Abstract
Maintaining pluripotency and indefinite self-renewal of embryonic stem cells requires a tight control of the expression of several key stemness factors, particularly Nanog and Oct4 transcription factors. The mammalian SWItch/Sucrose NonFermentable (SWI/SNF) complex contains Brg1 or Brm as its core subunit, along with Brg1-associated factors. Our previous studies have addressed chromatin-remodeling of the Oct4 gene locus in retinoic acid (RA)-treated embryonal carcinoma cell line P19, which involves receptor-interacting protein 140 (RIP140) for heterochromatinization on the proximal promoter region of this gene locus. However, the mechanism of RIP140 action in RA-triggered repressive chromatin-remodeling is unclear. The current study examines RA repression of the Nanog gene and compares the results with RA repression of the Oct4 gene on the chromatin level. The results show a loose nucleosome array on the Nanog gene promoter in undifferentiated embryonic stem cells. On RA treatment, the Nanog gene locus remodels specifically in the CR1 region of its proximal promoter, with the insertion of a nucleosome and compaction of this region. Further, RA induces coordinated chromatin-remodeling of both Nanog and Oct4 gene loci, which requires RA receptor-α, RIP140 and Brm. Finally, in these RA-triggered repressive chromatin-remodeling processes, lysine acetylation of RIP140 is critical for its recruiting Brm.
Highlights
Controlling the expression levels of Nanog and Oct4 transcription factors as well as other stemness factors is required for maintaining pluripotency and indefinite selfrenewal of embryonic stem cells (ESCs) [1,2,3]
We previously examined the chromatin conformation of the Oct4 gene promoter and its chromatin-remodeling in retinoic acid (RA)-induced differentiation of embryonal carcinoma P19 cells and reported heterochromatinization of the Oct4 gene locus in RAtreated cells, which involved receptor-interacting protein 140 (RIP140) [7]
Using RNA interference (RNAi), we found that RA repression of ESC markers Oct4, Nanog, SSEA1 and alkaline phosphatase (ALP) was effectively rescued by silencing RIP140 (Figure 1C), with the control, b-actin, remained relatively constant
Summary
Controlling the expression levels of Nanog and Oct transcription factors as well as other stemness factors is required for maintaining pluripotency and indefinite selfrenewal of embryonic stem cells (ESCs) [1,2,3]. Studies have shown that precise levels of Nanog and Oct are needed for ESCs to acquire their ground states and for maintaining their pluripotency [3,6]. We previously examined the chromatin conformation of the Oct gene promoter and its chromatin-remodeling in retinoic acid (RA)-induced differentiation of embryonal carcinoma P19 cells and reported heterochromatinization of the Oct gene locus in RAtreated cells, which involved receptor-interacting protein 140 (RIP140) [7]. This is consistent with the widely observed silencing of this gene locus in both differentiated ESC and P19 cells. The mechanism underlying RIP140’s action is not understood
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