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First published online 26 January 2005
doi: 10.1242/dev.01670

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LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism

University of Georgia, Rhodes Center, 425 River Road, Athens, GA 30602-2771, USA

View larger version (48K): [in a new window]   Fig. 1. Myc is a STAT3 target gene in ES cells. (A) LIF-maintained ES cells or embryoid bodies (-LIF) generated from ES cells were harvested and mRNA analysed by RT-PCR analysis using primers designed against Rex1, Oct4, Fgf5, brachyury, Myc and ß-actin. (B) Chromatin immunoprecipitation (ChIP) analysis with a FLAG monoclonal antibody was performed on crosslinked chromatin from a cell line expressing FLAG-tagged STAT3 (STAT3FLAG). Immunoprecipitated chromatin was then used to determine if STAT3FLAG bound the endogenous Myc promoter using a specific primer pair that flanks an E2F/E-box in the Myc P2 promoter (left panels). The same chromatin was used as a template in a PCR reaction using primer for the CDK1 promoter that is not implicated as a STAT3 target gene (right panel). Addition of FLAG antibody in ChIPs is as indicated (+). To control for the amount of total chromatin in the ChIP assay, equivalent amounts of total template (`input'), equivalent to 0.03% of total chromatin DNA used in ChIP assays, were used in a parallel PCR reaction using Myc or CDK1 specific primers. Immunoblot analysis: relative expression levels of total STAT3 in the transfected cell line is shown relative to endogenous STAT3 levels (vector alone transfectant). (C) STAT3 trans-activates the Myc gene in ES cells. LIF maintained STAT3-ER cells were deprived of LIF for 36 hours, stimulated with 4OHT (10 nM) or LIF (1x103 units/ml) and assayed 24 hours later by RT-PCR analysis.

View larger version (37K): [in a new window]   Fig. 2. Maintenance of Myc levels in ES cells requires suppression of T58 phosphorylation. (A) Top panel: whole cell lysates (40 µg total protein) from LIF maintained ES cells and embryoid bodies (-LIF; see A) were immunoblotted and probed with antibodies raised against Myc, phospho-T58 Myc (MycpT58) and GSK3ß; middle panel, extracts as in top panel were probed with anti-Oct4, and GSK3ß antibodies. GSK3ß kinase activity at corresponding times is shown. Bottom panel: quantitation of GSK3ß kinase activity in LIF-maintained ES cells and during differentiation was performed by phosphorimaging analysis and is depicted as a fold increase over initial levels in ES cells. Specificity of the assay was determined by immunoprecipitating GSK3ß from day 6 EB cell lysates and performing kinase reactions in the presence of MBP, GSK3 II inhibitor, ethanol as indicated by +. The presence of the GSK3ß antibody in the immunoprecipitation is as indicated (+). (B) ES cells maintained in LIF were treated with cycloheximide (+CHX, 10 µM) and at 30 minute intervals cells were harvested and whole cell lysates prepared. Following immunoblotting, extracts were probed with antibodies as indicated. Levels of Myc (white circles), Cdk2 (black squares) and cyclin D3 (black circles) are shown for each time point relative to pre-cycloheximide chase levels (t=0). (C) Whole-cell lysates were prepared from mycER LIF-maintained ES cells and day 1, day 2, day 3 and day 4 EBs (-LIF) that had been treated with proteosome inibitor MG132 (+, 5 µM) or ethanol alone (-) 3 hours prior to harvesting. After SDS PAGE and electroblotting onto a membrane, cell lysates (40 µg total protein) were then probed with antibodies raised against Myc and HDAC1/2. (D) Mutation of T58 (T58A) delays the downregulation of Myc protein following LIF withdrawal. mycER or mycT58AER levels were evaluated in LIF-maintained lines or in EBs generated by growth in the absence of LIF over 5 days (1-5) by immunoblot analysis using anti-Myc and anti-HDAC1 antibodies (loading control). (E) ES cells maintained in Wnt3a CM for five passages (15 days) were harvested or grown as EBs in the absence of CM for 1 or 7 days. Cell lysates were probed with antibodies as indicated and GSK3ß kinase assays performed as described in A but on extracts made from day 7 EBs. The specificity of kinase assays was confirmed as described in A.

View larger version (53K): [in a new window]   Fig. 3. Maintenance of stable Myc at physiological levels sustains self-renewal in the absence of LIF/Wnt3a CM. Myc expression was driven by the CAG promoter as a fusion with the steroid binding domain of the estrogen receptor (ER) for inducible activity and coupled to a puromycin resistance gene (puroR) by an internal ribosome entry site (IRES). (A) Flow cytometry profiles of SSEA1 reactivity on the surface of mycER or mycT58AER cells grown up to 12 days in the presence of LIF (+LIF) without 4OHT (-4OHT), or without LIF (-LIF) in the presence or absence of 4OHT. (B) Colony morphology of mycT58AER transfected ES cell colonies grown in the presence or absence of 4OHT 6 days after LIF withdrawal. (C) MycER (circles, squares) and mycT58AER ES cells (triangles) were grown in varying concentrations of LIF (0-1x103 units/ml), in the presence (black symbols) or absence (white symbols) of 4OHT. Colonies were assayed for AP activity every 3 days after LIF withdrawal in duplicate. MycER cells were also grown in the presence of 7.5 units/ml LIF with (closed square) or without (open square) 4OHT. (D) Immunoblot of cell lysate from the mycT58AER transfected D3 ES cell line showing levels of endogenous Myc, mycT58AER and Cdk2 after being probed simultaneously with anti-Myc and Cdk2 antibody, and then exposed to film for the same length of time. (E) RT-PCR analysis of Oct4, Rex1 and ß-actin transcripts in vector alone or mycT58AER cells grown for 14 days in the presence or absence of LIF and 4OHT.

View larger version (35K): [in a new window]   Fig. 4. MycT58AER-dependent self-renewal is independent of cell cycle and apoptotic effects and is reversible by withdrawal of 4OHT. (A) ES cells were plated at 5x105/ml in duplicate wells and scored at 12-hour intervals for 72 hours. (B) Flow cytometry analysis of PI stained cells. Top panel, mycT58AER cells + LIF (1x103 units/ml); middle panel, vector alone + LIF (1x103 units/ml); bottom panel, mycT58AER + 4OHT (5 nM). The percentage of cells in different cell cycle phases was determined using MultiCycle AV software (Phoenix Flow Systems). (C) Cell viability was determined by counting 1000 cells for Trypan Blue exclusion. (D) MycT58AER ES cells were maintained in the presence of 4OHT for 14 days (-LIF) then differentiated as embryoid bodies in either the absence or presence of 4OHT (-LIF). Cells were harvested for each of 7 days (1-7) and analysed for differentiation status as indicated. Upper panel, northern blot analysis: RNA samples probed for Oct4, brachyury, Fgf5, ornithine decarboxylase (Odc) and GAPDH. Lower panel, immunoblot (blot) analysis (blot): 40 µg total protein was resolved by SDS PAGE, transferred to a membrane then probed with anti-Myc and anti-Oct4 antibodies. (E) Colony-forming assays (performed in duplicate) on R1 and D3 ES cells passaged for 30 days in ESC media plus LIF (1x103 units/ml) or ESC media with 10 nM 4OHT. Cells were then plated at clonal density and after 6 days colony forming units (AP-positive colonies) were scored after growth under the conditions indicated (either maintained in ESC+LIF, or switched to ESC±LIF in the absence of 4OHT). Assays are shown as the mean of duplicate experiments.

View larger version (45K): [in a new window]   Fig. 5. Myc performs a role in self-renewal by blocking differentiation. (A) Myc40-178ER D3 cells were grown in the presence of LIF and in the absence or presence of 4OHT (25 nM) for up to 6 days. Colonies were then stained for AP activity and photographed at 20x magnification. (B) At corresponding sampling times (see A) the percentage of alkaline phosphatase positive colonies was determined. Data are the average of assays performed in triplicate and expressed as standard error of the mean. (C) The proportion of stem cells decreases following inactivation of Myc. Myc40-178ER D3 ES cells were grown for 6 days under the conditions indicated. Five-hundred cells were then plated at clonal density in ESC+LIF (1x103 units/ml) and colony forming units (AP-positive colony number) scored after a further 4 days growth (-4OHT). Data are shown of an experiment performed in duplicate. (D) mRNA levels for Oct4, Nanog, brachyury, GATA4 and ß-actin were evaluated by RT-PCR analysis in D3 myc40-178ER cells before and after addition of 4OHT.

View larger version (64K): [in a new window]   Fig. 6. Myc-maintained ES cells retain pluripotency. (A) MycT58AER R1 EGFP ES cells were maintained in the presence of 4OHT for 30 days, in the absence of LIF/Wnt3a CM and then grown as EB suspensions for up to 8 days by withdrawing 4OHT in the absence (top panel) or presence (bottom panel) of LIF. Cell lysates were subject to immunoblot analysis, probing with anti Myc, Oct4 and ß-tubulin antibodies. (B) MycT58AER ES cells grown in the presence of 4OHT for 30 days (as in A) were stained for AP activity or grown for an additional 7 days in the absence of 4OHT. (C) GFP marked R1 mycT58AER ES cells grown for 30 days in the presence of 4OHT (-LIF; as in A) then used for injection into blastocyst stage C57BL/6 embryos that were transferred into females and allowed to develop to 12.5 dpc, at which time they were analysed. Chimeric (bottom) and non-chimeric (top) embryos where EGFP R1 mycT58AER maintained ES cells had widely integrated into the three germ layers. (D) Dark field view of the same embryos. (E) Tissues were dissected from EGFP-positive embryos and analyzed at higher magnification. (F) The ability of mycT58AER ES cells to generate liveborn chimeras was confirmed by analysis of coat color. Host C57BL/6 embryos give rise to black coat color, whereas sv129 (R1 ES line) contribute to a sandy-colored coat. Chimeras shown were generated by contribution of LIF maintained (left) and 4OHT maintained (right) mycT58AER cells.

View larger version (19K): [in a new window]   Fig. 7. The relationship between LIF-STAT3, GSK3ß and Myc in maintenance of ES cell self-renewal. Following LIF withdrawal, Myc transcriptional activity collapses, GSK3ß is activated and Myc becomes phosphorylated at T58, triggering its ubiquitin (u)-mediated, proteosome-dependent degradation. `?' indicates the possible role of pathways involved in (1) suppression of GSK3ß activity or (2) pathways of transcriptional control that collaborate with LIF to control Myc transcription. The latter could involve signals generated by serum-derived growth factors. The involvement of LIF/STAT3-Myc independent pathways are also indicated.