I. RORgamma: The retinoid-related orphan receptor g (RORg) is a member of the nuclear receptor superfamily. To identify the physiological functions of RORg, mice deficient in RORg function were generated by targeted disruption. RORg KO mice lack peripheral and mesenteric lymph nodes and Peyer's patches indicating that RORg expression is indispensable for lymph node organogenesis. We examined the capacity of RORg-deficient mice to develop an adaptive immune response to antigen using ovalbumin (OVA)-induced inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking RORg, low-grade pulmonary inflammation was observed, characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal immunoglobulin activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, reduced numbers of CD4+ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN-g and IL-10 production was markedly greater in splenocytes from RORg-deficient mice following in vitro re-stimulation with OVA compared to wt splenocytes, and a shift towards a Th1 immune response was observed in sensitized/challenged RORg-deficient animals in vivo. These data reveal a critical role for RORg in the regulation of immunoglobulin production and Th1/Th2 balance in adaptive immunity.Retinoid-related orphan receptors alpha (RORg) and gamma (RORg) are both expressed in liver; however, their physiological functions in this tissue have not yet been clearly defined. The RORa1 and RORg1 isoforms, but not RORa4, show an oscillatory pattern of expression during circadian rhythm. To obtain insight into the physiological functions of ROR receptors in liver, we analyzed the gene expression profiles of livers from WT, RORa-deficient staggerer mice (RORasg/sg), RORg-/-, and RORasg/sgRORg-/- double knockout (DKO) mice by microarray analysis. DKO mice were generated to study functional redundancy between RORa and RORg. These analyses demonstrated that RORa and RORg affect the expression of a number of genes. RORa and RORg are particularly important in the regulation of genes encoding several Phase I and Phase II metabolic enzymes, including several 3b-hydroxysteroid dehydrogenases (Hsd3b), cytochrome P450 (Cyp) enzymes, and sulfotransferases. In addition, our results indicate that RORa and RORg each affect the expression of a specific set of genes but also exhibit functional redundancy. Our study shows that RORa and RORg receptors influence the regulation of several metabolic pathways, including those involved in the metabolism of steroids, bile acids, and xenobiotics, suggesting that RORs are important in the control of metabolic homeostasis. ? II. TAK1: The nuclear orphan receptor TAK1 functions as a positive as well as a negative regulator of transcription; however little is know about factors mediating its activity. Yeast two-hybrid analysis using the ligand binding domain of TAK1 as bait identified a novel TAK1-interacting protein, referred to as TIP27. TIP27 is a 27 kD nuclear protein that contains two zinc finger motifs. Confocal microscopy using TIP27 and several deletion mutants showed that TIP27 localized to the nucleus and that the carboxyl terminus containing a putative nuclear localization signal is important for its nuclear localization. TIP27 mRNA is expressed in several adult tissues but is most highly expressed in testis where it is induced at a specific stage of spermatogenesis. Mammalian two-hybrid analysis showed that TIP27 interacts specifically with TAK1 and not with several other nuclear receptors tested. Deletion mutation analysis determined that the region between Asp39 and Lys79 of TIP27, referred to as TAK1-interaction domain (TID), is critical for its interaction with TAK1. Moreover, it demonstrated that the TAK1-LBD from H3 till the carboxyl terminus is required for optimal interaction with TIP27. Pull-down assays demonstrated that TIP27 physically interacts with TAK1 and supported the importance of the TID. TIP27 is a strong repressor of DR1-dependent transcriptional activation by TAK1. This repression does not involve inhibition of TAK1 homodimerization or DR1 binding but appears to due to an inhibition of the recruitment of co-activators. Our studies indicate that TIP27 is an effective repressor of transcriptional activation by TAK1 and, therefore, may play a critical role in the regulation of several physiological functions by TAK1. Generation of TAK1 knockout mice revealed several phenotypes that are currently being investigated. The receptor-associated protein 80 (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIMs), was shown to interact with the estrogen receptor alpha in an agonist dependent manner. In addition, RAP80 was implicated in DNA repair. We demonstrated that following ionizing radiation (IR) and treatment with DNA-damaging agents RAP80 translocates to discrete nuclear foci that co-localize with those of ?-H2AX. The UIMs and the region between aa 204-304 are critical for the re-localization of RAP80 to IR-induced foci (IRIF). These observations suggest that RAP80 becomes part of a DNA-repair complex at the sites of IRIF. We also demonstrate that RAP80 forms a complex with the tumor repressor BRCA1 and that this interaction is mediated through the BRCT repeats of BRCA1. The UIMs are not required for the interaction of RAP80 with BRCA1. Knockdown of RAP80 in HEK293 cells significantly reduced DSB-induced homology-directed recombination (HDR). Moreover, inhibition RAP80 expression by siRNA increased radiosensitivity, whereas increased radioresistance was observed in human breast cancer MCF-7 cells over-expression of RAP80. Taken together, our data suggest that RAP80 plays an important role in DNA damage response signaling and HDR-mediated DSB repair. We further demonstrate that RAP80 can function as a substrate of the ataxia-telangiectasia mutated (ATM) protein kinase in vitro which phosphorylates RAP80 at Ser205 and Ser402. We show that this phosphorylation is not required for the migration of RAP80 to IRIF.
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