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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

Esrrb depletion results in TS cell differentiation.(a) Temporal expression dynamics of a number of selected TS cell genes as identified using RNA-seq analysis after 24 h and 4 days of treatment with the oestrogen-related receptor antagonist DES compared with untreated controls. (b) RT–QPCR showing expression of TS cell genes in TS cells treated for 24 h with DES compared with untreated controls. (c) Immunostaining showing downregulation of TS cell markers Eomes and Elf5 in TS cells treated for 24 h with Esrrb antagonist DES. Magnification bars, 100 μm. (d) Plot of differentially expressed genes identified using RNA-seq analysis after 3 and 24 h of PD03 exposure and 4 h and 4-day DES treatment. (e) Venn diagram showing overlap of genes deregulated on PD03 and DES treatments. (f) Western blot analysis showing depletion of Esrrb, Cdx2 and Eomes in Esrrb KD TS cell lines (KD-1, KD-2 and KD-3) compared with controls (scr-1 and scr-2; Supplementary Fig. 10b). (g) RT–QPCR analysis of Esrrb KD (KD-1, KD-2 and KD-3) and control (scr-1 and scr-2) TS cells. TS cell markers (Cdx2, Elf5, Eomes and Nr0b1) were downregulated in Esrrb-depleted cells, whereas differentiation markers (Syna, Cdkn1c, Prl2c2 and Prl3d1) were upregulated. Bars indicate the mean of three biological replicates±s.e.m. (h) Phase contrast microscope images of TS cells 5 days after transfection with Esrrb KD (KD-2) or scrambled control (scr-1) constructs. Esrrb KD lines were severely differentiated despite the presence of Fgf, whereas control lines formed tight, epithelial colonies. These images are representative for KD-1 and KD-2 Esrrb KD lines; KD-3 showed less severe phenotype in line with the reduced KD levels (Fig. 2f,g). Experiments were performed in biological triplicates. Magnification bar, 50 μm.
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f2: Esrrb depletion results in TS cell differentiation.(a) Temporal expression dynamics of a number of selected TS cell genes as identified using RNA-seq analysis after 24 h and 4 days of treatment with the oestrogen-related receptor antagonist DES compared with untreated controls. (b) RT–QPCR showing expression of TS cell genes in TS cells treated for 24 h with DES compared with untreated controls. (c) Immunostaining showing downregulation of TS cell markers Eomes and Elf5 in TS cells treated for 24 h with Esrrb antagonist DES. Magnification bars, 100 μm. (d) Plot of differentially expressed genes identified using RNA-seq analysis after 3 and 24 h of PD03 exposure and 4 h and 4-day DES treatment. (e) Venn diagram showing overlap of genes deregulated on PD03 and DES treatments. (f) Western blot analysis showing depletion of Esrrb, Cdx2 and Eomes in Esrrb KD TS cell lines (KD-1, KD-2 and KD-3) compared with controls (scr-1 and scr-2; Supplementary Fig. 10b). (g) RT–QPCR analysis of Esrrb KD (KD-1, KD-2 and KD-3) and control (scr-1 and scr-2) TS cells. TS cell markers (Cdx2, Elf5, Eomes and Nr0b1) were downregulated in Esrrb-depleted cells, whereas differentiation markers (Syna, Cdkn1c, Prl2c2 and Prl3d1) were upregulated. Bars indicate the mean of three biological replicates±s.e.m. (h) Phase contrast microscope images of TS cells 5 days after transfection with Esrrb KD (KD-2) or scrambled control (scr-1) constructs. Esrrb KD lines were severely differentiated despite the presence of Fgf, whereas control lines formed tight, epithelial colonies. These images are representative for KD-1 and KD-2 Esrrb KD lines; KD-3 showed less severe phenotype in line with the reduced KD levels (Fig. 2f,g). Experiments were performed in biological triplicates. Magnification bar, 50 μm.

Mentions: To gain first insights into which genes may be primary targets of Esrrb, we treated TS cells with the synthetic nonsteroidal oestrogen diethylstilbestrol (DES), an oestrogen-related receptor (Err) antagonist, for 24 h and 4 days. This compound interacts with all three Err isoforms Esrra, Esrrb and Esrrg but mainly acts through Esrrb in early development. It blocks co-activator binding and thus prevents transcriptional activity, and in vitro leads to TS cell differentiation19. Indeed, we observed morphological changes on DES treatment, indicative of TS cell differentiation. To obtain unbiased genome-wide coverage of transcriptional changes on short (24 h) and prolonged (4d) DES treatment, we performed RNA-seq and identified 654 differentially expressed genes. Numerous differentiation markers were upregulated including the family of placental lactogen genes characteristic for giant cells (Supplementary Data 2). Importantly, we found that transcripts of TS cell TFs Nr0b1, Zic3, Sox2, Eomes, Elf5 and Id2 were downregulated after 24 h of DES treatment, suggesting that they may be direct targets of Esrrb (Fig. 2a). We confirmed these findings by reverse transcriptase–quantitative polymerase chain reaction (RT–QPCR) and at the protein level by immunostaining for Eomes and Elf5 (Fig. 2b,c). Interestingly, when specifically examining the trajectories between control and 24 h DES treatment, other prominent TS cell regulators such as Cdx2 were less influenced during this immediate-response window (Fig. 2a). To further examine Esrrb as a primary mediator of TF induction by Fgf signalling in TS cells, we analysed the overlap of affected genes between the DES and PD03 RNA-seq data sets (Fig. 2d,e). Strikingly, we found that both DES and PD03 treatments had an impact on the same set of prominent stem cell genes Nr0b1, Zic3, Sox2, Id2, Cdx2, Eomes and Elf5 (Fig. 2d,e). Taken together, these data indicated that Fgf-Mek signalling regulates, via Esrrb, essential TFs such as Sox2, Cdx2, Eomes and Elf5 that sustain TS cell self-renewal.


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)

Esrrb depletion results in TS cell differentiation.(a) Temporal expression dynamics of a number of selected TS cell genes as identified using RNA-seq analysis after 24 h and 4 days of treatment with the oestrogen-related receptor antagonist DES compared with untreated controls. (b) RT–QPCR showing expression of TS cell genes in TS cells treated for 24 h with DES compared with untreated controls. (c) Immunostaining showing downregulation of TS cell markers Eomes and Elf5 in TS cells treated for 24 h with Esrrb antagonist DES. Magnification bars, 100 μm. (d) Plot of differentially expressed genes identified using RNA-seq analysis after 3 and 24 h of PD03 exposure and 4 h and 4-day DES treatment. (e) Venn diagram showing overlap of genes deregulated on PD03 and DES treatments. (f) Western blot analysis showing depletion of Esrrb, Cdx2 and Eomes in Esrrb KD TS cell lines (KD-1, KD-2 and KD-3) compared with controls (scr-1 and scr-2; Supplementary Fig. 10b). (g) RT–QPCR analysis of Esrrb KD (KD-1, KD-2 and KD-3) and control (scr-1 and scr-2) TS cells. TS cell markers (Cdx2, Elf5, Eomes and Nr0b1) were downregulated in Esrrb-depleted cells, whereas differentiation markers (Syna, Cdkn1c, Prl2c2 and Prl3d1) were upregulated. Bars indicate the mean of three biological replicates±s.e.m. (h) Phase contrast microscope images of TS cells 5 days after transfection with Esrrb KD (KD-2) or scrambled control (scr-1) constructs. Esrrb KD lines were severely differentiated despite the presence of Fgf, whereas control lines formed tight, epithelial colonies. These images are representative for KD-1 and KD-2 Esrrb KD lines; KD-3 showed less severe phenotype in line with the reduced KD levels (Fig. 2f,g). Experiments were performed in biological triplicates. Magnification bar, 50 μm.
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f2: Esrrb depletion results in TS cell differentiation.(a) Temporal expression dynamics of a number of selected TS cell genes as identified using RNA-seq analysis after 24 h and 4 days of treatment with the oestrogen-related receptor antagonist DES compared with untreated controls. (b) RT–QPCR showing expression of TS cell genes in TS cells treated for 24 h with DES compared with untreated controls. (c) Immunostaining showing downregulation of TS cell markers Eomes and Elf5 in TS cells treated for 24 h with Esrrb antagonist DES. Magnification bars, 100 μm. (d) Plot of differentially expressed genes identified using RNA-seq analysis after 3 and 24 h of PD03 exposure and 4 h and 4-day DES treatment. (e) Venn diagram showing overlap of genes deregulated on PD03 and DES treatments. (f) Western blot analysis showing depletion of Esrrb, Cdx2 and Eomes in Esrrb KD TS cell lines (KD-1, KD-2 and KD-3) compared with controls (scr-1 and scr-2; Supplementary Fig. 10b). (g) RT–QPCR analysis of Esrrb KD (KD-1, KD-2 and KD-3) and control (scr-1 and scr-2) TS cells. TS cell markers (Cdx2, Elf5, Eomes and Nr0b1) were downregulated in Esrrb-depleted cells, whereas differentiation markers (Syna, Cdkn1c, Prl2c2 and Prl3d1) were upregulated. Bars indicate the mean of three biological replicates±s.e.m. (h) Phase contrast microscope images of TS cells 5 days after transfection with Esrrb KD (KD-2) or scrambled control (scr-1) constructs. Esrrb KD lines were severely differentiated despite the presence of Fgf, whereas control lines formed tight, epithelial colonies. These images are representative for KD-1 and KD-2 Esrrb KD lines; KD-3 showed less severe phenotype in line with the reduced KD levels (Fig. 2f,g). Experiments were performed in biological triplicates. Magnification bar, 50 μm.
Mentions: To gain first insights into which genes may be primary targets of Esrrb, we treated TS cells with the synthetic nonsteroidal oestrogen diethylstilbestrol (DES), an oestrogen-related receptor (Err) antagonist, for 24 h and 4 days. This compound interacts with all three Err isoforms Esrra, Esrrb and Esrrg but mainly acts through Esrrb in early development. It blocks co-activator binding and thus prevents transcriptional activity, and in vitro leads to TS cell differentiation19. Indeed, we observed morphological changes on DES treatment, indicative of TS cell differentiation. To obtain unbiased genome-wide coverage of transcriptional changes on short (24 h) and prolonged (4d) DES treatment, we performed RNA-seq and identified 654 differentially expressed genes. Numerous differentiation markers were upregulated including the family of placental lactogen genes characteristic for giant cells (Supplementary Data 2). Importantly, we found that transcripts of TS cell TFs Nr0b1, Zic3, Sox2, Eomes, Elf5 and Id2 were downregulated after 24 h of DES treatment, suggesting that they may be direct targets of Esrrb (Fig. 2a). We confirmed these findings by reverse transcriptase–quantitative polymerase chain reaction (RT–QPCR) and at the protein level by immunostaining for Eomes and Elf5 (Fig. 2b,c). Interestingly, when specifically examining the trajectories between control and 24 h DES treatment, other prominent TS cell regulators such as Cdx2 were less influenced during this immediate-response window (Fig. 2a). To further examine Esrrb as a primary mediator of TF induction by Fgf signalling in TS cells, we analysed the overlap of affected genes between the DES and PD03 RNA-seq data sets (Fig. 2d,e). Strikingly, we found that both DES and PD03 treatments had an impact on the same set of prominent stem cell genes Nr0b1, Zic3, Sox2, Id2, Cdx2, Eomes and Elf5 (Fig. 2d,e). Taken together, these data indicated that Fgf-Mek signalling regulates, via Esrrb, essential TFs such as Sox2, Cdx2, Eomes and Elf5 that sustain TS cell self-renewal.

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