Modulation of PKM1/2 levels by steric blocking morpholinos alters the metabolic and pluripotent state of murine pluripotent stem cells

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described formative pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 mRNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the formative state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression towards a primed pluripotent state.

Single-Cell Multiomics Reveals Distinct Cell States at the Top of Human Hematopoietic Hierarchy

The advent of single cell (Sc) genomics has challenged the dogma of haematopoiesis as a tree-like structure of stepwise lineage commitment through distinct and increasingly restricted progenitor populations. Instead, analysis of ScRNA-seq has proposed that the earliest events in human hematopoietic stem cell (HSC) differentiation are characterized by only subtle molecular changes, with hematopoietic stem and progenitor cells (HSPCs) existing as a continuum of low-primed cell-states that gradually transition into a specific lineage (CLOUD-HSPCs). Here, we combine ScRNA-seq, ScATAC-seq and cell surface proteomics to dissect the heterogeneity of CLOUD-HSPCs at different stages of human life. Within CLOUD-HSPCs, pseudotime ordering of both mRNA and chromatin data revealed a bifurcation of megakaryocyte/erythroid and lympho/myeloid trajectories immediately downstream a subpopulation with an HSC-specific enhancer signature. Importantly, both HSCs and lineage-restricted progenitor populations could be prospectively isolated based on correlation of their molecular signatures with CD35 and CD11A expression, respectively. Moreover, we describe the changes that occur in this heterogeneity as hematopoiesis develops from neonatal to aged bone marrow, including an increase of HSCs and depletion of lympho-myeloid biased MPPs. Thus, this study dissects the heterogeneity of human CLOUD-HSPCs revealing distinct HSPC-states of relevance in homeostatic settings such as ageing.

Immune regulators SIB1 and LSD1 antagonistically regulate PhANGs via the GLK transcription factors

GOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs), indispensable for chloroplast biogenesis. We previously demonstrated that the salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN1 (SIB1), a transcription coregulator and positive regulator of SA-primed cell death, interacts with GLKs. The SIB1-GLK interaction raises the level of light-harvesting antenna proteins in the photosystem II, aggravating photoinhibition and singlet oxygen (1O2) burst. 1O2 then contributes to SA-primed cell death via EXECUTER1 (EX1, 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. We now reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLKs to repress their activities. Consistently, the overexpression of LSD1 represses the expression of PhANGs, but the loss of LSD1 increases their expression. The SA-induced SIB1 then counteractively interacts with GLKs, leading to EX1-mediated cell death. Collectively, we provide a working model that mutually exclusive SA-signaling components SIB1 and LSD1 antagonistically regulate GLKs to fine-tune the expression of PhANGs, priming SA-induced cell death, and sustaining 1O2 homeostasis, respectively.

PLANT NATRIURETIC PEPTIDE A antagonizes salicylic acid-primed cell death

ABSTRACTPeptide hormones perceived in the cell surface via receptor proteins enable cell-to-cell communication and act in multiple biological processes through the activation of intracellular signaling. Even though Arabidopsis is predicted to have more than 1,000 secreted peptides, the biological relevance of the majority of these is yet to be established. Here, we demonstrate that PLANT NATRIURETIC PEPTIDE A (PNP-A), a functional analog to vertebrate atrial natriuretic peptides, antagonizes the salicylic acid (SA)-mediated cell death in the Arabidopsis lesion-stimulating disease 1 (lsd1) mutant. While loss of PNP-A potentiates SA signaling, exogenous application of the PNP-A synthetic peptide or overexpression of PNP-A significantly compromises the SA-mediated cell death. Moreover, we identified a plasma membrane-localized receptor-like protein, which we name PNPAR (for PNP-A receptor), that binds PNP-A and is required to counteract SA responses. Our work identifies a novel peptide-receptor pair which modulates SA responses in Arabidopsis.