Abstract
TFCP2L1 (transcription factor CP2-like 1) is a transcriptional regulator critical for maintaining mouse and human embryonic stem cell (ESC) pluripotency. However, the direct TFCP2L1 target genes are uncharacterized. Here, using gene overexpression, immunoblotting, quantitative real-time PCR, ChIP, and reporter gene assays, we show that TFCP2L1 primarily induces estrogen-related receptor β (Esrrb) expression that supports mouse ESC identity and also selectively enhances Kruppel-like factor 4 (Klf4) expression and thereby promotes human ESC self-renewal. Specifically, we found that in mouse ESCs, TFCP2L1 binds directly to the Esrrb gene promoter and regulates its transcription. Esrrb knockdown impaired Tfcp2l1's ability to induce interleukin 6 family cytokine (leukemia inhibitory factor)-independent ESC self-renewal and to reprogram epiblast stem cells to naïve pluripotency. Conversely, Esrrb overexpression blocked differentiation induced by Tfcp2l1 down-regulation. Moreover, we identified Klf4 as a direct TFCP2L1 target in human ESCs, bypassing the requirement for activin A and basic fibroblast growth factor in short-term human ESC self-renewal. Enforced Klf4 expression recapitulated the self-renewal-promoting effect of Tfcp2l1, whereas Klf4 knockdown eliminated these effects and caused loss of colony-forming capability. These findings indicate that TFCP2L1 functions differently in naïve and primed pluripotency, insights that may help elucidate the different states of pluripotency.
Highlights
TFCP2L1 is a transcriptional regulator critical for maintaining mouse and human embryonic stem cell (ESC) pluripotency
The available human ESCs are more similar to mouse postimplantation epiblast-derived stem cells (EpiSCs) than to mouse ESCs in their self-renewal requirements and morphology and represent a “primed” pluripotency state [3, 7, 8]. mESC self-renewal can be maintained in two distinct culture systems: serum-containing medium supplemented with leukemia inhibitor factor (LIF) [9, 10] and serum-free N2B27 medium supplemented with two small molecule inhibitors (2i), CHIR99021 and PD0325901 [11]
Enforced expression of transcription factor CP2-like 1 (Tfcp2l1) promotes self-renewal, whereas its suppression leads to human ESCs (hESCs) differentiation toward endoderm and mesoderm specification [22, 24]. These findings suggest that the self-renewal–promoting function of Tfcp2l1 is conserved in mESCs and hESCs
Summary
TFCP2L1 (transcription factor CP2-like 1) is a transcriptional regulator critical for maintaining mouse and human embryonic stem cell (ESC) pluripotency. It is unclear whether Tfcp2l1 functions through direct activation of a selective pluripotent factor To resolve this issue, we sought to identify genes directly regulated by Tfcp2l1 in mouse and human ESCs mainly based on gain- and loss-of-function analyses. We sought to identify genes directly regulated by Tfcp2l1 in mouse and human ESCs mainly based on gain- and loss-of-function analyses These analyses identified Esrrb and Klf as two direct targets of Tfcp2l1 that are capable of mediating the self-renewal– promoting effects of Tfcp2l1 in mESCs and hESCs, respectively
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