Psat1-generated α-ketoglutarate and glutamine promote muscle stem cell activation and regeneration.

By satisfying bioenergetic demands, generating biomass, and providing metabolites serving as cofactors for chromatin modifiers, metabolism regulates adult stem cell biology. Here, we report that a branch of glycolysis, the serine biosynthesis pathway (SBP), is activated in regenerating muscle stem cells (MuSCs). Gene inactivation and metabolomics revealed that Psat1, one of the three SBP enzymes, controls MuSC activation and expansion of myogenic progenitors through production of the metabolite α-ketoglutarate (α-KG) and α-KG-generated glutamine. Psat1 ablation resulted in defective expansion of MuSCs and impaired regeneration. Psat1, α-KG, and glutamine were reduced in MuSCs of old mice. α-KG or glutamine re-established appropriate muscle regeneration of adult conditional Psat1 -/- mice and of old mice. These findings contribute insights into the metabolic role of Psat1 during muscle regeneration and suggest α-KG and glutamine as potential therapeutic interventions to ameliorate muscle regeneration during aging.

Protocols to generate and isolate mouse myogenic progenitors both in vitro and in vivo.

Mouse embryonic stem cells (mESCs) can be directed to acquire cell-lineage-specific genetic programs and phenotypes by stepwise exposure to defined factors, allowing the development of in vitro models for studying disease and tissue generation. In this protocol, we describe the use of cultured mESCs to generate presomitic-like mesoderm cells undergoing further specification towards myogenic progenitors (MPs). Further, we describe here a procedure to obtain, dissect, and fluorescence-activated cell sorting (FACS)-isolate somitic cells from genetically labeled Pax7+/Cre; Rosa26YFP/+ embryos. For complete details on the use and execution of this protocol, please refer to Khateb et al.1.

Polycomb Ezh1 maintains murine muscle stem cell quiescence through non-canonical regulation of Notch signaling.

Organismal homeostasis and regeneration are predicated on committed stem cells that can reside for long periods in a mitotically dormant but reversible cell-cycle arrest state defined as quiescence. Premature escape from quiescence is detrimental, as it results in stem cell depletion, with consequent defective tissue homeostasis and regeneration. Here, we report that Polycomb Ezh1 confers quiescence to murine muscle stem cells (MuSCs) through a non-canonical function. In the absence of Ezh1, MuSCs spontaneously exit quiescence. Following repeated injuries, the MuSC pool is progressively depleted, resulting in failure to sustain proper muscle regeneration. Rather than regulating repressive histone H3K27 methylation, Ezh1 maintains gene expression of the Notch signaling pathway in MuSCs. Selective genetic reconstitution of the Notch signaling corrects stem cell number and re-establishes quiescence of Ezh1-/- MuSCs.

Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism.

BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation. METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs. RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity. CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells.

Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I.

BACKGROUND: Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the 'gatekeeper' mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. METHODS: The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients. RESULTS: Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. CONCLUSIONS: Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.

Transcriptomics, regulatory syntax, and enhancer identification in mesoderm-induced ESCs at single-cell resolution.

Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.

The Third Intron of IRF8 Is a Cell-Type-Specific Chromatin Priming Element during Mouse Embryonal Stem Cell Differentiation.

Interferon regulatory factor 8 (IRF8) is a nuclear transcription factor that plays a key role in the hierarchical differentiation of hematopoietic stem cells toward monocyte/dendritic cell lineages. Therefore, its expression is mainly limited to bone marrow-derived cells. The molecular mechanisms governing this cell-type-restricted expression have been described. However, the molecular mechanisms that are responsible for its silencing in non-hematopoietic cells are elusive. Recently, we demonstrated a role for IRF8 third intron in restricting its expression in non-hematopoietic cells. Furthermore, we showed that this intron alone is sufficient to promote repressed chromatin a cell-type-specific manner. Here we demonstrate the effect of the IRF8 third intron on chromatin conformation during murine embryonal stem cell differentiation. Using genome editing, we provide data showing that the third intron plays a key role in priming the chromatin state of the IRF8 locus during cell differentiation. It mediates dual regulatory effects in a cell-type-specific mode. It acts as a repressor element governing chromatin state of the IRF8 locus during embryonal stem cell differentiation to cardiomyocytes that are expression-restrictive cells. Conversely, it functions as an activator element that is essential for open chromatin structure during the differentiation of these cells to dendritic cells that are expression-permissive cells. Together, these results point to the role of IRF8 third intron as a cell-type-specific chromatin priming element during embryonal stem cell differentiation. These data add another layer to our understanding of the molecular mechanisms governing misexpression of a cell-type-specific gene such as IRF8.

The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells.

Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron's properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation.