Search

PGC-1α is a key transcription coactivator regulating energy metabolism in a tissue-specific manner. PGC-1α expression is tightly regulated, it is a highly labile protein, and it interacts with various proteins--the known attributes of intrinsically disordered proteins (IDPs). In this study, we characterize PGC-1α as an IDP and demonstrate that it is susceptible to 20S proteasomal degradation by default. We further demonstrate that PGC-1α degradation is inhibited by NQO1, a 20S gatekeeper protein. NQO1 binds and protects PGC-1α from degradation in an NADH-dependent manner. Using different cellular physiological settings, we also demonstrate that NQO1-mediated PGC-1α protection plays an important role in controlling both basal and physiologically induced PGC-1α protein level and activity. Our findings link NQO1, a cellular redox sensor, to the metabolite-sensing network that tunes PGC-1α expression and activity in regulating energy metabolism.

The short-lived proto-oncoprotein c-Fos is a component of the activator protein 1 (AP-1) transcription factor. A large region of c-Fos is intrinsically unstructured and susceptible to a recently characterized proteasomal ubiquitin-independent degradation (UID) pathway. UID is active by a default mechanism that is inhibited by NAD(P)H:quinone oxidoreductase 1 (NQO1), a 20S proteasome gatekeeper. Here, we show that NQO1 binds and induces robust c-Fos accumulation by blocking the UID pathway. c-Jun, a partner of c-Fos, also protects c-Fos from proteasomal degradation by default. Our findings suggest that NQO1 protects monomeric c-Fos from proteasomal UID, a function that is fulfilled later by c-Jun. We show that this process regulates c-Fos homeostasis (proteostasis) in response to serum stimulation, phosphorylation, nuclear translocation, and transcription activity. In addition, we show that NQO1 is important to ensure immediate c-Fos accumulation in response to serum, since a delayed response was observed under low NQO1 expression. These data suggest that in vivo, protein unstructured regions determine the kinetics and the homeostasis of regulatory proteins. Our data provide evidence for another layer of regulation of key regulatory proteins that functions at the level of protein degradation and is designed to ensure optimal formation of functional complexes such as AP-1.

The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage.

Previous studies of human hepatitis B virus (HBV) transcription revealed the requirement of two enhancer elements. Enhancer I (EnhI) is located upstream of the X promoter and is targeted by multiple activators, including basic leucine zipper proteins, and enhancer II (EnhII) is located upstream to the PreCore promoter and is targeted mainly by nuclear receptors (NRs). The mode of interplay between these enhancers and their unique contributions in regulating HBV transcription remained obscure. By using time course analysis we revealed that the HBV transcripts are categorized into early and late groups. Chang (CCL-13) cells are impaired in expression of the late transcripts. This could be corrected by overexpressing EnhII activators, such as hepatocyte nuclear factor 4 alpha, the retinoid X receptor alpha, and the peroxisome proliferator-activated receptor alpha, suggesting that in Chang cells EnhI but not EnhII is active. Replacing the 5'-end EnhI sequence with a synthetic Gal4 response (UAS) DNA fragment ceased the production of the early transcripts. Under this condition NR overexpression poorly activated EnhII. However, activation of the UAS by Gal4-p53 restored both the expression of the early transcripts and the EnhII response to NRs. Thus, a functional EnhI is required for activation of EnhII. We found a major difference between Gal4-p53 and Gal4-VP16 behavior. Gal4-p53 activated the early transcripts, while Gal4-VP16 inhibited the early transcripts but activated the late transcripts. These findings indicate that the composition of the EnhI binding proteins may play a role in early to late switching. Our data provides strong evidence for the role of EnhI in regulating global and temporal HBV gene expression.

A variant polyadenylation signal, which is conserved and employed by mammalian hepadnaviruses, has a sequence resembling that of the TATA box. We report here that this composite box manifests all the promoter characteristics. It binds effectively TATA-binding protein with TFIIB and TFIIA in a synergistic manner. This capacity, however, is lost when the box is converted to a canonical and simple poly(A) signal. Furthermore, we show that it has promoter activity and supports transcription of reporter genes preferentially in liver-derived cells, a characteristic behavior of the hepatitis B virus (HBV) promoters. In addition, we show that the HBV noncanonical poly(A) signal supports transcription initiation from the viral genome, suggesting that it is a genuine promoter, possibly of the polymerase/reverse transcriptase gene. Finally, we found that this deviant poly(A) signal is crucial for HBV replication since a viral mutant with a canonical poly(A) box is impaired in replication. Our data, therefore, raise the interesting and novel possibility that a composite poly(A) box might have a dual function. At the level of DNA it functions as a promoter to initiate transcription, whereas at the level of RNA it serves as a poly(A) signal to process RNA. An interesting outcome of this strategy of gene expression is that it provides a novel mechanism for the synthesis of an approximately genome length transcript.

The physical and functional link between adhesion molecules and the cytoskeletal network suggests that the cytoskeleton might mediate the transduction of cell-to-cell contact signals, which often regulate growth and differentiation in an antagonistic manner. Depolymerization of the cytoskeleton in confluent cell cultures is reportedly sufficient to initiate DNA synthesis. Here we show that depolymerization of the cytoskeleton is also sufficient to repress differentiation-specific gene expression. Glutamine synthetase is a glia-specific differentiation marker gene whose expression in the retinal tissue is regulated by glucocorticoids and is ultimately dependent on glia-neuron cell contacts. Depolymerization of the actin or microtubule network in cells of the intact retina mimics the effects of cell separation, repressing glutamine synthetase induction by a mechanism that involves induction of c-Jun and inhibition of glucocorticoid receptor transcriptional activity. Depolymerization of the cytoskeleton activates JNK and p38 mitogen-activated protein kinase and induces c-Jun expression by a signaling pathway that depends on tyrosine kinase activity. Induction of c-Jun expression is restricted to Müller glial cells, the only cells in the tissue that express glutamine synthetase and maintain the ability to proliferate upon cell separation. Our results suggest that the cytoskeletal network might play a part in the transduction of cell contact signals to the nucleus.

pX, the hepatitis B virus (HBV)-encoded regulator, coactivates transcription through an unknown mechanism. pX interacts with several components of the transcription machinery, including certain activators, TFIIB, TFIIH, and the RNA polymerase II (POLII) enzyme. We show that pX localizes in the nucleus and coimmunoprecipitates with TFIIB from nuclear extracts. We used TFIIB mutants inactive in binding either POLII or TATA binding protein to study the role of TFIIB-pX interaction in transcription coactivation. pX was able to bind the former type of TFIIB mutant and not the latter. Neither of these sets of TFIIB mutants supports transcription. Remarkably, the latter TFIIB mutants fully block pX activity, suggesting the role of TFIIB in pX-mediated coactivation. By contrast, in the presence of pX, TFIIB mutants with disrupted POLII binding acquire the wild-type phenotype, both in vivo and in vitro. These results suggest that pX may establish the otherwise inefficient TFIIB mutant-POLII interaction, by acting as a molecular bridge. Collectively, our results demonstrate that TFIIB is the in vivo target of pX.