No abstract

Ubiquitination plays a major role in protein degradation. Although phosphorylation-dependent ubiquitination is well known for the regulation of protein stability, methylation-dependent ubiquitination machinery has not been characterized. Here, we provide evidence that methylation-dependent ubiquitination is carried out by damage-specific DNA binding protein 1 (DDB1)/cullin4 (CUL4) E3 ubiquitin ligase complex and a DDB1-CUL4-associated factor 1 (DCAF1) adaptor, which recognizes monomethylated substrates. Molecular modeling and binding affinity studies reveal that the putative chromo domain of DCAF1 directly recognizes monomethylated substrates, whereas critical binding pocket mutations of the DCAF1 chromo domain ablated the binding from the monomethylated substrates. Further, we discovered that enhancer of zeste homolog 2 (EZH2) methyltransferase has distinct substrate specificities for histone H3K27 and nonhistones exemplified by an orphan nuclear receptor, RORα. We propose that EZH2-DCAF1/DDB1/CUL4 represents a previously unrecognized methylation-dependent ubiquitination machinery specifically recognizing "methyl degron"; through this, nonhistone protein stability can be dynamically regulated in a methylation-dependent manner.

No abstract

Wnt family members play diverse roles in development and disease. Noncanonical Wnt ligands can inhibit canonical Wnt signaling depending on the cellular context; however, the underlying mechanism of this antagonism remains poorly understood. Here we identify a specific mechanism of orphan nuclear receptor RORalpha-mediated inhibition of canonical Wnt signaling in colon cancer. Wnt5a/PKCalpha-dependent phosphorylation on serine residue 35 of RORalpha is crucial to link RORalpha to Wnt/beta-catenin signaling, which exerts inhibitory function of the expression of Wnt/beta-catenin target genes. Intriguingly, there is a significant correlation of reduction of RORalpha phosphorylation in colorectal tumor cases compared to their normal counterpart, providing the clinical relevance of the findings. Our data provide evidence for a role of RORalpha, functioning at the crossroads between the canonical and the noncanonical Wnt signaling pathways, in mediating transrepression of the Wnt/beta-catenin target genes, thereby providing new approaches for the development of therapeutic agents for human cancers.

The retinoic acid receptor-related orphan receptor-α (RORα) is an important regulator of various biological processes, including cerebellum development, circadian rhythm and cancer. Here, we show that hepatic RORα controls lipid homeostasis by negatively regulating transcriptional activity of peroxisome proliferators-activated receptor-γ (PPARγ) that mediates hepatic lipid metabolism. Liver-specific Rorα-deficient mice develop hepatic steatosis, obesity and insulin resistance when challenged with a high-fat diet (HFD). Global transcriptome analysis reveals that liver-specific deletion of Rorα leads to the dysregulation of PPARγ signaling and increases hepatic glucose and lipid metabolism. RORα specifically binds and recruits histone deacetylase 3 (HDAC3) to PPARγ target promoters for the transcriptional repression of PPARγ. PPARγ antagonism restores metabolic homeostasis in HFD-fed liver-specific Rorα deficient mice. Our data indicate that RORα has a pivotal role in the regulation of hepatic lipid homeostasis. Therapeutic strategies designed to modulate RORα activity may be beneficial for the treatment of metabolic disorders.

A critical component of the DNA damage response is the p53 tumor suppressor, and aberrant p53 function leads to uncontrolled cell proliferation and malignancy. Several molecules have been shown to regulate p53 stability; however, genome-wide systemic approaches for determining the affected, specific downstream target genes have not been extensively studied. Here, we first identified an orphan nuclear receptor, RORα, as a direct target gene of p53, which contains functional p53 response elements. The functional consequences of DNA damage-induced RORα are to stabilize p53 and activate p53 transcription in a HAUSP/Usp7-dependent manner. Interestingly, microarray analysis revealed that RORα-mediated p53 stabilization leads to the activation of a subset of p53 target genes that are specifically involved in apoptosis. We further confirmed that RORα enhances p53-dependent, in vivo apoptotic function in the Drosophila model system. Together, we determined that RORα is a p53 regulator that exerts its role in increased apoptosis via p53.

Retinoic acid-related orphan receptor α (RORα) functions as a transcription factor for various biological processes, including circadian rhythm, cancer, and metabolism. Here, we generate intestinal epithelial cell (IEC)-specific RORα-deficient (RORαΔIEC) mice and find that RORα is crucial for maintaining intestinal homeostasis by attenuating nuclear factor κB (NF-κB) transcriptional activity. RORαΔIEC mice exhibit excessive intestinal inflammation and highly activated inflammatory responses in the dextran sulfate sodium (DSS) mouse colitis model. Transcriptome analysis reveals that deletion of RORα leads to up-regulation of NF-κB target genes in IECs. Chromatin immunoprecipitation analysis reveals corecruitment of RORα and histone deacetylase 3 (HDAC3) on NF-κB target promoters and subsequent dismissal of CREB binding protein (CBP) and bromodomain-containing protein 4 (BRD4) for transcriptional repression. Together, we demonstrate that RORα/HDAC3-mediated attenuation of NF-κB signaling controls the balance of inflammatory responses, and therapeutic strategies targeting this epigenetic regulation could be beneficial to the treatment of chronic inflammatory diseases, including inflammatory bowel disease (IBD).

The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.