ABSTRACT
The transcriptional network acting downstream of LIF, WNT and MAPK-ERK to stabilize mouse embryonic stem cells (ESCs) in their naive state has been extensively characterized. However, the upstream factors regulating these three signaling pathways remain largely uncharted. PR-domain-containing proteins (PRDMs) are zinc-finger sequence-specific chromatin factors that have essential roles in embryonic development and cell fate decisions. Here we characterize the transcriptional regulator PRDM15, which acts independently of PRDM14 to regulate the naive state of mouse ESCs. Mechanistically, PRDM15 modulates WNT and MAPK-ERK signaling by directly promoting the expression of Rspo1 (R-spondin1) and Spry1 (Sprouty1). Consistent with these findings, CRISPR-Cas9-mediated disruption of PRDM15-binding sites in the Rspo1 and Spry1 promoters recapitulates PRDM15 depletion, both in terms of local chromatin organization and the transcriptional modulation of these genes. Collectively, our findings uncover an essential role for PRDM15 as a chromatin factor that modulates the transcription of upstream regulators of WNT and MAPK-ERK signaling to safeguard naive pluripotency.
Subject(s)
DNA-Binding Proteins/genetics , Embryonic Stem Cells/metabolism , Gene Expression Regulation , MAP Kinase Signaling System/genetics , Transcription Factors/genetics , Wnt Signaling Pathway/genetics , Animals , Blotting, Western , Cell Line , Cell Self Renewal/genetics , Cells, Cultured , Cellular Reprogramming/genetics , DNA-Binding Proteins/metabolism , Fluorescent Antibody Technique , Gene Expression Profiling/methods , Humans , Induced Pluripotent Stem Cells/metabolism , Mice, Knockout , Mice, Nude , Mice, Transgenic , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolismABSTRACT
Hutchinson-Gilford progeria (HGPS) is a premature ageing syndrome caused by a mutation in LMNA, resulting in a truncated form of lamin A called progerin. Progerin triggers loss of the heterochromatic marker H3K27me3, and premature senescence, which is prevented by telomerase. However, the mechanism how progerin causes disease remains unclear. Here, we describe an inducible cellular system to model HGPS and find that LAP2α (lamina-associated polypeptide-α) interacts with lamin A, while its interaction with progerin is significantly reduced. Super-resolution microscopy revealed that over 50% of telomeres localize to the lamina and that LAP2α association with telomeres is impaired in HGPS. This impaired interaction is central to HGPS since increasing LAP2α levels rescues progerin-induced proliferation defects and loss of H3K27me3, whereas lowering LAP2 levels exacerbates progerin-induced defects. These findings provide novel insights into the pathophysiology underlying HGPS, and how the nuclear lamina regulates proliferation and chromatin organization.
Subject(s)
DNA-Binding Proteins/metabolism , Lamin Type A/metabolism , Membrane Proteins/metabolism , Progeria/pathology , Telomere/metabolism , Humans , Microscopy , Protein BindingABSTRACT
Embryonic stem (ES) cells were first derived from inner cell mass (ICM) explants of preimplantation stage mouse blastocysts some 30 years ago. ES cells are of primary interest as they are used to genetically modify the genome of mice via gene targeting. Although many founder ES lines have been established, there is still a need to obtain new ES lines or their derivatives, often from new mutant mouse lines, to study the function of a mutated gene in different cell types. Existing methods for isolating ES cell lines are inefficient. Here, we describe a reproducible, efficient, and economical method to derive ES cells from different mouse strains using a defined serum-free, serum replacement (KO-SR) media, with 50-85% efficiency. We have derived over 100 ES lines, which when karyotyped>70% were euploid. Two of these lines, when tested, produced germ-line chimeras. We also present procedures for the routine maintenance and karyotyping of the ES cells.
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Cell Separation/methods , Embryonic Stem Cells , Animals , Blastocyst/cytology , Cell Culture Techniques/instrumentation , Cell Line , Cryopreservation/methods , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Female , Fibroblasts/cytology , Fibroblasts/physiology , Germ Cells/cytology , Karyotyping , Male , Mice , Mice, Inbred C57BL , PregnancyABSTRACT
Since the discovery of the prototypical Sprouty (Spry) protein in Drosophila, there has been an effort to determine how these novel modulators of the Ras/MAP-kinase pathway function. A clue to their mechanism of action comes from the several highly conserved sequences within all the currently known Spry isoforms: an approximately 110-residue cysteine-rich sequence in the C-terminal half that directs Spry proteins to a concentration of signaling proteins at the plasma membrane; a small motif surrounding a tyrosine residue (Y55 in human Spry2) that is responsible for interaction with other proteins. In cultured mammalian cells, hSpry2 inhibits epidermal growth factor receptor (EGFR) endocytosis and subsequently sustains the activation of MAP kinase but negatively regulates the same pathway following stimulation of fibroblast growth factor receptors (FGFRs). Current evidence indicates that Cbl is a key protein that interacts directly with Spry2 following activation of receptor tyrosine kinases (RTKs). It appears to be the ability of Cbl to interact as an E3 ubiquitin ligase on specific target proteins and as a docking protein in other contexts that dictates the differential effects Spry2 has on the Ras/MAP-kinase pathway following EGFR and FGFR activation.
