Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination

Abstract

Embryonic stem (ES) cells are under precise control of both intrinsic self-renewal gene regulatory network and extrinsic growth factor-triggered signaling cascades. How external signaling pathways connect to core self-renewal transcriptional circuits is largely unknown. To probe this, we chose BMP signaling, which is previously recognized as a master control for both self-renewal and lineage commitment of murine ES cells. Here, we mapped target gene promoter occupancy of SMAD1/5 and SMAD4 on a genome-wide scale and found that they associate with a large group of developmental regulators that are enriched for H3K27 trimethylation and H3K4 trimethylation bivalent marks and are repressed in the self-renewing state, whereas they are rapidly induced upon differentiation. Smad knockdown experiments further indicate that SMAD-mediated BMP signaling is largely required for differentiation-related processes rather than directly influencing self-renewal. Among the SMAD-associated genes, we further identified Dpysl2 (previously known as Crmp2) and the H3K27 demethylase Kdm6b (previously known as Jmjd3) as BMP4-modulated early neural differentiation regulators. Combined with computational analysis, our results suggest that SMAD-mediated BMP signaling balances self-renewal versus differentiation by modulating a set of developmental regulators.

miR-23a, miR-24 and miR-27a protect differentiating ESCs from BMP4-induced apoptosis

“…Accordingly, sequence analysis of the genomic region upstream the miR-cluster identified many consensus binding sites for Smads. 4 These data indicated that transcription of the miR-23a cluster gene is regulated by BMP4.…”

“…3 This effect is mediated by the regulation of many direct targets of Smad1, 5 and 8, the transcription factors downstream of the BMP4 receptor. 4 BMP4 also contributes to govern early steps of differentiation. First, BMP4 acts by regulating ESC-epiblast transition and then by suppressing neural differentiation and promoting non-neural lineage formation.…”

Smad2 mediates Activin/Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells

“…To obtain a more precise picture of the roles of Smad2-mediated Activin/Nodal signaling in mouse ES cell differentiation, we adopted a more direct differentiation system, in which ES cells were cultured as monolayer in a defined N2B27-supplemented medium without feeder, serum or serum replacement [21]. The cells were differentiated toward mesendoderm with Activin treatment for 4-5 days; otherwise the cells underwent neuroectoderm differentiation in the absence of Activin (data not shown).…”

“…Genomic DNA immunoprecipitated by Smad2 was then amplified, labeled with fluorophores and finally detected on the Agilent mouse promoter arrays, consisting of ~60-base pair (bp) probes at ~200-bp intervals covering from upstream −5.5 kilobases (kb) to downstream +2.5 kb promoter regions relative to the transcriptional start sites (TSS) for ~17 000 annotated genes of the mouse genome. Binding sites were defined by the method described previously [21], which was shown to obtain a highly credible data set with a falsepositive rate of less than 20% as validated by ChIP-PCR and other criteria. Seven hundred ninety-two genes harboring Smad2 binding sites were identified (Supplementary information, Table S1).…”

“…Distinct from human ES cells, mouse ES cells employ BMP signaling to promote self-renewal in combination with leukemia inhibitory factor (LIF) signaling mainly by inhibiting neural differentiation [20,21]. TGF-β signaling was recognized as being inert in undifferentiated mouse ES cells probably due to low expression of the TGF-β type II receptor [22,23].…”