Project description:One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight in the function of this unique histone variant in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells. Examination of the histone variant macroH2A1 in mouse Embryonic stem cells
Project description:One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight in the function of this unique histone variant in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.
Project description:Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states,termed commitment, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.
Project description:Hematopoietic stem cells (HSCs) must balance self-renewal and lineage differentiation to regenerate the hematopoietic system throughout life. HSCs exhibit lineage-associated gene expression that keeps them responsive to demands of mature blood production. However, it is not known whether this process, termed lineage priming, directly influences HSC self-renewal. We investigated the link between stemness and lineage priming by attenuating the early lymphoid transcription factor E47 through ID2 over-expression (OE). Transcriptional profiling of ID2 OE HSCs showed down regulation of B-cell factors including EBF1 and FOXO1 with a concomitant increase in stemness programs and myeloerythroid factors including CEBPA and GATA1. This resulted in myeloid commitment bias from the earliest stages of differentiation. HSC self-renewal was strongly affected by this lineage perturbation resulting in an 11-fold expansion of HSCs. Thus, early lymphoid transcription factors antagonize human HSC self-renewal, providing a direct link between differentiation program priming and the maintenance of stem cell self-renewal. Three independent lineage depleted CB samples were transduced with P-CTRL or P-ID2 and injected into 5 mice (30 mice total). From every group of 5 mice, human lin- cells were isolated and GFP+CD34+CD38-CD45RA- HSPCs were sorted by FACS.
Project description:Hematopoietic stem cells (HSCs) must balance self-renewal and lineage differentiation to regenerate the hematopoietic system throughout life. HSCs exhibit lineage-associated gene expression that keeps them responsive to demands of mature blood production. However, it is not known whether this process, termed lineage priming, directly influences HSC self-renewal. We investigated the link between stemness and lineage priming by attenuating the early lymphoid transcription factor E47 through ID2 over-expression (OE). Transcriptional profiling of ID2 OE HSCs showed down regulation of B-cell factors including EBF1 and FOXO1 with a concomitant increase in stemness programs and myeloerythroid factors including CEBPA and GATA1. This resulted in myeloid commitment bias from the earliest stages of differentiation. HSC self-renewal was strongly affected by this lineage perturbation resulting in an 11-fold expansion of HSCs. Thus, early lymphoid transcription factors antagonize human HSC self-renewal, providing a direct link between differentiation program priming and the maintenance of stem cell self-renewal.
Project description:Osteoporosis, a prevalent age-related bone disorder, stems from imbalanced osteoclast/osteoblast activity and skewed mesenchymal stem cell (MSC) lineage commitment. Although lactate is linked to osteoporosis pathogenesis, the role of protein lactylation in MSC osteogenic differentiation remains unelucidated. This experiment explored protein lactylation during early MSC osteogenic differentiation, focusing on histone variant MacroH2A1. Proteomics identified 375–318 lactylated proteins across undifferentiated, osteogenic differentiation Day 3 (OB3), and adipogenic differentiation Day 3 (AD3) MSCs; MacroH2A1 lactylation at lysine 134 (K134) was downregulated in OB3. MacroH2A1(K134Ala) mutation (disrupting lactylation) enhanced MSC osteogenesis. In ovariectomized mice, AAV-MacroH2A1(K134Ala) increased femoral trabecular bone mass and reduced marrow adipose tissue. This experiment demonstrates that MacroH2A1 K134 lactylation regulates MSC osteogenic commitment via epigenetic and transcriptional modulation, offering mechanistic insights and a potential therapeutic target for osteoporosis.
Project description:Osteoporosis, a prevalent age-related bone disorder, stems from imbalanced osteoclast/osteoblast activity and skewed mesenchymal stem cell (MSC) lineage commitment. Although lactate is linked to osteoporosis pathogenesis, the role of protein lactylation in MSC osteogenic differentiation remains unelucidated. This experiment explored protein lactylation during early MSC osteogenic differentiation, focusing on histone variant MacroH2A1. Proteomics identified 375–318 lactylated proteins across undifferentiated, osteogenic differentiation Day 3 (OB3), and adipogenic differentiation Day 3 (AD3) MSCs; MacroH2A1 lactylation at lysine 134 (K134) was downregulated in OB3. MacroH2A1(K134Ala) mutation (disrupting lactylation) enhanced MSC osteogenesis. In ovariectomized mice, AAV-MacroH2A1(K134Ala) increased femoral trabecular bone mass and reduced marrow adipose tissue. This experiment demonstrates that MacroH2A1 K134 lactylation regulates MSC osteogenic commitment via epigenetic and transcriptional modulation, offering mechanistic insights and a potential therapeutic target for osteoporosis.
Project description:The Nucleosome Remodeling and Deacetylase (NuRD) complex plays an important role in gene expression regulation, stem cell self-renewal, and lineage commitment. Yet little is known about the dynamics of NuRD during cellular differentiation. Here, we study these dynamics using genome-wide profiling and quantitative interaction proteomics in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). The genomic targets of NuRD are highly dynamic during differentiation, with most binding occurring at cell-type specific promoters and enhancers. We identify ZFP296 as a novel, ESC-specific NuRD interactor that also interacts with the SIN3A complex. ChIP-sequencing in Zfp296 knockout (KO) ESCs reveals decreased NuRD binding both genome-wide and at ZFP296 binding sites, although this has little effect on the transcriptome. Nevertheless, Zfp296 KO ESCs exhibit delayed induction of lineage-specific markers upon differentiation to embryoid bodies. In summary, we identify an ESC-specific NuRD interacting protein which regulates genome-wide NuRD binding and cellular differentiation.