SUMOylation of Tr2 orphan receptor involves Pml and fine-tunes Oct4 expression in stem cells

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.     

The expression level of the orphan nuclear receptor GCNF (germ cell nuclear factor) is critical for neuronal differentiation

The germ cell nuclear factor (GCNF) is essential for normal embryonic development and gametogenesis. To test the prediction that GCNF is additionally required for neuronal differentiation, we used the mouse embryonal carcinoma cell line PCC7-Mz1, which represents an advantageous model to study neuronal cells from the stage of fate choice until the acquirement of functional competence. We generated stable transfectants that express gcnf sense or antisense RNA under the control of a tetracycline-regulated promoter. After retinoic acid-induced withdrawal from the cell cycle, sense clones developed a neuron network with changed properties, and the time course of neuron maturation was shortened. Consistent with these data, differentiation of neuronal precursor cells was impaired in antisense cultures. This involved a delay in 1) the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells and stem cells of the central nervous system, and 2) up-regulation of the somatodendritic protein microtubule-associated protein 2 and the synaptic vesicle protein synaptophysin. Neuronal cells in the antisense cultures acquired functional competence, although with a significant delay. Our data propose that the level of GCNF is critical for differentiation and maturation of neuronal precursor cells.     

Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain

For most ligand-dependent nuclear receptors, the status of endogenous ligand modulates the relative affinities for corepressor and coactivator complexes. It is less clear what parameters modulate the switch between corepressor and coactivator for the orphan receptors. Our previous work demonstrated that hepatocyte nuclear factor 4alpha1 (HNF4alpha1, NR2A1) interacts with the p160 coactivator GRIP1 and the cointegrators CBP and p300 in the absence of exogenously added ligand and that removal of the F domain enhances these interactions. Here, we utilized transient-transfection analysis to demonstrate repression of HNF4alpha1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4alpha-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4alpha1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4alpha1 in vitro and repress HNF4alpha1 activity in vivo, although the isolated F domain itself failed to interact with SMRT. Surprisingly, loss of both the A/B and F domains restored full repression by SMRT, suggesting involvement of both domains in the SMRT interaction. Finally, we show that when coexpressed along with HNF4alpha1 and GRIP1, CBP, or p300, SMRT can titer out HNF4alpha1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4alpha can functionally interact with both a coactivator and a corepressor without altering the status of any putative ligand and that the presence of the F domain may play a role in discriminating between the different coregulators.     

Analysis of protein dimerization and ligand binding of orphan receptor HNF4alpha

Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1).     

Treatment of tumor cells with histone deacetylase inhibitors results in altered recruitment of methyl-CpG binding proteins to a methylated CpG island

When human cancer cells with silencing of the CDH1 gene associated with CpG island methylation and histone deacetylation were treated with histone deacetylase inhibitors, alteration in recruitment of methyl-CpG binding proteins (MBPs) to the methylated CDH1-CpG island was observed, as well as altered histone acetylation status. This change was independent of the histone deacetylase inhibitor used. These results suggest that histone hyperacetylation provides a more open chromatin structure conformation for the recruitment of additional MBPs.     

Differential roles for MBD2 and MBD3 at methylated CpG islands,active promoters and binding to exon sequences

The heterogeneous collection of nucleosome remodelling and deacetylation (NuRD) complexes can be grouped into the MBD2- or MBD3-containing complexes MBD2–NuRD and MBD3–NuRD. MBD2 is known to bind to methylated CpG sequences in vitro in contrast to MBD3. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here, we show that MBD2–NuRD, in contrast to MBD3–NuRD, converts open chromatin with euchromatic histone modifications into tightly compacted chromatin with repressive histone marks. Genome-wide, a strong enrichment for MBD2 at methylated CpG sequences is found, whereas CpGs bound by MBD3 are devoid of methylation. MBD2-bound genes are generally lower expressed as compared with MBD3-bound genes. When depleting cells for MBD2, the MBD2-bound genes increase their activity, whereas MBD2 plus MBD3-bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes.     

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.