Arabidopsis mRNA export factor MOS11: molecular interactions and role in abiotic stress responses.

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.

Nrf2 pre-recruitment at Enhancer 2 is a hallmark of H2 O2 -induced epigenetic transcriptional memory in the HMOX1 gene in human umbilical artery endothelial cells.

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.

The RALF signaling pathway regulates cell wall integrity during pollen tube growth in maize.

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.

The chromatin - triple helix connection.

Mammalian genomes are extensively transcribed, producing a large number of coding and non-coding transcripts. A large fraction of the nuclear RNAs is physically associated with chromatin, functioning in gene activation and silencing, shaping higher-order genome organisation, such as involvement in long-range enhancer-promoter interactions, transcription hubs, heterochromatin, nuclear bodies and phase transitions. Different mechanisms allow the tethering of these chromatin-associated RNAs (caRNA) to chromosomes, including RNA binding proteins, the RNA polymerases and R-loops. In this review, we focus on the sequence-specific targeting of RNA to DNA by forming triple helical structures and describe its interplay with chromatin. It turns out that nucleosome positioning at triple helix target sites and the nucleosome itself are essential factors in determining the formation and stability of triple helices. The histone H3-tail plays a critical role in triple helix stabilisation, and the role of its epigenetic modifications in this process is discussed.

Characterization of the nuclear import of the human CHD4-NuRD complex.

Chromatin remodeling enzymes form large multiprotein complexes that play central roles in regulating access to the genome. Here, we characterize the nuclear import of the human CHD4 protein. We show that CHD4 enters the nucleus by means of several importin-α proteins (1, 5, 6 and 7), but independently of importin ß1. Importin α1 directly interacts with a monopartite 'KRKR'-motif in the N-terminus of CHD4 (amino acids 304-307). However, alanine mutagenesis of this motif only leads to an ∼50% reduction in nuclear localization of CHD4, implying that there are additional import mechanisms. Interestingly, we could show that CHD4 was already associated with the nucleosome remodeling deacetylase (NuRD) core subunits, such as MTA2, HDAC1 and RbAp46 (also known as RBBP7), in the cytoplasm, suggesting an assembly of the NuRD core complex before nuclear import. We propose that, in addition to the importin-α-dependent nuclear localization signal, CHD4 is dragged into the nucleus by a 'piggyback' mechanism using the import signals of the associated NuRD subunits.

Elongation factor 1 is a component of the Arabidopsis RNA polymerase II elongation complex and associates with a subset of transcribed genes.

Elongation factors modulate the efficiency of mRNA synthesis by RNA polymerase II (RNAPII) in the context of chromatin, thus contributing to implement proper gene expression programmes. The zinc-finger protein elongation factor 1 (ELF1) is a conserved transcript elongation factor (TEF), whose molecular function so far has not been studied in plants. Using biochemical approaches, we examined the interaction of Arabidopsis ELF1 with DNA and histones in vitro and with the RNAPII elongation complex in vivo. In addition, cytological assays demonstrated the nuclear localisation of the protein, and by means of double-mutant analyses, interplay with genes encoding other elongation factors was explored. The genome-wide distribution of ELF1 was addressed by chromatin immunoprecipitation. ELF1 isolated from Arabidopsis cells robustly copurified with RNAPII and various other elongation factors including SPT4-SPT5, SPT6, IWS1, FACT and PAF1C. Analysis of a CRISPR-Cas9-mediated gene editing mutant of ELF1 revealed distinct genetic interactions with mutants deficient in other elongation factors. Moreover, ELF1 associated with genomic regions actively transcribed by RNAPII. However, ELF1 occupied only c. 33% of the RNAPII transcribed loci with preference for inducible rather than constitutively expressed genes. Collectively, these results establish that Arabidopsis ELF1 shares several characteristic attributes with RNAPII TEFs.

Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro.

Nuclear eukaryotic RNA polymerases (RNAPs) transcribe a chromatin template in vivo. Since the basic unit of chromatin, the nucleosome, renders the DNA largely inaccessible, RNAPs have to overcome the nucleosomal barrier for efficient RNA synthesis. Gaining mechanistical insights in the transcription of chromatin templates will be essential to understand the complex process of eukaryotic gene expression. In this article we describe the use of defined in vitro transcription systems for comparative analysis of highly purified RNAPs I-III from S. cerevisiae (hereafter called yeast) transcribing in vitro reconstituted nucleosomal templates. We also provide a protocol to study promoter-dependent RNAP I transcription of purified native 35S ribosomal RNA (rRNA) gene chromatin.

Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae.

In archaea and bacteria the major classes of RNAs are synthesized by one DNA-dependent RNA polymerase (RNAP). In contrast, most eukaryotes have three highly specialized RNAPs to transcribe the nuclear genome. RNAP I synthesizes almost exclusively ribosomal (r)RNA, RNAP II synthesizes mRNA as well as many noncoding RNAs involved in RNA processing or RNA silencing pathways and RNAP III synthesizes mainly tRNA and 5S rRNA. This review discusses functional differences of the three nuclear core RNAPs in the yeast S. cerevisiae with a particular focus on RNAP I transcription of nucleolar ribosomal (r)DNA chromatin.

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Phosphorylation of the FACT histone chaperone subunit SPT16 affects chromatin at RNA polymerase II transcriptional start sites in Arabidopsis.

The heterodimeric histone chaperone FACT, consisting of SSRP1 and SPT16, contributes to dynamic nucleosome rearrangements during various DNA-dependent processes including transcription. In search of post-translational modifications that may regulate the activity of FACT, SSRP1 and SPT16 were isolated from Arabidopsis cells and analysed by mass spectrometry. Four acetylated lysine residues could be mapped within the basic C-terminal region of SSRP1, while three phosphorylated serine/threonine residues were identified in the acidic C-terminal region of SPT16. Mutational analysis of the SSRP1 acetylation sites revealed only mild effects. However, phosphorylation of SPT16 that is catalysed by protein kinase CK2, modulates histone interactions. A non-phosphorylatable version of SPT16 displayed reduced histone binding and proved inactive in complementing the growth and developmental phenotypes of spt16 mutant plants. In plants expressing the non-phosphorylatable SPT16 version we detected at a subset of genes enrichment of histone H3 directly upstream of RNA polymerase II transcriptional start sites (TSSs) in a region that usually is nucleosome-depleted. This suggests that some genes require phosphorylation of the SPT16 acidic region for establishing the correct nucleosome occupancy at the TSS of active genes.

Peculiarities of Plasmodium falciparum Gene Regulation and Chromatin Structure.

The highly complex life cycle of the human malaria parasite, Plasmodium falciparum, is based on an orchestrated and tightly regulated gene expression program. In general, eukaryotic transcription regulation is determined by a combination of sequence-specific transcription factors binding to regulatory DNA elements and the packaging of DNA into chromatin as an additional layer. The accessibility of regulatory DNA elements is controlled by the nucleosome occupancy and changes of their positions by an active process called nucleosome remodeling. These epigenetic mechanisms are poorly explored in P. falciparum. The parasite genome is characterized by an extraordinarily high AT-content and the distinct architecture of functional elements, and chromatin-related proteins also exhibit high sequence divergence compared to other eukaryotes. Together with the distinct biochemical properties of nucleosomes, these features suggest substantial differences in chromatin-dependent regulation. Here, we highlight the peculiarities of epigenetic mechanisms in P. falciparum, addressing chromatin structure and dynamics with respect to their impact on transcriptional control. We focus on the specialized chromatin remodeling enzymes and discuss their essential function in P. falciparum gene regulation.

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology.

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.

Sequence and functional differences in the ATPase domains of CHD3 and SNF2H promise potential for selective regulability and drugability.

Chromatin remodelers use the energy of ATP hydrolysis to regulate chromatin dynamics. Their impact for development and disease requires strict enzymatic control. Here, we address the differential regulability of the ATPase domain of hSNF2H and hCHD3, exhibiting similar substrate affinities and enzymatic activities. Both enzymes are comparably strongly inhibited in their ATP hydrolysis activity by the competitive ATPase inhibitor ADP. However, the nucleosome remodeling activity of SNF2H is more strongly affected than that of CHD3. Beside ADP, also IP6 inhibits the nucleosome translocation of both enzymes to varying degrees, following a competitive inhibition mode at CHD3, but not at SNF2H. Our observations are further substantiated by mutating conserved Q- and K-residues of ATPase domain motifs. The variants still bind both substrates and exhibit a wild-type similar, basal ATP hydrolysis. Apart from three CHD3 variants, none of the variants can translocate nucleosomes, suggesting for the first time that the basal ATPase activity of CHD3 is sufficient for nucleosome remodeling. Together with the ADP data, our results propose a more efficient coupling of ATP hydrolysis and remodeling in CHD3. This aspect correlates with findings that CHD3 nucleosome translocation is visible at much lower ATP concentrations than SNF2H. We propose sequence differences between the ATPase domains of both enzymes as an explanation for the functional differences and suggest that aa interactions, including the conserved Q- and K-residues distinctly regulate ATPase-dependent functions of both proteins. Our data emphasize the benefits of remodeler ATPase domains for selective drugability and/or regulability of chromatin dynamics.

ARMADILLO REPEAT ONLY proteins confine Rho GTPase signalling to polar growth sites.

Polar growth requires the precise tuning of Rho GTPase signalling at distinct plasma membrane domains. The activity of Rho of plant (ROP) GTPases is regulated by the opposing action of guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Whereas plant-specific ROPGEFs have been shown to be embedded in higher-level regulatory mechanisms involving membrane-bound receptor-like kinases, the regulation of GAPs has remained enigmatic. Here, we show that three Arabidopsis ARMADILLO REPEAT ONLY (ARO) proteins are essential for the stabilization of growth sites in root hair cells and trichomes. AROs interact with ROP1 enhancer GAPs (RENGAPs) and bind to the plasma membrane via a conserved polybasic region at the ARO amino terminus. The ectopic spreading of ROP2 in aro2/3/4 mutant root hair cells and the preferential interaction of AROs with active ROPs and anionic phospholipids suggests that AROs recruit RENGAPs into complexes with ROPs to confine ROP signalling to distinct membrane regions.

Analyzing RNA-DNA Triplex Formation in Chromatin.

A significant fraction of non-coding RNAs (ncRNAs) is associated with chromatin, shown to regulate gene expression and to organize nuclear architecture. Mechanisms of direct and indirect RNA-chromatin interactions have been described, including the sequence-specific formation of triple helix structures. Triplexes are formed by the sequence-specific binding of RNA to the bases located in the major groove of DNA. We recently showed that triplexes do exist in the context of cellular chromatin and that these structures are stabilized by the histone H3 tail of adjacent nucleosomes. The in vitro characterization of the specificity and binding affinity of triplex sequences next to nucleosomes are essential parameters to identify potential sites of RNA-chromatin interaction in vivo. Here we provide a detailed protocol to determine the influence of nucleosome positioning on triple helix formation. This assay allows the comparative quantification of triplex formation and specificity for triplex targeting sequences relative to the spatial nucleosome position.

Super-resolution in situ analysis of active ribosomal DNA chromatin organization in the nucleolus.

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.

RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure.

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I-specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

Author Correction: Mechanisms governing the pioneering and redistribution capabilities of the non-classical pioneer PU.1.

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

Mechanisms governing the pioneering and redistribution capabilities of the non-classical pioneer PU.1.

Establishing gene regulatory networks during differentiation or reprogramming requires master or pioneer transcription factors (TFs) such as PU.1, a prototype master TF of hematopoietic lineage differentiation. To systematically determine molecular features that control its activity, here we analyze DNA-binding in vitro and genome-wide in vivo across different cell types with native or ectopic PU.1 expression. Although PU.1, in contrast to classical pioneer factors, is unable to access nucleosomal target sites in vitro, ectopic induction of PU.1 leads to the extensive remodeling of chromatin and redistribution of partner TFs. De novo chromatin access, stable binding, and redistribution of partner TFs both require PU.1's N-terminal acidic activation domain and its ability to recruit SWI/SNF remodeling complexes, suggesting that the latter may collect and distribute co-associated TFs in conjunction with the non-classical pioneer TF PU.1.