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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.


Nr0b1 (=Dax1) and Integrator as Esrrb interactors in TS cells.(a) Esrrb-3xFlag immunoprecipitates analysed using western blot probed with anti-Nr0b1 antibody (Supplementary Fig. 10d). (b) Venn diagram showing the overlap between Esrrb and Nr0b1 ChIP-seq peaks in TS cells. (c) Esrrb- and Nr0b1-binding profiles at the Cdx2 and (d) Cdh1 loci. (e) Esrrb-3xFlag immunoprecipitates analysed using western blot probed with anti-Integrator1 (Ints1) and anti-Integrator 9 (Ints9) antibodies, confirming the prominent interaction between Esrrb and Integrator complex components (Supplementary Fig. 10e). (f) Endogenous anti-Ints9 immunoprecipitates analysed by western blot with anti-Esrrb antibody shows that Esrrb interacts with Ints9 (Supplementary Fig. 10f). (g) Context-dependent function of Esrrb in TS cells may be in part mediated by the interaction with the Integrator complex. (h) Comparison of Esrrb-centred self-renewal networks in TS cells and ES cells. Although Esrrb expression is driven by distinct signalling pathways in TS (Fgf/Mek) and ES (LIF/Gsk inhibition) cells, some interacting partners are shared (for example, transcription factors (TFs) Nr0b1, Tfcp2lf1 and the NuRD complex), whereas others are stem cell-specific (for example, Integrator or Mediator complexes). Similarly, Esrrb commonly binds a shared set of genes in both stem cell types, in addition to TS- and ES-cell-specific targets.
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f5: Nr0b1 (=Dax1) and Integrator as Esrrb interactors in TS cells.(a) Esrrb-3xFlag immunoprecipitates analysed using western blot probed with anti-Nr0b1 antibody (Supplementary Fig. 10d). (b) Venn diagram showing the overlap between Esrrb and Nr0b1 ChIP-seq peaks in TS cells. (c) Esrrb- and Nr0b1-binding profiles at the Cdx2 and (d) Cdh1 loci. (e) Esrrb-3xFlag immunoprecipitates analysed using western blot probed with anti-Integrator1 (Ints1) and anti-Integrator 9 (Ints9) antibodies, confirming the prominent interaction between Esrrb and Integrator complex components (Supplementary Fig. 10e). (f) Endogenous anti-Ints9 immunoprecipitates analysed by western blot with anti-Esrrb antibody shows that Esrrb interacts with Ints9 (Supplementary Fig. 10f). (g) Context-dependent function of Esrrb in TS cells may be in part mediated by the interaction with the Integrator complex. (h) Comparison of Esrrb-centred self-renewal networks in TS cells and ES cells. Although Esrrb expression is driven by distinct signalling pathways in TS (Fgf/Mek) and ES (LIF/Gsk inhibition) cells, some interacting partners are shared (for example, transcription factors (TFs) Nr0b1, Tfcp2lf1 and the NuRD complex), whereas others are stem cell-specific (for example, Integrator or Mediator complexes). Similarly, Esrrb commonly binds a shared set of genes in both stem cell types, in addition to TS- and ES-cell-specific targets.

Mentions: Besides interactors involved in epigenetic regulation of transcription, we identified also TFs and cofactor complexes that directly interact with RNAPII (Table 1; Fig. 4a). Similar to some shared epigenetic complexes, we found that the TFs Nr0b1, Esrra, Tf2l1, Zfp462 and others overlapped with the Esrrb interactome in ES cells, thereby further validating our immunoprecipitation (IP) LC-MS/MS analysis (Fig. 4a). Since Nr0b1 has been found to have an important role in ES cell self-renewal, we confirmed by co-immunoprecipitation that it also interacts with Esrrb in TS cells (Fig. 5a). ChIP-seq analysis for Nr0b1 in TS cells showed binding overlap with Esrrb on a subset of essential TS cell-specific (for example, Cdx2 and Tfap2) and general developmental loci (Lin28a and Cdh1; Fig. 5b–d; Supplementary Fig. 8a; Supplementary Data 8). As with Esrrb before, we observed that Nr0b1 binding in TS and ES cells showed a small overlap, with only 52 Esrrb/Nr0b1 co-bound regions shared between ES and TS cells (Supplementary Fig. 8b, Supplementary Data 8). These detailed novel data on the context-specific wiring of transcriptional networks are supported also by the limited overlap of Tfcp2l1, another TF that complexes with Esrrb in both TS and ES cells, with Esrrb TS cell peaks (Supplementary Fig. 8c; Supplementary Data 8).


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)

Nr0b1 (=Dax1) and Integrator as Esrrb interactors in TS cells.(a) Esrrb-3xFlag immunoprecipitates analysed using western blot probed with anti-Nr0b1 antibody (Supplementary Fig. 10d). (b) Venn diagram showing the overlap between Esrrb and Nr0b1 ChIP-seq peaks in TS cells. (c) Esrrb- and Nr0b1-binding profiles at the Cdx2 and (d) Cdh1 loci. (e) Esrrb-3xFlag immunoprecipitates analysed using western blot probed with anti-Integrator1 (Ints1) and anti-Integrator 9 (Ints9) antibodies, confirming the prominent interaction between Esrrb and Integrator complex components (Supplementary Fig. 10e). (f) Endogenous anti-Ints9 immunoprecipitates analysed by western blot with anti-Esrrb antibody shows that Esrrb interacts with Ints9 (Supplementary Fig. 10f). (g) Context-dependent function of Esrrb in TS cells may be in part mediated by the interaction with the Integrator complex. (h) Comparison of Esrrb-centred self-renewal networks in TS cells and ES cells. Although Esrrb expression is driven by distinct signalling pathways in TS (Fgf/Mek) and ES (LIF/Gsk inhibition) cells, some interacting partners are shared (for example, transcription factors (TFs) Nr0b1, Tfcp2lf1 and the NuRD complex), whereas others are stem cell-specific (for example, Integrator or Mediator complexes). Similarly, Esrrb commonly binds a shared set of genes in both stem cell types, in addition to TS- and ES-cell-specific targets.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525203&req=5

f5: Nr0b1 (=Dax1) and Integrator as Esrrb interactors in TS cells.(a) Esrrb-3xFlag immunoprecipitates analysed using western blot probed with anti-Nr0b1 antibody (Supplementary Fig. 10d). (b) Venn diagram showing the overlap between Esrrb and Nr0b1 ChIP-seq peaks in TS cells. (c) Esrrb- and Nr0b1-binding profiles at the Cdx2 and (d) Cdh1 loci. (e) Esrrb-3xFlag immunoprecipitates analysed using western blot probed with anti-Integrator1 (Ints1) and anti-Integrator 9 (Ints9) antibodies, confirming the prominent interaction between Esrrb and Integrator complex components (Supplementary Fig. 10e). (f) Endogenous anti-Ints9 immunoprecipitates analysed by western blot with anti-Esrrb antibody shows that Esrrb interacts with Ints9 (Supplementary Fig. 10f). (g) Context-dependent function of Esrrb in TS cells may be in part mediated by the interaction with the Integrator complex. (h) Comparison of Esrrb-centred self-renewal networks in TS cells and ES cells. Although Esrrb expression is driven by distinct signalling pathways in TS (Fgf/Mek) and ES (LIF/Gsk inhibition) cells, some interacting partners are shared (for example, transcription factors (TFs) Nr0b1, Tfcp2lf1 and the NuRD complex), whereas others are stem cell-specific (for example, Integrator or Mediator complexes). Similarly, Esrrb commonly binds a shared set of genes in both stem cell types, in addition to TS- and ES-cell-specific targets.
Mentions: Besides interactors involved in epigenetic regulation of transcription, we identified also TFs and cofactor complexes that directly interact with RNAPII (Table 1; Fig. 4a). Similar to some shared epigenetic complexes, we found that the TFs Nr0b1, Esrra, Tf2l1, Zfp462 and others overlapped with the Esrrb interactome in ES cells, thereby further validating our immunoprecipitation (IP) LC-MS/MS analysis (Fig. 4a). Since Nr0b1 has been found to have an important role in ES cell self-renewal, we confirmed by co-immunoprecipitation that it also interacts with Esrrb in TS cells (Fig. 5a). ChIP-seq analysis for Nr0b1 in TS cells showed binding overlap with Esrrb on a subset of essential TS cell-specific (for example, Cdx2 and Tfap2) and general developmental loci (Lin28a and Cdh1; Fig. 5b–d; Supplementary Fig. 8a; Supplementary Data 8). As with Esrrb before, we observed that Nr0b1 binding in TS and ES cells showed a small overlap, with only 52 Esrrb/Nr0b1 co-bound regions shared between ES and TS cells (Supplementary Fig. 8b, Supplementary Data 8). These detailed novel data on the context-specific wiring of transcriptional networks are supported also by the limited overlap of Tfcp2l1, another TF that complexes with Esrrb in both TS and ES cells, with Esrrb TS cell peaks (Supplementary Fig. 8c; Supplementary Data 8).

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.