We previously reported an intricate mechanism underlying the homeostasis of Oct4 expression in normally proliferating stem cell culture of P19, mediated by SUMOylation of orphan nuclear receptor TR2. In the present study, we identify a signaling pathway initiated from the nongenomic activity of all-trans retinoic acid (atRA) to stimulate complex formation of extracellular signal-regulated kinase 2 (ERK2) with its upstream kinase, mitogen-activated protein kinase kinase (MEK). The activated ERK2 phosphorylates threonine-210 (Thr-210) of TR2, stimulating its subsequent SUMOylation. Dephosphorylated TR2 recruits coactivator PCAF and functions as an activator for its target gene Oct4. Upon phosphorylation at Thr-210, TR2 increasingly associates with promyelocytic leukemia (PML) nuclear bodies, becomes SUMOylated, and recruits corepressor RIP140 to act as a repressor for its target, Oct4. To normally proliferating P19 stem cell culture, exposure to a physiological concentration of atRA triggers a rapid nongenomic signaling cascade to suppress Oct4 gene and regulate cell proliferation.
The cellular retinoic acid binding protein I gene is induced by thyroid hormone (T3) through a T3 response element (TRE) approximately 1 kb upstream of the basal promoter. The upstream region is organized into a positioned nucleosomal array with the N1 nucleosome spanning the GC box region. T3 induces apparent interactions between chromatin segments containing the TRE and the GC box regions and the sliding of upstream nucleosomes toward N1 with concomitant N1 remodeling. Concurrently, the chromatin-remodeling factor BRM is replaced by BRG1 and histones are hyperacetylated. All these events are abolished in Med1/Trap220 null cells, indicating a key role for TRAP/Mediator in these processes. A MED1/TRAP220-containing Mediator complex constitutively occupies the GC box region but not the TRE, serving as a nexus for distal and proximal factors. This indicates new TRAP/Mediator functions in facilitating ultimate recruitment and function of RNA polymerase II and the general transcription machinery.
The Tr2 orphan nuclear receptor can be SUMOylated, resulting in the replacement of coregulators recruited to the regulatory region of its endogenous target gene, Oct4. UnSUMOylated Tr2 activates Oct4, enhancing embryonal carcinoma-cell proliferation, and is localized to the promyelocytic leukemia (Pml) nuclear bodies. When its abundance is elevated, Tr2 is SUMOylated at Lys238 and seems to be released from the nuclear bodies to act as a repressor. SUMOylation of Tr2 induces an exchange of its coregulators: corepressor Rip140 replaces coactivator Pcaf, which switches Tr2 from an activator to a repressor. This involves dynamic partitioning of Tr2 into Pml-containing and Pml-free pools. These results support a model where SUMOylation-dependent partitioning and differential coregulator recruitment contribute to the maintenance of a homeostatic supply of activating, as opposed to repressive, Tr2, thus fine-tuning Oct4 expression and regulating stem-cell proliferation.
Promyelocytic leukemia (Pml) protein is required for Oct4 gene expression and the maintenance of its open chromatin conformation in stem cells. In proliferating stem cells, Pml-nuclear body, along with transcription factors TR2, SF1 and Sp1 and Brg1-dependent chromatin remodeling complex (BRGC), associates with conserved region 1 (CR1) of this promoter to maintain a nucleosome-free region for gene activity. Retinoic acid (RA) rapidly down-regulates Pml, resulting in the replacement of BRGC with Brm-containing remodeling complex (BRMC), disassociation of SF1 and SP1, retaining of TR2, recruitment of RIP140, G9a and HP1γ, and sequential insertion of two nucleosomes on CR1 that progressively displays repressive heterochromatin marks. This study demonstrates a functional role for Pml in maintaining a specific open chromatin conformation of the Oct4 promoter region for its constant expression in stem cells; and illustrates the mechanism underlying RA-induced chromatin remodeling of Oct4 gene in differentiating cells, in which Pml plays a critical role. The study also demonstrates a novel mode of chromatin remodeling which occurs by repositioning and sequentially inserting nucleosomes into a specific region of the gene promoter to compact the chromatin in differentiating cells.
Excessive and/or persistent activation of calcium-calmodulin protein kinase II (CaMKII) is detrimental in acute and chronic cardiac injury. However, intrinsic regulators of CaMKII activity are poorly understood. We find that cellular retinoic acid-binding protein 1 (CRABP1) directly interacts with CaMKII and uncover a functional role for CRABP1 in regulating CaMKII activation. We generated -null mice (CKO) in C57BL/6J background for pathophysiological studies. CKO mice develop hypertrophy as adults, exhibiting significant left ventricular dilation with reduced ejection fraction at the baseline cardiac function. Interestingly, CKO mice have elevated basal CaMKII phosphorylation at T287, and phosphorylation on its substrate phospholamban (PLN) at T17. Acute isoproterenol (ISO) challenge (80 mg/kg two doses in 1 day) causes more severe apoptosis and necrosis in CKO hearts, and treatment with a CaMKII inhibitor KN-93 protects CKO mice from this injury. Chronic (30 mg/kg/day) ISO challenge also significantly increases hypertrophy and fibrosis in CKO mice as compared to WT. In wild-type mice, CRABP1 expression is increased in early stages of ISO challenge and eventually reduces to the basal level. Mechanistically, CRABP1 directly inhibits CaMKII by competing with calmodulin (CaM) for CaMKII interaction. This study demonstrates increased susceptibility of CKO mice to ISO-induced acute and chronic cardiac injury due to, at least in part, elevated CaMKII activity. Deleting results in reduced baseline cardiac function and aggravated damage challenged with acute and persistent β-adrenergic stimulation. This is the first report of a physiological role of CRABP1 as an endogenous regulator of CaMKII, which protects the heart from ISO-induced damage.
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