Critical roles of transcriptional coactivator MED1 in the formation and function of mouse adipose tissues.

The MED1 subunit has been shown to mediate ligand-dependent binding of the Mediator coactivator complex to multiple nuclear receptors, including the adipogenic PPARγ, and to play an essential role in ectopic PPARγ-induced adipogenesis of mouse embryonic fibroblasts. However, the precise roles of MED1, and its various domains, at various stages of adipogenesis and in adipose tissue have been unclear. Here, after establishing requirements for MED1, including specific domains, for differentiation of 3T3L1 cells and both primary white and brown preadipocytes, we used multiple genetic approaches to assess requirements for MED1 in adipocyte formation, maintenance, and function in mice. We show that MED1 is indeed essential for the differentiation and/or function of both brown and white adipocytes, as its absence in these cells leads to, respectively, defective brown fat function and lipodystrophy. This work establishes MED1 as an essential transcriptional coactivator that ensures homeostatic functions of adipocytes.

Transcriptional down-regulation of metabolic genes by Gdown1 ablation induces quiescent cell re-entry into the cell cycle.

Liver regeneration and metabolism are highly interconnected. Here, we show that hepatocyte-specific ablation of RNA polymerase II (Pol II)-associated Gdown1 leads to down-regulation of highly expressed genes involved in plasma protein synthesis and metabolism, a concomitant cell cycle re-entry associated with induction of cell cycle-related genes (including cyclin D1), and up-regulation of p21 through activation of p53 signaling. In the absence of p53, Gdown1-deficient hepatocytes show a severe dysregulation of cell cycle progression, with incomplete mitoses, and a premalignant-like transformation. Mechanistically, Gdown1 is associated with elongating Pol II on the highly expressed genes and its ablation leads to reduced Pol II recruitment to these genes, suggesting that Pol II redistribution may facilitate hepatocyte re-entry into the cell cycle. These results establish an important physiological function for a Pol II regulatory factor (Gdown1) in the maintenance of normal liver cell transcription through constraints on cell cycle re-entry of quiescent hepatocytes.

DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs.

The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice. Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFß superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.

Transcriptional regulation by Pol II(G) involving mediator and competitive interactions of Gdown1 and TFIIF with Pol II.

Pol II(G) is a distinct form of RNA polymerase II that contains the tightly associated Gdown1 polypeptide (encoded by POLR2M). Unlike Pol II, Pol II(G) is highly dependent upon Mediator for robust activator-dependent transcription in a biochemically defined in vitro system. Here, in vitro studies show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, thereby inhibiting an essential function of TFIIF in preinitiation complex assembly, but also that Mediator can actually facilitate Pol II(G) binding to the promoter prior to subsequent Mediator functions. Complementary ChIP and RNAi analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where it appears to restrict transcription. These and other results suggest that Pol II(G) may act to modulate some genes while simultaneously, as a poised (noninitiated) polymerase, setting the stage for Mediator-dependent enhancement of their activity.

Structural basis for evolutionarily conserved interactions between TFIIS and Paf1C.

The elongation factor TFIIS interacts with Paf1C complex to facilitate processive transcription by Pol II. We here determined the crystal structure of the trypanosoma TFIIS LW domain in a complex with the LFG motif of Leo1, as well as the structures of apo-form TFIIS LW domains from trypanosoma, yeast and human. We revealed that all three TFIIS LW domains possess a conserved hydrophobic core that mediates their interactions with Leo1. Intriguingly, the structural study revealed that trypanosoma Leo1 binding induces the TFIIS LW domain to undergo a conformational change reflected in the length and orientation of α6 helix that is absent in the yeast and human counterparts. These differences explain the higher binding affinity of the TFIIS LW domain interacting with Leo1 in trypanosoma than in yeast and human, and indicate species-specific variations in the interactions. Importantly, the interactions between the TFIIS LW domain and an LFG motif of Leo1 were found to be critical for TFIIS to anchor the entire Paf1C complex. Thus, in addition to revealing a detailed structural basis for the TFIIS-Paf1C interaction, our studies also shed light on the origin and evolution of the roles of TFIIS and Paf1C complex in regulation of transcription elongation.

Regulation of hepatocyte cell cycle re-entry by RNA polymerase II-associated Gdown1.

Liver is the central organ responsible for whole-body metabolism, and its constituent hepatocytes are the major players that carry out liver functions. Although they are highly differentiated and rarely divide, hepatocytes re-enter the cell cycle following hepatic loss due to liver damage or injury. However, the exact molecular mechanisms underlying cell cycle re-entry remain undefined. Gdown1 is an RNA polymerase II (Pol II)-associated protein that has been linked to the function of the Mediator transcriptional coactivator complex. We recently found that Gdown1 ablation in mouse liver leads to down-regulation of highly expressed liver-specific genes and a concomitant cell cycle re-entry associated with the induction of cell cycle-related genes. Unexpectedly, in view of a previously documented inhibitory effect on transcription initiation by Pol II in vitro, we found that Gdown1 is associated with elongating Pol II on the highly expressed genes and that its ablation leads to a reduced Pol II occupancy that correlates with the reduced expression of these genes. Based on these observations, we discuss the in vitro and in vivo functions of Gdown1 and consider mechanisms by which the dysregulated Pol II recruitment associated with Gdown1 loss might induce quiescent cell re-entry into the cell cycle.

Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.

Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function.

Identification of target genes for EWS/ATF-1 chimeric transcription factor.

Chromatin immunoprecipitation is a useful technique to detect in vivo direct interaction between any transcription factor and its binding site on genomic DNA. We applied this skill to identify the direct target gene for EWS/ATF-1 by coupling with a GFP reporter assay. This novel approach isolated 62 of cloned DNA fragments responding upon EWS/ATF-1 expression and 16 of 62 clones included putative ATF-1 binding sites. Further analysis revealed that six of the cloned fragments included possible regulatory regions of ATM, GPP34, ARNT2, NKX6.1, NYD-SP28 and POSH. Most of these clones upregulated reporter activity by overexpression of EWS/ATF-1, suggesting that putative ATF-1 binding sites in these clones are functional elements for ATF-1 in vivo. Consistently, endogenous expression of these genes was upregulated by EWS/ATF-1. Interestingly, the clone containing the promoter region of POSH, which is known to be a strong inducer of apoptosis, repressed reporter activity by overexpression of EWS/ATF-1. Correspondingly, EWS/ATF-1 expression decreased endogenous POSH expression, suggesting that six isolated genes may be involved in direct regulation by EWS/ATF-1. Moreover, induction of POSH brought apoptotic cell death to KAS, the clear cell sarcoma (CCS) cell line, suggesting that repressed expression of POSH in CCS may be relevant to the normal signaling pathway in apoptosis.

Regulation of sigma factor competition by the alarmone ppGpp.

Many regulons controlled by alternative sigma factors, including sigma(S) and sigma(32), are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of sigma(S)-dependent promoters because underproduction of sigma(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-sigma(70)) restored expression from sigma(S)-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type sigma(70) to compete with sigma(32) for core binding and the mutant sigma(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type sigma(70). Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp. Consistently, the fraction of sigma(S) and sigma(32) bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.

The osmotic stress response and virulence in pyelonephritis isolates of Escherichia coli: contributions of RpoS, ProP, ProU and other systems.

Trehalose synthesis (RpoS-dependent) and betaine uptake mediated by transporters ProP and ProU contribute to the osmotolerance of Escherichia coli K-12. Pyelonephritis isolates CFT073 and HU734 were similar and diminished in osmotolerance, respectively, compared to E. coli K-12. The roles of RpoS, ProP and ProU in osmoregulation and urovirulence were assessed for these isolates. Strain HU734 expressed an RpoS variant which had low activity and a C-terminal extension. This bacterium accumulated very little trehalose and had poor stationary-phase thermotolerance. For E. coli CFT073, introduction of an rpoS deletion impaired trehalose accumulation, osmotolerance and stationary-phase thermotolerance. The rpoS defects accounted for the difference in osmotolerance between these strains in minimal medium of very high osmolality (1.4 mol kg(-1)) but not in medium of lower osmolality (0.4 mol kg(-1)). The slow growth of both pyelonephritis isolates in high-osmolality medium was stimulated by glycine betaine (GB) and deletion of proP and/or proU impaired GB uptake. An HU734 derivative lacking both proP and proU retained osmoprotective GB uptake activity that could be attributed to system BetU, which is not present in strain K-12 or CFT073. BetU transported GB (K(m), 22 microM) and proline betaine. High-osmolality human urine (0.92 mol kg(-1)) included membrane-permeant osmolyte urea (0.44 M) plus other constituents which contributed an osmolality of only approximately 0.4 mol kg(-1). Strains HU734 and CFT073 showed correspondingly low GB uptake activities after cultivation in this urine. Deletion of proP and proU slowed the growth of E. coli HU734 in this high-osmolality human urine (which contains betaines) but had little impact on its colonization of the murine urinary tract after transurethral inoculation. By contrast, deletion of rpoS, proP and proU had no effect on the very rapid growth of CFT073 in high-osmolality urine or on its experimental colonization of the murine urinary tract. RpoS-dependent gene expression is not essential for growth in human urine or colonization of the murine urinary tract. Additional osmoregulatory systems, some not present in E. coli K-12 (e.g. BetU), may facilitate growth of pyelonephritis isolates in human urine and colonization of mammalian urinary tracts. The contributions of systems ProP and ProU to urinary tract colonization cannot be definitively assessed until all such systems are identified.