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The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in Arabidopsis thaliana but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the crwn1crwn4 double mutant. Copper-associated (CA) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of CA gene expression and that CRWN1 interacts with the CA gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions., The nuclear lamina regulates chromatin organization and gene positioning. Here the authors show that CROWDED NUCLEI proteins contribute to the meshwork lamina structure in Arabidopsis nuclei and regulate copper tolerance by promoting lamina association and expression of copper response genes.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8–SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors., Plants regulate phosphate homeostasis via the interaction of PHR transcription factors with SPX receptors bound to inositol pyrophosphate signaling molecules. Here the authors show that inositol pyrophosphate-bound SPX interacts with the coiled-coil domain of PHR, which regulates the oligomerization and activity of the transcription factor.

The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.

Genome-wide studies reveal that transcription by RNA polymerase II (Pol II) is dynamically regulated. To obtain a comprehensive view of a single transcription cycle, we switched on transcription of five long human genes (>100 kbp) with tumor necrosis factor-alpha (TNFalpha) and monitored (using microarrays, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation) the appearance of nascent RNA, changes in binding of Pol II and two insulators (the cohesin subunit RAD21 and the CCCTC-binding factor CTCF), and modifications of histone H3. Activation triggers a wave of transcription that sweeps along the genes at approximately 3.1 kbp/min; splicing occurs cotranscriptionally, a major checkpoint acts several kilobases downstream of the transcription start site to regulate polymerase transit, and Pol II tends to stall at cohesin/CTCF binding sites.

The migration of cortical γ-aminobutyric acidergic interneurons has been extensively studied in rodent embryos, whereas few studies have documented their postnatal migration. Combining in vivo analysis together with time-lapse imaging on cortical slices, we explored the origin and migration of cortical interneurons during the first weeks of postnatal life. Strikingly, we observed that a large pool of GAD65-GFP-positive cells accumulate in the dorsal white matter region during the first postnatal week. Part of these cells divides and expresses the transcription factor paired box 6 indicating the presence of local transient amplifying precursors. The vast majority of these cells are immature interneurons expressing the neuronal marker doublecortin and partly the calcium-binding protein calretinin. Time-lapse imaging reveals that GAD65-GFP-positive neurons migrate from the white matter pool into the overlying anterior cingulate cortex (aCC). Some interneurons in the postnatal aCC express the same immature neuronal markers suggesting ongoing migration of calretinin-positive interneurons. Finally, bromodeoxyuridine incorporation experiments confirm that a small fraction of interneurons located in the aCC are generated during the early postnatal period. These results altogether reveal that at postnatal ages, the dorsal white matter contains a pool of interneuron precursors that divide and migrate into the aCC.

Chronic exposure to elevated levels of glucose and fatty acids leads to dysfunction of pancreatic β-cells by mechanisms that are only partly understood. The transcription factor peroxisome proliferator-activated receptor α (PPARα) is an important regulator of genes involved in fatty acid metabolism and has been shown to protect against lipid-induced β-cell dysfunction. We and others have previously shown that expression of the PPARα gene in β-cells is rapidly repressed by glucose. Here we show that the PPARα gene is transcribed from five alternative transcription start sites, resulting in three alternative first exons that are spliced to exon 2. Expression of all PPARα transcripts is repressed by glucose both in insulinoma cells and in isolated pancreatic islets. The observation that the dynamics of glucose repression of PPARα transcription are very similar to those of glucose activation of target genes by the carbohydrate response element-binding protein (ChREBP) prompted us to investigate the potential role of ChREBP in the regulation of PPARα expression. We show that a constitutively active ChREBP lacking the N-terminal domain efficiently represses PPARα expression in insulinoma cells and in rodent and human islets. In addition, we demonstrate that siRNA-mediated knockdown of ChREBP abrogates glucose repression of PPARα expression as well as induction of well established ChREBP target genes in insulinoma cells. In conclusion, this work shows that ChREBP is a critical and direct mediator of glucose repression of PPARα gene expression in pancreatic β-cells, suggesting that ChREBP may be important for glucose suppression of the fatty acid oxidation capacity of β-cells.

RNA-directed modification of histones is essential for the maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation is an additional factor regulating inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. In this study, we analysed the mechanism of gene silencing mediated by MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana. Transcript profiling revealed that the majority of up-regulated loci in mom1 carry sequences related to transposons and homologous to the 24-nt siRNAs accumulated in wild-type plants that are the hallmarks of RNA-directed DNA methylation (RdDM). Analysis of a single-copy gene, SUPPRESSOR OF drm1 drm2 cmt3 (SDC), revealed that mom1 activates SDC with concomitant reduction of di-methylated histone H3 lysine 9 (H3K9me2) at the tandem repeats in the promoter region without changes in siRNA accumulation and cytosine methylation. The reduction of H3K9me2 is not observed in regions flanking the tandem repeats. The results suggest that MOM1 transduces RdDM signals to repressive histone modification in the core region of RdDM.

Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5' incision by ERCC1-XPF precedes the 3' incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.

The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCLs). Constitutive expression of BCL6 mediates lymphomagenesis through aberrant proliferation, survival, and differentiation blockade. Binding of BCL6 to the SMRT/N-CoR corepressors mediates the BCL6 survival effect in DLBCL. Although the basis for differentiation blockade is unknown in DLBCL, recent data suggest that BCL6 binding to the MTA3 corepressor might be involved. We report that BCL6 and MTA3 are coexpressed in normal germinal center B cells and DLBCL. Depletion of MTA3 in DLBCL cells induced a differentiation-related BCL6 target gene (PRDM1), but not target genes involved in survival. Accordingly, MTA3 and PRDM1 expression are mutually exclusive in germinal center B cells. We performed chromatin immunoprecipitation (ChIP)–on-chip mapping of the PRDM1 locus, identifying a novel BCL6 binding site on intron 3 of the PRDM1 gene, and show that BCL6 recruits MTA3 to this site. In DLBCL cells, MTA3 depletion induced plasmacytic differentiation but did not decrease viability of DLBCL cells. However, MTA3 depletion synergized with a specific BCL6 inhibitor that blocks SMRT/N-CoR binding to decrease DLBCL viability. Taken together, these results show that BCL6 regulates distinct transcriptional programs through the SMRT/N-CoR and MTA3 corepressors, respectively, and provides a basis for combinatorial therapeutic targeting of BCL6.

Attribution of the most probable functions to proteins identified by proteomics is a significant challenge that requires extensive literature analysis. We have developed a system for automated prediction of implicit and explicit biologically meaningful functions for a proteomics study of the nucleolus. This approach uses a set of vocabulary terms to map and integrate the information from the entire MEDLINE database. Based on a combination of cross-species sequence homology searches and the corresponding literature, our approach facilitated the direct association between sequence data and information from biological texts describing function. Comparison of our automated functional assignment to manual annotation demonstrated our method to be highly effective. To establish the sensitivity, we defined the functional subtleties within a family containing a highly conserved sequence. Clustering of the DEAD-box protein family of RNA helicases confirmed that these proteins shared similar morphology although functional subfamilies were accurately identified by our approach. We visualized the nucleolar proteome in terms of protein functions using multi-dimensional scaling, showing functional associations between nucleolar proteins that were not previously realized. Finally, by clustering the functional properties of the established nucleolar proteins, we predicted novel nucleolar proteins. Subsequently, nonproteomics studies confirmed the predictions of previously unidentified nucleolar proteins.

A lentiviral vector expressing a mutant huntingtin protein (htt171-82Q) was used to generate a chronic model of Huntington's disease (HD) in rat primary striatal cultures. In this model, the majority of neurons expressed the transgene so that Western blot analysis and flow cytometry measurement could complement immunohistological evaluation. Mutant huntingtin produced a slowly progressing pathology characterized after 1 month by the appearance of neuritic aggregates followed by intranuclear inclusions, morphological anomalies of neurites, loss of neurofilament 160, increased expression in stress response protein Hsp70, and later loss of neuronal markers such as NeuN and MAP-2. At 2 months post-infection, a significant increase in TUNEL-positive cells confirmed actual striatal cell loss. Interestingly, cortical cultures infected with the same vector showed no sign of neuronal dysfunction despite accumulation of numerous inclusions. We finally examined whether the trophic factors CNTF and BDNF that were found neuroprotective in acute HD models could prevent striatal degeneration in a chronic model. Results demonstrated that both agents were neuroprotective without modifying inclusion formation. The present study demonstrates that viral vectors coding for mutant htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary striatal cultures.

As a component of chromatin-modifying complexes with histone acetyltransferase (HAT) activity, TRRAP has been shown to be involved in various cellular processes including gene transcription and oncogenic transformation. Inactivation of Trrap, the murine ortholog of TRRAP, in mice revealed its function in development and cell cycle progression. However, the underlying mechanism is unknown. Here, we show that the loss of Trrap in mammalian cells leads to chromosome missegregation, mitotic exit failure and compromised mitotic checkpoint. These mitotic checkpoint defects are caused by defective Trrap-mediated transcription of the mitotic checkpoint proteins Mad1 and Mad2. The mode of regulation by Trrap involves acetylation of histones H4 and H3 at the gene promoter of these mitotic players. Trrap associated with the HAT Tip60 and PCAF at the Mad1 and Mad2 promoters in a cell cycle-dependent manner and Trrap depletion abolished recruitment of these HATs. Finally, ectopic expression of Mad1 and Mad2 fully restores the mitotic checkpoint in Trrap-deficient cells. These results demonstrate that Trrap controls the mitotic checkpoint integrity by specifically regulating Mad1 and Mad2 genes.

Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.

The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.

Liver glucose metabolism plays a central role in glucose homeostasis and may also regulate feeding and energy expenditure. Here we assessed the impact of glucose transporter 2 (Glut2) gene inactivation in adult mouse liver (LG2KO mice). Loss of Glut2 suppressed hepatic glucose uptake but not glucose output. In the fasted state, expression of carbohydrate-responsive element-binding protein (ChREBP) and its glycolytic and lipogenic target genes was abnormally elevated. Feeding, energy expenditure, and insulin sensitivity were identical in LG2KO and control mice. Glucose tolerance was initially normal after Glut2 inactivation, but LG2KO mice exhibited progressive impairment of glucose-stimulated insulin secretion even though β cell mass and insulin content remained normal. Liver transcript profiling revealed a coordinated downregulation of cholesterol biosynthesis genes in LG2KO mice that was associated with reduced hepatic cholesterol in fasted mice and reduced bile acids (BAs) in feces, with a similar trend in plasma. We showed that chronic BAs or farnesoid X receptor (FXR) agonist treatment of primary islets increases glucose-stimulated insulin secretion, an effect not seen in islets from Fxr(-/-) mice. Collectively, our data show that glucose sensing by the liver controls β cell glucose competence and suggest BAs as a potential mechanistic link.

Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs.

Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized.

The mammalian circadian timing system consists of a master pacemaker in neurons of the suprachiasmatic nucleus (SCN) and clocks of a similar molecular makeup in most peripheral body cells. Peripheral oscillators are self-sustained and cell autonomous, but they have to be synchronized by the SCN to ensure phase coherence within the organism. In principle, the rhythmic expression of genes in peripheral organs could thus be driven not only by local oscillators, but also by circadian systemic signals. To discriminate between these mechanisms, we engineered a mouse strain with a conditionally active liver clock, in which REV-ERBα represses the transcription of the essential core clock gene Bmal1 in a doxycycline-dependent manner. We examined circadian liver gene expression genome-wide in mice in which hepatocyte oscillators were either running or arrested, and found that the rhythmic transcription of most genes depended on functional hepatocyte clocks. However, we discovered 31 genes, including the core clock gene mPer2, whose expression oscillated robustly irrespective of whether the liver clock was running or not. By contrast, in liver explants cultured in vitro, circadian cycles of mPer2::luciferase bioluminescence could only be observed when hepatocyte oscillators were operational. Hence, the circadian cycles observed in the liver of intact animals without functional hepatocyte oscillators were likely generated by systemic signals. The finding that rhythmic mPer2 expression can be driven by both systemic cues and local oscillators suggests a plausible mechanism for the phase entrainment of subsidiary clocks in peripheral organs., Author Summary In contrast to previously held belief, molecular circadian oscillators are not restricted to specialized pacemaker tissues, such as the brain's suprachiasmatic nucleus (SCN), but exist in virtually all body cells. Although the circadian clocks operative in peripheral cell types are as robust as those residing in SCN neurons, they quickly become desynchronized in vitro due to variations in period length. Hence, in intact animals, the phase coherence between peripheral oscillators must be established by daily signals generated by the SCN master clock. Although the hierarchy between master and slave oscillators is now well established, the respective roles of these clocks in governing the circadian transcription program in a given organ have never been examined. In principle, the circadian expression of genes in a peripheral tissue could be driven either by cyclic systemic cues, by peripheral oscillators, or by both. In order to discriminate between genes regulated by local oscillators and systemic cues in liver, we generated mice in which hepatocyte clocks can be turned on and off at will. These studies suggest that 90% of the circadian transcription program in the liver is abolished or strongly attenuated when hepatocyte clocks are turned off, indicating that the expression of most circadian liver genes is orchestrated by local cellular clocks. The remaining 10% of cyclically expressed liver genes continue to be transcribed in a robustly circadian fashion in the absence of functional hepatocyte oscillators. These genes, which unexpectedly include the bona fide clock gene mPer2, must therefore be regulated by oscillating systemic signals, such as hormones, metabolites, or body temperature. Although temperature rhythms display only modest amplitudes, they appear to play a significant role in the phase entrainment of mPer2 transcription., Research on mice engineered with an inducible liver clock enabled identification of some genes with expression controlled by the local clock, and other genes (includingmPer2) that maintained circadian oscillations thanks to cues from the SCN.

Plants fend off potentially damaging ultraviolet (UV)-B radiation by synthesizing and accumulating UV-B-absorbing flavonols that function as sunscreens. Regulation of this biosynthetic pathway is largely transcriptional and controlled by a network of transcription factors, among which the PRODUCTION OF FLAVONOL GLYCOSIDES (PFG) family of R2R3-MYB transcription factors was recently identified with a pivotal function. Here, we describe the response of Arabidopsis seedlings to narrow-band UV-B radiation at the level of phenylpropanoid pathway genes using whole-genome transcriptional profiling and identify the corresponding flavonol glycosides accumulating under UV-B. We further show that the bZIP transcriptional regulator ELONGATED HYPOCOTYL5 (HY5) is required for the transcriptional activation of the PFG1/MYB12 and PFG3/MYB111 genes under UV-B and visible light. A synthetic protein composed of HY5 with the VP16 activation domain is sufficient to activate PFG1/MYB12 expression in planta. However, even though myb11 myb12 myb111 triple mutants have strongly reduced CHS levels in darkness as well as in constant light, neither light- nor UV-B-inducibility seems impaired. Notwithstanding this, absence of the three PFG family transcription factors results in reduced UV-B tolerance, whereas PFG1/MYB12 overexpression leads to an increased tolerance. Thus, our data suggest that HY5-dependent regulation of PFG gene expression contributes to the establishment of UV-B tolerance.

The Spatial gene is expressed in highly polarized cell types such as testis germ cells, brain neurons and thymic epithelial cells (TEC). Its expression was documented in testis and brain but poorly characterized in thymus. Here, we characterize for the first time Spatial-expressing TEC throughout ontogeny and adult mouse thymus. Spatial is expressed in thymic-fated domain by embryonic day E10.5 and persists in subcapsular, cortical, medullary epithelial cells and in MTS24+ progenitor TEC. Using mouse strains in which thymocyte development is blocked at various stages, we show that Spatial expression is independent of thymocyte-derived signals during thymus organogenesis. Analyses on purified thymic cell subsets show that Spatial short isoforms are expressed in cortical TEC (cTEC) and mature medullary TEC (mTEC). Spatial long isoforms were detected in the same TEC population. Spatial presents a nuclear distribution specific to mature mTEC expressing UEA1 and Aire. Aire- and RANKL-deficientmice revealed that Spatial expression is drastically reduced in the thymus of these mutants. These findings reveal a critical function of Aire in regulating Spatial expression, which is compatible with promiscuous Spatial gene expression. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA., Funded by: Institut National de la Santé et de la Recherche Médicale (INSERM).

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1alpha, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1beta (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1alpha and identify Ser247 as a major site of in vitro modification by casein kinase 1delta (CK1delta). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1alpha, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1delta under hypoxic conditions. Conversely, overexpression of CK1delta or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1alpha activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1alpha at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1alpha and ARNT PAS-B domains. We suggest that modification of HIF-1alpha by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits. J Cell Sci

It is commonly observed that onset or release of transcriptional gene silencing (TGS) correlates with alteration of repressive epigenetic marks. The TGS regulator MOM1 in Arabidopsis is exceptional since it regulates transcription in intermediate heterochromatin with only minor changes in epigenetic marks. We have isolated an enhancer of the mom1 mutation that points towards regulatory interplay between MOM1 and RNA polymerase-V (Pol-V). Pol-V transcribes heterochromatic loci, which seems to be required for maintenance of their silencing; however, it is still not clear how Pol-V is targeted to heterochromatin. We now provide evidence that Pol-V is required for MOM1-mediated suppression of transcription at a subset of its chromosomal targets. Thus, Pol-V genetically interacts with MOM1 in the control of gene silencing. Interestingly, functional cooperation of MOM1 and Pol-V not only broadens the range of the controlled loci in comparison to each individual factor, but also determines the degree of TGS.

International audience; Monitoring Editor: Thomas Sommer The ubiquitin-associated (UBA) domain of the mRNA nuclear export receptor Mex67 helps coordinating transcription elongation and nuclear export by interacting both with ubiquitin conjugates and specific targets, such as Hpr1, a component of the THO complex. Here we analyzed substrate specificity and ubiquitin selectivity of the Mex67 UBA domain. UBA-Mex67 is formed by three helices arranged in a classical UBA fold plus a fourth helix, H4. Deletion or mutation of helix H4 strengthens the interaction between UBA-Mex67 and ubiquitin, but decreases its affinity for Hpr1. Interaction with Hpr1 is required for Mex67 UBA domain to bind poly-ubiquitin, possibly by inducing an H4-dependent conformational change. In vivo, deletion of helix H4 reduces cotranscriptional recruitment of Mex67 on activated genes and also shows a mRNA export defect. Based on these results, we propose that H4 functions as a molecular switch that coordinates the interaction of Mex67 with ubiquitin bound to specific substrates, defines the selectivity of the Mex67 UBA domain for poly-ubiquitin and prevents its binding to nonspecific substrates.

Chromatin modifications at core histones including acetylation, methylation, phosphorylation and ubiquitination play an important role in diverse biological processes. Acetylation of specific lysine residues within the N terminus tails of core histones is arguably the most studied histone modification; however, its precise roles in different cellular processes and how it is disrupted in human diseases remain poorly understood. In the last decade, a number of histone acetyltransferases (HATs) enzymes responsible for histone acetylation, has been identified and functional studies have begun to unravel their biological functions. The activity of many HATs is dependent on HAT complexes, the multiprotein assemblies that contain one HAT catalytic subunit, adapter proteins, several other molecules of unknown function and a large protein called TRansformation/tRanscription domain-Associated Protein (TRRAP). As a common component of many HAT complexes, TRRAP appears to be responsible for the recruitment of these complexes to chromatin during transcription, replication and DNA repair. Recent studies have shed new light on the role of TRRAP in HAT complexes as well as mechanisms by which it mediates diverse cellular processes. Thus, TRRAP appears to be responsible for a concerted and context-dependent recruitment of HATs and coordination of distinct chromatin-based processes, suggesting that its deregulation may contribute to diseases. In this review, we summarize recent developments in our understanding of the function of TRRAP and TRRAP-containing HAT complexes in normal cellular processes and speculate on the mechanism underlying abnormal events that may lead to human diseases such as cancer.

The mRNA nuclear export receptor Mex67/Mtr2 is recruited to mRNAs through RNA-binding adaptors, including components of the THO/TREX complex that couple transcription to mRNA export. Here we show that the ubiquitin-associated (UBA) domain of Mex67 is not only required for proper nuclear export of mRNA but also contributes to recruitment of Mex67 to transcribing genes. Our results reveal that the UBA domain of Mex67 directly interacts with polyubiquitin chains and with Hpr1, a component of the THO/TREX complex, which is regulated by ubiquitylation in a transcription-dependent manner. This interaction transiently protects Hpr1 from ubiquitin/proteasome-mediated degradation and thereby coordinates recruitment of the mRNA export machinery with transcription and early messenger ribonucleoproteins assembly.

To identify a growth-promoting activity related to retinoblastoma-interacting-zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products. To identify a growth-promoting activity related to retinoblastoma- interacting-zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products. © 2006 American Chemical Society.

Transcription activation of some Saccharomyces cerevisiae genes is paralleled by their repositioning to the nuclear periphery, but the mechanism underlying gene anchoring is poorly defined. We show that the nuclear pore complex-associated Mlp1p and the shuttling mRNA export receptor Mex67p contribute to the stable association of the activated GAL10 and HSP104 genes with the nuclear periphery. However, we find no obligatory link between gene positioning and gene expression. Furthermore, gene anchoring correlates with the cotranscriptional recruitment of Mex67p to transcribing genes. Notably, the association of Mex67p with chromatin is not mediated by RNA. Interestingly, a mutant GAL2 gene lacking the coding region is still able to recruit Mex67p upon transcriptional activation and to relocate to the nuclear periphery. Together these data suggest that, at least for GAL2, nascent messenger ribonucleoprotein does not play a major role in gene anchoring and that the early recruitment of Mex67p contributes to gene repositioning by virtue of an RNA-independent process.

International audience; Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

The mRNA export adaptor Yra1p/REF contributes to nascent mRNP assembly and recruitment of the export receptor Mex67p. yra1 mutants exhibit mRNA export defects and a decrease in LacZ reporter and certain endogenous transcripts. The loss of Mlp1p/Mlp2p, two TPR-like proteins attached to nuclear pores, rescues LacZ mRNA levels and increases their appearance in the cytoplasm, without restoring bulk poly(A)+ RNA export. Chromatin immunoprecipitation, FISH and pulse-chase experiments indicate that Mlps downregulate LacZ mRNA synthesis in a yra1 mutant strain. Microarray analyses reveal that Mlp2p also reduces a subset of cellular transcripts in the yra1 mutant. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlps rescues the growth defect of yra1 and nab2 but not other mRNA export mutants. We propose that Nab2p and Yra1p are required for proper mRNP docking to the Mlp platform. Defects in Yra1p prevent mRNPs from crossing the Mlp gate and this block negatively feeds back on the transcription of a subset of genes, suggesting that Mlps link mRNA transcription and export.

The translationally controlled tumor protein (TPT1, also known as TCTP) is a highly conserved, abundantly expressed protein found in mammals as well as in a wide range of other organisms of both the animal and plant kingdom. Initially considered as a growth-related protein, later studies showed TPT1 is endowed with multiple biological activities, including calcium binding. The present study aimed to evaluate the expression of TPT1 in the human placenta and to examine the functional role of the protein in the calcium binding and homeostasis of trophoblast cells. Samples were analyzed by Western blot, reverse transcription-polymerase chain reaction and immunohistochemistry. The effect of TPT1 knockdown by small interfering RNA (siRNA) on calcium uptake and buffering was assessed in the HTR-8/SVneo cell line. TPT1 protein and mRNA were detected in first-trimester and term placenta. In the tissue, TPT1 was localized in the villous trophoblast. TPT1 expression significantly increased during gestation, with the higher protein and mRNA levels reached at term. Recombinant placental TPT1 bound calcium in vitro, while downregulation of the protein levels by siRNA in HTR-8/SVneo cells was associated with a reduced cellular calcium-uptake activity and buffering capacity. These data demonstrate, for the first time, the expression of TPT1 in the human placenta and support a direct role of the protein in placental calcium transport.

Conditionally immortalized human cells are valuable substrates for basic biologic studies, as well as for the production of specific proteins and for the creation of bioartificial organs. We previously demonstrated that the lentivector-mediated transduction of immortalizing genes into human primary cells is an efficient method for obtaining such cell lines. Here, we used human muscle satellite cells as model targets to examine the impact of the transduced genes on the genotypic and phenotypic characteristics of the immortalized cells. The most commonly used immortalizing gene, the SV40 large T antigen (T-Ag), was extremely efficient at inducing the continuous growth of primary myoblasts, but the resulting cells rapidly accumulated major chromosomal aberrations and exhibited profound phenotypic changes. In contrast, the constitutive expression of telomerase and Bmi-1 in satellite cells from a control individual and from a patient suffering from Duchenne's muscular dystrophy yielded cell lines that remained diploid and conserved their growth factor dependence for proliferation. However, despite the absence of detectable cytogenetic abnormalities, clones derived from satellite cells of a control individual exhibited a differentiation block in vitro. In contrast, a Duchenne-derived cell line exhibited all the phenotypic characteristics of its primary parent, including an ability to differentiate fully into myotubes when placed in proper culture conditions. This cell line should constitute a useful reagent for a wide range of studies aimed at this disease.

0022-3034 (Print) Comparative Study Journal Article Research Support, Non-U.S. Gov't; The PGal4 transposon inserted upstream of the pan-neural gene prospero (pros) causes several neural and behavioral defects in the Voila(1) strain. The precise excision of the transposon simultaneously rescued all these defects whereas its unprecise excision created new pros(V) alleles, including the null allele pros(V17). Here, we describe the relationship between the genetic structure of pros locus, larval locomotion, and larval gustatory response. These two behaviors showed varying degrees of variation depending upon the pros allele. We also found a good relation between behavioral alteration, the level of Pros protein in the embryo, and the degree of disorganization in the larval neuromuscular junction. These data suggest that the complete development of the nervous system requires a full complement of Pros, and that a gradual decrease in the levels of this protein can proportionally alter the development and the function of the nervous system.

Glucagon gene expression in the endocrine pancreas is controlled by three islet-specific elements (G3, G2, and G4) and theα -cell-specific element G1. Two proteins interacting with G1 have previously been identified as Pax6 and Cdx2/3. We identify here the third yet uncharacterized complex on G1 as hepatocyte nuclear factor 3 (HNF-3)β, a member of the HNF-3/forkhead transcription family, which plays an important role in the development of endoderm-related organs. HNF-3 has been previously demonstrated to interact with the G2 element and to be crucial for glucagon gene expression; we thus define a second binding site for this transcription on the glucagon gene promoter. We demonstrate that both HNF-3α and -β produced in heterologous cells can interact with similar affinities to either the G1 or G2 element. Pax6, which binds to an overlapping site on G1, exhibited a greater affinity as compared with HNF-3α or -β. We show that both HNF-3β and -α can transactivate glucagon gene transcription through the G2 and G1 elements. However, HNF-3 via its transactivating domains specifically impaired Pax6-mediated transactivation of the glucagon promoter but had no effect on transactivation by Cdx2/3. We suggest that HNF-3 may play a dual role on glucagon gene transcription by 1) inhibiting the transactivation potential of Pax6 on the G1 and G3 elements and 2) direct activation through G1 and G2.

Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

The yeast HIS3 promoter region contains two functionally distinct TATA elements, TC and TR, that are responsible respectively for initiation from the +1 and +13 sites. Both TC and TR support basal HIS3 transcription and require the TATA binding protein TFIID, but only TR responds to transcriptional activation by GCN4 and GAL4. By selecting for yeast strains that increase transcription by a GCN4 derivative with a defective activation domain, we have isolated a temperature-sensitive mutation in CDC39, a previously defined gene implicated in cell-cycle control and the pheromone response. This cdc39-2 mutation causes increased basal transcription of many, but not all genes, as well as increased transcriptional activation by GCN4 and GAL4. Surprisingly, basal HIS3 transcription from the +1 initiation site is strongly increased, while initiation from the +13 site is barely affected. Thus, unlike acidic activator proteins that function through TR, CDC39 preferentially affects transcription mediated by TC. CDC39 is an essential gene that encodes a very large nuclear protein (2108 amino acids) containing two glutamine-rich regions. These observations suggest that CDC39 negatively regulates transcription either by affecting the general RNA polymerase II machinery or by altering chromatin structure.