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The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei. [ABSTRACT FROM AUTHOR]

Previous studies have established that the Snf2h-containing chromatin remodeling complex NoRC mediates epigenetic silencing of a subset of rRNA genes (rDNA) by recruiting enzymatic activities that modify histones and methylate DNA. Here we have analyzed nucleosome positions at the murine rDNA promoter and show that active and silent rDNA copies are characterized not only by specific epigenetic marks but also by differently positioned nucleosomes. At active genes the promoter-bound nucleosome covers nucleotides from −157 to −2, whereas at silent genes the nucleosome is positioned 25 nucleotides further downstream. We provide evidence that NoRC is the molecular machine that shifts the promoter-bound nucleosome downstream of the transcription start site into a translational position that is unfavorable for transcription complex formation. [ABSTRACT FROM AUTHOR]

DNA packaged into chromatin is the physiologically relevant substrate for all DNA-dependent processes inside the nuclei of eukaryotic cells. The highly compacted structures of DNA and histones is inherently repressive for all DNA-dependent processes. Active mechanisms exist to generate dynamic chromatin, which maintains two apparently contradictory functions: tight compaction and free accessibility of DNA. The dynamics of chromatin higher order folding and the influence of chromatin structure on transcription will be described in this paper. Chromatin dynamics and higher order folding may be the key regulators of not only transcription but of all DNA-dependent processes in the nucleus. [ABSTRACT FROM AUTHOR]

Nucleosome remodelling complexes CHRAC and ACF contribute to chromatin dynamics by converting chemical energy into sliding of histone octamers on DNA. Their shared ATPase subunit ISWI binds DNA at the sites of entry into the nucleosome. A prevalent model assumes that DNA distortions catatysed by ISWI are converted into relocation of DNA relative to a histone octamer. HMGB1, one of the most abundant nuclear non-histone proteins, binds with preference to distorted DNA. We have now found that transient interaction of HMGB1 with nucteosomal tinker DNA overlapping ISWI-binding sites enhances the ability of ACE to bind nucteosomal DNA and accelerates the sliding activity of limiting concentrations of remodelling factor. By contrast, an HMGB1 mutant with increased binding affinity was inhibitory. These observations are consistent with a rote for HMGB1 as a DNA chaperone facilitating the rate-limiting DNA distortion during nucleosome remodelling. [ABSTRACT FROM AUTHOR]

Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. We have tested a panel of dMi-2 deletion mutants in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to dHP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. Our results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein-DNA interactions. Furthermore, we identify DNA binding as a novel chromodomain-associated activity. [ABSTRACT FROM AUTHOR]

Transcription by RNA potymerase I on nucteosomat templates requires binding of the transcription termination factor TTF-I to a cognate site 160 bp upstream of the transcription start site. Binding of TTF-I is accompanied by changes in the chromatin architecture which suggests that TTF-I recruits a remodeling activity to the rDNA promoter. We have cloned a cDNA that encodes TIPS (TTF-I-interacting protein 5), a 205 kDa protein that shares a number of important protein domains with WSTF (Williams syndrome transcription factor) and hAcf1/WCRF180, the largest subunits of human chromatin remodeling complexes hCHRAC and WCRF. TIPS co-localizes with the basal RNA polymerase I transcription factor UBF in the nucleolus and is associated with SNF2h. The cellular TIP5-SNF2h complex, termed NoRC (nucleotar remodeling complex), induces nucteosome sliding in an ATP- and histone H4 tail-dependent fashion. The results suggest that NoRC is a novel nucleotar chromatin remodeling machine that may serve a rote in the regulation of the rDNA locus. [ABSTRACT FROM AUTHOR]

The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP‐dependent 'nucleosome remodelling' activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. We have now identified the p175 subunit of CHRAC as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI qualitatively by altering the directionality of nucleosome movements and the histone 'tail' requirements of the reaction. The Acf1–ISWI heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC‐14 and CHRAC‐16. Whether topoisomerase II is an integral subunit has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be molecularly defined as a complex consisting of ISWI, Acf1, CHRAC‐14 and CHRAC‐16. [ABSTRACT FROM AUTHOR]

Mi-2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP-dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. We have compared the remodelling activity of recombinant Drosophila Mi-2 (dMi-2) with that of recombinant ISWI. Both proteins are nucleosome­stimulated ATPases and promote nucleosome mobilization. However, dMi-2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. We have used antibodies to immobilize a complex containing dMi-2 and the dRPD3 histone deacetylase from Drosophila embryo extracts. This complex shares the nucleosome­stimulated ATPase and nucleosome mobilization properties of recombinant dMi-2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish dMi-2 from both SWI2/SNF2 and ISWI, defining a new family of ATP-dependent remodelling machines. [ABSTRACT FROM AUTHOR]

Transcription of ribosomal genes assembled into chromatin requires binding of the transcription termination factor TTF-I to the promoter-proximal terminator T0. To analyze the mechanism of TTFI-mediated transcriptional activation, we have used mutant templates with altered sequence, polarity and distance of T0 with respect to the transcription start site. Transcription activation by TTF-I is chromatin specific and requires the precise positioning of the terminator relative to the promoter. Whereas termination by TTF-I depends on the correct orientation of a terminator, TTF-I-mediated transcriptional activation is orientation independent. TTF-I can bind to nucleosomal DNA in the absence of enzymatic activities that destabilize nucleosome structure. Chromatin-bound TTF-I synergizes with ATP-dependent cofactors present in extracts of Drosophila embryos and mouse cells to position a nucleosome over the rDNA promoter and the transcription start site. Nucleosome positioning correlates tightly with the activation of rDNA transcription. We suggest that transcriptional activation by TTF-I is a stepwise process involving the creation of a defined promoter architecture and that the positioning of a nucleosome is compatible with, if not a prerequisite for, transcription initiation from rDNA chromatin. [ABSTRACT FROM AUTHOR]

Eukaryotic ribosomal gene promoters are preceded by a terminator element which is recognized by the transcription termination factor TTF-I. We have studied the function of this promoter-proximal terminator and show that binding of TTF-I is the key event which leads to ATP-dependent nucleosome remodeling and transcriptional activation of mouse rDNA pre-assembled into chromatin. We have analyzed TTF-I mutants for their ability to bind to free or nucleosomal DNA, and show that the DNA binding domain of TTF-I on its own is not sufficient for interaction with chromatin, indicating that specific protein features exist that endow a transcription factor with chromatin binding and remodeling properties. This first analysis of RNA polymerase I transcription in chromatin provides a clue for the function of the upstream terminator and establishes a dual role for TTF-I both as a termination factor and a chromatin-specific transcription activator. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies. [ABSTRACT FROM AUTHOR]

Summary: Transcription and export (TREX) is a multi‐subunit complex that links synthesis, processing and export of mRNAs. It interacts with the RNA helicase UAP56 and export factors such as MOS11 and ALYs to facilitate nucleocytosolic transport of mRNAs. Plant MOS11 is a conserved, but sparsely researched RNA‐binding export factor, related to yeast Tho1 and mammalian CIP29/SARNP.Using biochemical approaches, the domains of Arabidopsis thaliana MOS11 required for interaction with UAP56 and RNA‐binding were identified. Further analyses revealed marked genetic interactions between MOS11 and ALY genes. Cell fractionation in combination with transcript profiling demonstrated that MOS11 is required for export of a subset of mRNAs that are shorter and more GC‐rich than MOS11‐independent transcripts.The central α‐helical domain of MOS11 proved essential for physical interaction with UAP56 and for RNA‐binding. MOS11 is involved in the nucleocytosolic transport of mRNAs that are upregulated under stress conditions and accordingly mos11 mutant plants turned out to be sensitive to elevated NaCl concentrations and heat stress.Collectively, our analyses identify functional interaction domains of MOS11. In addition, the results establish that mRNA export is critically involved in the plant response to stress conditions and that MOS11 plays a prominent role at this. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) have been shown to modulate gene expression and are involved in the initiation and progression of various cancer types. Despite the wealth of studies describing transcriptome changes upon lncRNA knockdown, there is limited information describing lncRNA-mediated effects on regulatory elements (REs) modulating gene expression. In this study, we investigated how the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA regulates primary target genes using time-resolved MALAT1 knockdown followed by parallel RNA-seq and ATAC-seq assays. The results revealed that MALAT1 primarily regulates specific protein-coding genes and a substantial decrease in the accessibility downstream of the NR4A1 gene that was associated with a decreased NR4A1 expression. Moreover, the presence of an NR4A1-downstream RE was demonstrated by CRISPR-i assays to define a functional MALAT1/NR4A1 axis. By analyzing TCGA data, we identified a positive correlation between NR4A1 expression and NR4A1-downstream RE accessibility in breast cancer but not in pancreatic cancer. Accordingly, this regulatory mechanism was experimentally validated in breast cancer cells (MCF7) but not in pancreatic duct epithelial carcinoma (PANC1) cells. Therefore, our results demonstrated that MALAT1 is involved in a molecular mechanism that fine-tunes NR4A1 expression by modulating the accessibility of a downstream RE in a cell type-specific manner. [ABSTRACT FROM AUTHOR]

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis (Arabidopsis thaliana). To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize (Zea mays). We show that Clade IB ZmRALF2/3 mutations, but not Clade III ZmRALF1/5 mutations, cause cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located in the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution patterns leading to altered cell wall organization and thickness culminating in pollen tube burst. Clade IB, but not Clade III ZmRALFs, strongly interact as ligands with the pollen-specific Catharanthus roseus RLK1-like (CrRLK1L) receptor kinases Z. mays FERONIA-like (ZmFERL) 4/7/9, LORELEI-like glycosylphosphatidylinositol-anchor (LLG) proteins Z. mays LLG 1 and 2 (ZmLLG1/2), and Z. mays pollen extension-like (PEX) cell wall proteins ZmPEX2/4. Notably, ZmFERL4 outcompetes ZmLLG2 and ZmPEX2 outcompetes ZmFERL4 for ZmRALF2 binding. Based on these data, we suggest that Clade IB RALFs act in a dual role as cell wall components and extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in maize and probably other plants. Pollen-specific RAPID ALKALINIZATION FACTORs interact with receptor kinases, GPI-anchored proteins, and cell wall proteins to regulate cell wall integrity during pollen tube growth in maize. [ABSTRACT FROM AUTHOR]

Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is the first key step of ribosome biogenesis. While the molecular mechanisms of rRNA transcription regulation have been elucidated in great detail, the functional organization of the multicopy rRNA gene clusters (rDNA) in the nucleolus is less well understood. Here we apply super-resolution 3D structured illumination microscopy (3D-SIM) to investigate the spatial organization of transcriptionally competent active rDNA chromatin at size scales well below the diffraction limit by optical microscopy. We identify active rDNA chromatin units exhibiting uniformly ring-shaped conformations with diameters of ~240 nm in mouse and ~170 nm in human fibroblasts, consistent with rDNA looping. The active rDNA chromatin units are clearly separated from each other and from the surrounding areas of rRNA processing. Simultaneous imaging of all active genes bound by Pol I and the architectural chromatin protein Upstream Binding Transcription Factor (UBF) reveals a random spatial orientation of regular repeats of rDNA coding sequences within the nucleoli. These observations imply rDNA looping and exclude potential formation of systematic spatial assemblies of the well-ordered repetitive arrays of transcription units. Collectively, this study uncovers key features of the 3D organization of active rDNA chromatin units and their nucleolar clusters providing a spatial framework of nucleolar chromatin organization at unprecedented detail. [ABSTRACT FROM AUTHOR]