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Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells.

Latos PA, Goncalves A, Oxley D, Mohammed H, Turro E, Hemberger M - Nat Commun (2015)

Bottom Line: In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes.Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex.Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

View Article: PubMed Central - PubMed

Affiliation: 1] Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK [2] Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.

ABSTRACT
Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

No MeSH data available.


Related in: MedlinePlus

Effects of Fgf/Erk signalling inhibition on TS cell transcription factors.(a) Western blot analysis showing the absence of phosphorylated Erk1/2 in cells treated with Mek inhibitor PD0325901 (‘PD03') for 3, 12 and 24 h compared with untreated controls; levels of total Erk1/2 remained unchanged. Esrrb was reduced after 3 h of PD03 treatment and nearly absent after 12 h of PD03 treatment (Supplementary Fig. 10a). (b) RT–QPCR showing expression of TS cell markers in TS cells treated with PD03 for 3, 24 and 48 h compared with untreated controls. Esrrb was the most rapidly downregulated gene. Bars represent the mean of three biological replicates±s.e.m. (c) Immunostaining of Fgf-responsive transcription factors Cdx2, Elf5 and Eomes in TS cells treated with PD03 for 24 h and untreated controls. Magnification bars, 100 μm. (d) RNA-seq analysis after 3 and 24 h of PD03 treatment compared with untreated controls identified a total of 399 deregulated genes at high-confidence (posterior probability score >0.95) that could be grouped into early (significantly changed, using these parameters, after 3 h) and late (after 24 h) responders. Several example genes are indicated. (e) Temporal expression dynamics of a number of selected TS cell genes as identified using RNA-seq analysis. Note that Esrrb stands out as the most rapidly downregulated TS cell transcription factor also in this genome-wide approach.
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f1: Effects of Fgf/Erk signalling inhibition on TS cell transcription factors.(a) Western blot analysis showing the absence of phosphorylated Erk1/2 in cells treated with Mek inhibitor PD0325901 (‘PD03') for 3, 12 and 24 h compared with untreated controls; levels of total Erk1/2 remained unchanged. Esrrb was reduced after 3 h of PD03 treatment and nearly absent after 12 h of PD03 treatment (Supplementary Fig. 10a). (b) RT–QPCR showing expression of TS cell markers in TS cells treated with PD03 for 3, 24 and 48 h compared with untreated controls. Esrrb was the most rapidly downregulated gene. Bars represent the mean of three biological replicates±s.e.m. (c) Immunostaining of Fgf-responsive transcription factors Cdx2, Elf5 and Eomes in TS cells treated with PD03 for 24 h and untreated controls. Magnification bars, 100 μm. (d) RNA-seq analysis after 3 and 24 h of PD03 treatment compared with untreated controls identified a total of 399 deregulated genes at high-confidence (posterior probability score >0.95) that could be grouped into early (significantly changed, using these parameters, after 3 h) and late (after 24 h) responders. Several example genes are indicated. (e) Temporal expression dynamics of a number of selected TS cell genes as identified using RNA-seq analysis. Note that Esrrb stands out as the most rapidly downregulated TS cell transcription factor also in this genome-wide approach.

Mentions: Derivation and maintenance of TS cells depend on the presence of Fgf signalling224. Numerous gene knockout experiments identified the mitogen-activated kinase Mek/Erk branch of the Fgf signalling pathway as predominantly active in both TS cells and extraembryonic ectoderm1825262728. Therefore, we first tested changes in expression of key TS cell TFs on Mek/Erk inhibition using the Mek inhibitor PD0325901 (‘PD03'; Fig. 1a). Among the candidate TFs we examined after 3–48 h of treatment, Esrrb was the fastest and most profoundly downregulated gene, followed closely by Sox2, in line with a recent report18 (Fig. 1b). Some TFs implicated in TS cell maintenance including Eomes, Elf5 and Cdx2 were also downregulated on Mek inhibition albeit at a slower pace, whereas the expression of others such as Ets2 or Tfap2c remained unchanged. These data were confirmed by immunostaining for some of the most prominent TS cell TFs, namely Cdx2, Elf5, Eomes and Tfap2c (Fig. 1c; Supplementary Fig. 1a). To further refine this analysis and to obtain an unbiased genome-wide coverage of the immediate-early-response genes of Mek inhibition in TS cells, we performed RNA sequencing (RNA-seq) analysis after 3 and 24 h of PD03 treatment. This global expression analysis identified in total 399 genes that were deregulated after 3 and 24 h by Fgf signalling (Fig. 1d; Supplementary Data 1). The majority of these genes were induced by Erk activation as 240 of them were downregulated on Mek inhibition, while only 159 genes were upregulated using stringent confidence parameters (Fig. 1d,e; Supplementary Data 1). Functional gene annotation analysis using MouseMine confirmed that affected genes were specifically enriched for extraembryonic (trophoblast) tissue development, as well as for embryonic lethality and transcriptional control in particular for the downregulated genes (Supplementary Fig. 1b,c). Of particular note were the dynamics of downregulation on Mek inhibition; thus, we identified 38 early responders that were downregulated, but only 10 that were upregulated (Fig. 1d). Notably, of the known TS cell TFs, this analysis confirmed Esrrb as the earliest, most rapidly silenced gene on PD03 treatment (Fig. 1e). These results provided a comprehensive overview of Fgf-regulated genes in TS cells and identified many potential candidates with a role in trophoblast development.


Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells.

Latos PA, Goncalves A, Oxley D, Mohammed H, Turro E, Hemberger M - Nat Commun (2015)

Effects of Fgf/Erk signalling inhibition on TS cell transcription factors.(a) Western blot analysis showing the absence of phosphorylated Erk1/2 in cells treated with Mek inhibitor PD0325901 (‘PD03') for 3, 12 and 24 h compared with untreated controls; levels of total Erk1/2 remained unchanged. Esrrb was reduced after 3 h of PD03 treatment and nearly absent after 12 h of PD03 treatment (Supplementary Fig. 10a). (b) RT–QPCR showing expression of TS cell markers in TS cells treated with PD03 for 3, 24 and 48 h compared with untreated controls. Esrrb was the most rapidly downregulated gene. Bars represent the mean of three biological replicates±s.e.m. (c) Immunostaining of Fgf-responsive transcription factors Cdx2, Elf5 and Eomes in TS cells treated with PD03 for 24 h and untreated controls. Magnification bars, 100 μm. (d) RNA-seq analysis after 3 and 24 h of PD03 treatment compared with untreated controls identified a total of 399 deregulated genes at high-confidence (posterior probability score >0.95) that could be grouped into early (significantly changed, using these parameters, after 3 h) and late (after 24 h) responders. Several example genes are indicated. (e) Temporal expression dynamics of a number of selected TS cell genes as identified using RNA-seq analysis. Note that Esrrb stands out as the most rapidly downregulated TS cell transcription factor also in this genome-wide approach.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525203&req=5

f1: Effects of Fgf/Erk signalling inhibition on TS cell transcription factors.(a) Western blot analysis showing the absence of phosphorylated Erk1/2 in cells treated with Mek inhibitor PD0325901 (‘PD03') for 3, 12 and 24 h compared with untreated controls; levels of total Erk1/2 remained unchanged. Esrrb was reduced after 3 h of PD03 treatment and nearly absent after 12 h of PD03 treatment (Supplementary Fig. 10a). (b) RT–QPCR showing expression of TS cell markers in TS cells treated with PD03 for 3, 24 and 48 h compared with untreated controls. Esrrb was the most rapidly downregulated gene. Bars represent the mean of three biological replicates±s.e.m. (c) Immunostaining of Fgf-responsive transcription factors Cdx2, Elf5 and Eomes in TS cells treated with PD03 for 24 h and untreated controls. Magnification bars, 100 μm. (d) RNA-seq analysis after 3 and 24 h of PD03 treatment compared with untreated controls identified a total of 399 deregulated genes at high-confidence (posterior probability score >0.95) that could be grouped into early (significantly changed, using these parameters, after 3 h) and late (after 24 h) responders. Several example genes are indicated. (e) Temporal expression dynamics of a number of selected TS cell genes as identified using RNA-seq analysis. Note that Esrrb stands out as the most rapidly downregulated TS cell transcription factor also in this genome-wide approach.
Mentions: Derivation and maintenance of TS cells depend on the presence of Fgf signalling224. Numerous gene knockout experiments identified the mitogen-activated kinase Mek/Erk branch of the Fgf signalling pathway as predominantly active in both TS cells and extraembryonic ectoderm1825262728. Therefore, we first tested changes in expression of key TS cell TFs on Mek/Erk inhibition using the Mek inhibitor PD0325901 (‘PD03'; Fig. 1a). Among the candidate TFs we examined after 3–48 h of treatment, Esrrb was the fastest and most profoundly downregulated gene, followed closely by Sox2, in line with a recent report18 (Fig. 1b). Some TFs implicated in TS cell maintenance including Eomes, Elf5 and Cdx2 were also downregulated on Mek inhibition albeit at a slower pace, whereas the expression of others such as Ets2 or Tfap2c remained unchanged. These data were confirmed by immunostaining for some of the most prominent TS cell TFs, namely Cdx2, Elf5, Eomes and Tfap2c (Fig. 1c; Supplementary Fig. 1a). To further refine this analysis and to obtain an unbiased genome-wide coverage of the immediate-early-response genes of Mek inhibition in TS cells, we performed RNA sequencing (RNA-seq) analysis after 3 and 24 h of PD03 treatment. This global expression analysis identified in total 399 genes that were deregulated after 3 and 24 h by Fgf signalling (Fig. 1d; Supplementary Data 1). The majority of these genes were induced by Erk activation as 240 of them were downregulated on Mek inhibition, while only 159 genes were upregulated using stringent confidence parameters (Fig. 1d,e; Supplementary Data 1). Functional gene annotation analysis using MouseMine confirmed that affected genes were specifically enriched for extraembryonic (trophoblast) tissue development, as well as for embryonic lethality and transcriptional control in particular for the downregulated genes (Supplementary Fig. 1b,c). Of particular note were the dynamics of downregulation on Mek inhibition; thus, we identified 38 early responders that were downregulated, but only 10 that were upregulated (Fig. 1d). Notably, of the known TS cell TFs, this analysis confirmed Esrrb as the earliest, most rapidly silenced gene on PD03 treatment (Fig. 1e). These results provided a comprehensive overview of Fgf-regulated genes in TS cells and identified many potential candidates with a role in trophoblast development.

Bottom Line: In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes.Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex.Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

View Article: PubMed Central - PubMed

Affiliation: 1] Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK [2] Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.

ABSTRACT
Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

No MeSH data available.


Related in: MedlinePlus