate Nodal signaling-directed transcription of Lefty1/2. We showed that TRIM33 puncta formation in mESCs depends on PML and specific assembly of PML NBs. Moreover, TRIM33 and PML co-regulate Lefty1/2 expression in mESCs. In addition, both PML and mESCs-specific PML NBs are required for TRIM33 recruitment at Lefty1/2 loci. Remarkably, PML NBs directly associate with the Lefty1/2 loci in mESCs. Finally, a TurboID proximity labeling experiment confirmed that TRIM33 is highly enriched in the mESCs-specific PML NBs. Thus, our study provides the mechanistic insight about TRIM33 condensate in regulating Nodal signaling-directed transcription in mESCs, it also reveals that PML NBs recruit distinct sets of client proteins in cell context dependent manner.
Project description:PML nuclear body (PML NB) recruits different client proteins under different cell context. We used TurboID proximity labeling (PL) method followed by MS to determine the composition of PML NBs in mESCs, differentiated cells, and NaAsO2-treated mESCs.
Project description:Because PML-RARA-positive acute promyelocytic leukemia (APL) is a morphologically differentiated leukemia, much speculation has been made about whether its leukemic cell of origin might be committed myeloid precursor (e.g., a promyelocyte) vs. a hematopoietic stem/progenitor cell (HSPC). We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice) are detected in the purified KLS cells of these mice (Kit+Lin-Sca+ cells, which are highly enriched for HSPCs), and this expression results in biological effects in multi-lineage competitive repopulation assays. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice, it does not significantly alter the transcriptional signature of these cells, or induce their self-renewal. In sum, these results suggest that in murine models, PML-RARA acts primarily to affect the function of multi-potent progenitor cells, rather than promyelocytes. Since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in our mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients. Bone marrow from individual mice expressing PML-RARA from the murine Ctg locus (mCG-PR) and littermate controls was harvested from both femurs and tibia. Standard cell lysis was performed, and total RNA was extracted from the cells and analyzed using the Affymetrix Mouse Exon 1.0 ST platform.
Project description:Promyelocytic leukemia (PML) body is a phase-separated nuclear structure composed of various proteins including several chromatin regulators, and physically associates with chromatin, implying its crucial roles for particular genome functions. To investigate functional roles of PML bodies in chromatin organization, we conducted ATAC-seq with wild-type and PML KO mESCs.
Project description:TRIM33 is a chromatin reader required for nodal signaling during mesendoderm differentiation, although differences in its function between mouse embryonic stem cells (mESCs) and differentiated mesendoderm cells are unknown. Here, we found that TRIM33 co-condenses with PML nuclear bodies (NBs) via liquid-liquid phase separation specifically in mESCs to mediate nodal signaling-directed transcription of Lefty1/2. Our findings show that TRIM33 puncta formation depends on the presence of PML NBs. TurboID proximity labeling further revealed that PML NBs recruit distinct sets of client proteins in NaAsO2-treated, untreated mESCs, and differentiated cells. TRIM33 and PML co-regulate Lefty1/2 expression, while PML NBs directly associate with the Lefty1/2 loci and regulate a gene cluster that includes Lefty1/2 loci specifically in mESCs. Moreover, TRIM33 association with chromatin depends on PML NBs. Thus, PML NBs serve as a hub for transcriptional regulation of Lefty1/2 by TRIM33 and other pluripotency factors in mESCs.
Project description:TRIM33 is a chromatin reader required for nodal signaling during mesendoderm differentiation, although differences in its function between mouse embryonic stem cells (mESCs) and differentiated mesendoderm cells are unknown. Here, we found that TRIM33 co-condenses with PML nuclear bodies (NBs) via liquid-liquid phase separation specifically in mESCs to mediate nodal signaling-directed transcription of Lefty1/2. Our findings show that TRIM33 puncta formation depends on the presence of PML NBs. TurboID proximity labeling further revealed that PML NBs recruit distinct sets of client proteins in NaAsO2-treated, untreated mESCs, and differentiated cells. TRIM33 and PML co-regulate Lefty1/2 expression, while PML NBs directly associate with the Lefty1/2 loci and regulate a gene cluster that includes Lefty1/2 loci specifically in mESCs. Moreover, TRIM33 association with chromatin depends on PML NBs. Thus, PML NBs serve as a hub for transcriptional regulation of Lefty1/2 by TRIM33 and other pluripotency factors in mESCs.
Project description:Promyelocytic leukemia (PML) body is a phase-separated nuclear structure composed of various proteins including several chromatin regulators, and physically associates with chromatin, implying its crucial roles for particular genome functions. To investigate roles of PML bodies in transcriptional regulation, we conducted ChIP-seq analysis for histone modifications, including H3K4me3, H3K27ac, H3K27me3, and H3K9ac with wild-type and PML knockout mESCs.