Multilayered Regulatory Network Research Articles

Engineered Protease-Responsive RNA-Binding Proteins (RBPs) to Expand the Toolbox of Synthetic Circuits in Mammalian Cells.

Genetically encoded sensor-actuator circuits aim at reprogramming cellular functions and are inspired by intracellular networks: from the input signal (sensor) to the desired output response (actuator). In the last years, circuits with posttranscriptional regulation of gene expression have aroused great interest for their potential in the biomedical space. Posttranscriptional modulation can be achieved with ribozymes, riboswitches (simple regulatory elements based on RNA secondary structures), noncoding RNAs, and RNA-binding proteins (RBPs). RBPs are proteins that recognize specific motifs on the mRNA target inducing mRNA decay or translation inhibition. The use of RBPs deriving from different species in mammalian cells has allowed to create sophisticated and multilayered regulatory networks, addressing the previous limitation of regulatory orthogonal parts that can be assembled in synthetic devices. In this chapter, we describe the engineering and tests of protease-responsive RNA-binding proteins (L7Ae and MS2-cNOT7) to expand the toolbox of synthetic circuits in mammalian cells.

The Genome-Wide Characterization of Alternative Splicing and RNA Editing in the Development of Coprinopsis cinerea.

Coprinopsis cinerea is one of the model species used in fungal developmental studies. This mushroom-forming Basidiomycetes fungus has several developmental destinies in response to changing environments, with dynamic developmental regulations of the organism. Although the gene expression in C. cinerea development has already been profiled broadly, previous studies have only focused on a specific stage or process of fungal development. A comprehensive perspective across different developmental paths is lacking, and a global view on the dynamic transcriptional regulations in the life cycle and the developmental paths is far from complete. In addition, knowledge on co- and post-transcriptional modifications in this fungus remains rare. In this study, we investigated the transcriptional changes and modifications in C. cinerea during the processes of spore germination, vegetative growth, oidiation, sclerotia formation, and fruiting body formation by inducing different developmental paths of the organism and profiling the transcriptomes using the high-throughput sequencing method. Transition in the identity and abundance of expressed genes drive the physiological and morphological alterations of the organism, including metabolism and multicellularity construction. Moreover, stage- and tissue-specific alternative splicing and RNA editing took place and functioned in C. cinerea. These modifications were negatively correlated to the conservation features of genes and could provide extra plasticity to the transcriptome during fungal development. We suggest that C. cinerea applies different molecular strategies in its developmental regulation, including shifts in expressed gene sets, diversifications of genetic information, and reversible diversifications of RNA molecules. Such features would increase the fungal adaptability in the rapidly changing environment, especially in the transition of developmental programs and the maintenance and balance of genetic and transcriptomic divergence. The multi-layer regulatory network of gene expression serves as the molecular basis of the functioning of developmental regulation.

Exogenous 6-BA inhibited hypocotyl elongation under darkness in Picea crassifolia Kom revealed by transcriptome profiling.

Hypocotyl elongation is an important process in plant growth and development, and is under hormonal regulatory signaling pathways. In our study, exogenous 6-BA significantly inhibited Picea crassifolia hypocotyl elongation more than ethylene in the dark, indicating the existence of different regulatory strategies in conifers, therefore, the P. crassifolia transcriptome was studied to explore the responsive genes and their regulatory pathways for exogenous N6-benzyladenine (6-BA) inhibition of hypocotyl elongation using RNA-Sequencing approach. We present the first transcriptome assembly of P. crassifolia obtained from 24.38 Gb clean data. With lowly-expressed and short contigs excluded, the assembly contains roughly 130,612 unigenes with an N50 length of 1,278 bp. Differential expression analysis found 3,629 differentially expressed genes (DEGs) and found that the differential expression fold of genes was mainly concentrated between 2 and 8 (1 ≤ log2FoldChange ≤ 3). Functional annotation showed that the GO term with the highest number of enriched genes (83 unigenes) was the shoot system development (GO: 0048367) and the KEGG category, plant hormone signal transduction (ko04075), was enriched 30 unigenes. Further analysis revealed that several cytokinin dehydrogenase genes (PcCTD1, PcCTD3 and PcCTD6) catabolized cytokinins, while xyloglucan endotransglucosylase hydrolase gene (PcXTH31), WALLS ARE THIN 1-like gene (PcWAT1-1) and Small auxin-induced gene (PcSAUR15) were strongly repressed thus synergistically completing the inhibition of hypocotyl elongation in P. crassifolia. Besides, PcbHLH149, PcMYB44 and PcERF14 were predicted to be potential core TFs that may form a multi-layered regulatory network with the above proteins for the regulation of hypocotyl growth.

Alternative Splicing in the Regulatory Circuit of Plant Temperature Response.

As sessile organisms, plants have evolved complex mechanisms to rapidly respond to ever-changing ambient temperatures. Temperature response in plants is modulated by a multilayer regulatory network, including transcriptional and post-transcriptional regulations. Alternative splicing (AS) is an essential post-transcriptional regulatory mechanism. Extensive studies have confirmed its key role in plant temperature response, from adjustment to diurnal and seasonal temperature changes to response to extreme temperatures, which has been well documented by previous reviews. As a key node in the temperature response regulatory network, AS can be modulated by various upstream regulations, such as chromatin modification, transcription rate, RNA binding proteins, RNA structure and RNA modifications. Meanwhile, a number of downstream mechanisms are affected by AS, such as nonsense-mediated mRNA decay (NMD) pathway, translation efficiency and production of different protein variants. In this review, we focus on the links between splicing regulation and other mechanisms in plant temperature response. Recent advances regarding how AS is regulated and the following consequences in gene functional modulation in plant temperature response will be discussed. Substantial evidence suggests that a multilayer regulatory network integrating AS in plant temperature response has been unveiled.

Emerging RUNX2-Mediated Gene Regulatory Mechanisms Consisting of Multi-Layered Regulatory Networks in Skeletal Development.

Skeletal development is tightly coordinated by chondrocytes and osteoblasts, which are derived from skeletal progenitors, and distinct cell-type gene regulatory programs underlie the specification and differentiation of cells. Runt-related transcription factor 2 (Runx2) is essential to chondrocyte hypertrophy and osteoblast differentiation. Genetic studies have revealed the biological functions of Runx2 and its involvement in skeletal genetic diseases. Meanwhile, molecular biology has provided a framework for our understanding of RUNX2-mediated transactivation at a limited number of cis-regulatory elements. Furthermore, studies using next-generation sequencing (NGS) have provided information on RUNX2-mediated gene regulation at the genome level and novel insights into the multiple layers of gene regulatory mechanisms, including the modes of action of RUNX2, chromatin accessibility, the concept of pioneer factors and phase separation, and three-dimensional chromatin organization. In this review, I summarize the emerging RUNX2-mediated regulatory mechanism from a multi-layer perspective and discuss future perspectives for applications in the treatment of skeletal diseases.

Identification of key genes and biological regulatory mechanisms in diabetic nephropathy: Meta-analysis of gene expression datasets

BackgroundDiabetic nephropathy (DN) which refers to the cases with biopsy proven kidney lesions, is one of the main complications of diabetes all around the world; however, the underlying biological changes causing DN remain to be understood. Studying the alterations in gene expression profiles could give a holistic view of the molecular pathogenicity of DN and aid to discover key molecules as potential therapeutic targets. Here, we performed a meta-analysis study that included microarray gene expression profiles coming from glomerular samples of DN patients in order to acquire a list of consensus Differentially Expressed Genes (meta-DEGs) correlated with DN. MethodsAfter quality control and normalization steps, five gene expression datasets (GES1009, GSE30528, GSE47183, GSE104948, and GSE93804) were entered into the meta-analysis. The meta-analysis was performed by random effect size method and the meta-DEGs were put through network analysis and different pathway enrichment analyses steps. MiRTarBase and TRRUST databases were utilized to predict the meta-DEGs related miRNAs and transcription factors. A co-regulatory network including DEGs, transcription factors and miRNAs was constructed by Cytoscape, and top molecules were identified based on centrality scores in the network. ResultsThe identified meta-DEGs were 1364 DEGs including 665 downregulated and 669 upregulated DEGs. The results of pathway enrichment analysis showed, “immune system”, “extracellular matrix organization”, “hemostasis”, “signal transduction”, and “platelet activation” to be the top enriched terms with involvement of the meta-DEGs. After construction of the multilayer regulatory network, several top DEGs (TP53, MYC, BTG2, VEGFA, PTEN, etc.), as well as top miRNAs (miR-335, miR-16, miR-17, miR-20a, and miR-93), and transcription factors (SP1, STAT3, NF-KB1, RELA, E2F1), were introduced as potential therapeutic targets in DN. Among the regulatory molecules, miR-335-5p and SP1 were the most interactive miRNA and transcription factor molecules with the highest degree scores in the constructed network. ConclusionBy performing a meta-analysis of available DN-related transcriptomics datasets, we reached a consensus list of DEGs for this complicated disorder. Further enrichment and network analyses steps revealed the involved pathways in the DN pathogenesis and marked the most potential therapeutic targets in this disease.

Identification of key genes and biological regulatory mechanisms in diabetic nephropathy: Meta-analysis of gene expression datasets

BackgroundDiabetic nephropathy (DN) which refers to the cases with biopsy proven kidney lesions, is one of the main complications of diabetes all around the world; however, the underlying biological changes causing DN remain to be understood. Studying the alterations in gene expression profiles could give a holistic view of the molecular pathogenicity of DN and aid to discover key molecules as potential therapeutic targets. Here, we performed a meta-analysis study that included microarray gene expression profiles coming from glomerular samples of DN patients in order to acquire a list of consensus Differentially Expressed Genes (meta-DEGs) correlated with DN. MethodsAfter quality control and normalization steps, five gene expression datasets (GES1009, GSE30528, GSE47183, GSE104948, and GSE93804) were entered into the meta-analysis. The meta-analysis was performed by random effect size method and the meta-DEGs were put through network analysis and different pathway enrichment analyses steps. MiRTarBase and TRRUST databases were utilized to predict the meta-DEGs related miRNAs and transcription factors. A co-regulatory network including DEGs, transcription factors and miRNAs was constructed by Cytoscape, and top molecules were identified based on centrality scores in the network. ResultsThe identified meta-DEGs were 1364 DEGs including 665 downregulated and 669 upregulated DEGs. The results of pathway enrichment analysis showed, “immune system”, “extracellular matrix organization”, “hemostasis”, “signal transduction”, and “platelet activation” to be the top enriched terms with involvement of the meta-DEGs. After construction of the multilayer regulatory network, several top DEGs (TP53, MYC, BTG2, VEGFA, PTEN, etc.), as well as top miRNAs (miR-335, miR-16, miR-17, miR-20a, and miR-93), and transcription factors (SP1, STAT3, NF-KB1, RELA, E2F1), were introduced as potential therapeutic targets in DN. Among the regulatory molecules, miR-335-5p and SP1 were the most interactive miRNA and transcription factor molecules with the highest degree scores in the constructed network. ConclusionBy performing a meta-analysis of available DN-related transcriptomics datasets, we reached a consensus list of DEGs for this complicated disorder. Further enrichment and network analyses steps revealed the involved pathways in the DN pathogenesis and marked the most potential therapeutic targets in this disease.

Single-cell transcriptome highlights a multilayer regulatory network on an invasive trajectory within colorectal cancer progression

PurposeColorectal cancer (CRC) is one of the most common and fatal gastrointestinal malignancies, in which cancer stem cells (CSCs) were identified to enable tumor heterogeneity and initiate tumor formation. However, the process from CSCs to invasion cells is unconfirmed.MethodsSeveral bioinformatics methods, including clustering, pseudotime analysis, gene set variation analysis and gene ontology enrichment, were used to construct a path of gradual transformation of CSCs to invasive cells, called “stem-to-invasion path”. A large amount of signaling interactions were collated to build the multilayer regulatory network. Kaplan–Meier curve and time-dependent ROC method were applied to reveal prognostic values.ResultsWe validated the heterogeneity of cells in the tumor microenvironment and revealed the presence of malignant epithelial cells with high invasive potential within primary colonic carcinomas. Next, the “stem-to-invasion path” was identified through constructing a branching trajectory with cancer cells arranged in order. A multilayer regulatory network considered as the vital factor involved in acquiring invasion characteristics underlying the path was built to elucidate the interactions between tumor cell and tumor-associated microenvironment. Then we further identified a novel combinatorial biomarker that can be used to assess the prognosis for CRC patients, and validated its predictive robustness on the independent dataset.ConclusionOur work provides new insights into the acquisition of invasive potential in primary tumor cells, as well as potential therapeutic targets for CRC invasiveness, which may be useful for the cancer research and clinical treatment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00432-022-04020-2.

Grape Transcriptome Response to Powdery Mildew Infection: Comparative Transcriptome Profiling of Chinese Wild Grapes Provides Insights Into Powdery Mildew Resistance.

Erysiphe necator, the fungal pathogen of grape powdery mildew disease, poses a great threat to the grape market and the wine industry. To better understand the molecular basis of grape responses to E. necator, we performed comparative transcriptome profiling on two Chinese wild grape accessions with varying degrees of resistance to E. necator. At 6-, 24-, and 96-h postinoculation of E. necator, 2,856, 2,678, and 1,542 differentially expressed genes (DEGs) were identified in the susceptible accession Vitis pseudoreticulata 'Hunan-1', and at those same time points, 1,921, 2,498, and 3,249 DEGs, respectively, were identified in the resistant accession V. quinquangularis 'Shang-24'. 'Hunan-1' had a substantially larger fraction of down-regulated genes than 'Shang-24' at every infection stage. Analysis of DEGs revealed that up-regulated genes were mostly associated with defense response and disease resistance-related metabolite biosynthesis, and such signaling genes were significantly suppressed in 'Hunan-1'. Interestingly, fatty acid biosynthesis- and elongation-related genes were suppressed by the fungus in the 'Shang-24' accession but somehow induced in the 'Hunan-1' accession, consistent with the concept that E. necator is likely to be a fatty acid auxotroph that requires lipids from the host. Moreover, genes involved in biosynthesis and signaling of phytohormones, such as jasmonic acid and cytokinin, as well as genes encoding protein kinases and nucleotide-binding domain leucine-rich repeat proteins, differentially responded to E. necator in the two wild grapes. The variation of gene regulation associated with nutrient uptake by the fungus and with signaling transduction and pathogen recognition suggests a multilayered regulatory network that works in concert to assist in the establishment of fungal pathogen infections.

Comprehensive analysis of IgA nephropathy expression profiles: identification of potential biomarkers and therapeutic agents

BackgroundIgA nephropathy (IgAN) is a kidney disease recognized by the presence of IgA antibody depositions in kidneys. The underlying mechanisms of this complicated disease are remained to be explored and still, there is an urgent need for the discovery of noninvasive biomarkers for its diagnosis. In this investigation, an integrative approach was applied to mRNA and miRNA expression profiles in PBMCs to discover a gene signature and novel potential targets/biomarkers in IgAN.MethodsDatasets were selected from gene expression omnibus database. After quality control checking, two datasets were analyzed by Limma to identify differentially expressed genes/miRNAs (DEGs and DEmiRs). Following identification of DEmiR-target genes and data integration, intersecting mRNAs were subjected to different bioinformatic analyses. The intersecting mRNAs, DEmiRs, related transcription factors (from TRRUST database), and long-non coding RNAs (from LncTarD database) were used for the construction of a multilayer regulatory network via Cytoscape.Result“GSE25590” (miRNA) and “GSE73953” (mRNA) datasets were analyzed and after integration, 628 intersecting mRNAs were identified. The mRNAs were mainly associated with “Innate immune system”, “Apoptosis”, as well as “NGF signaling” pathways. A multilayer regulatory network was constructed and several hub-DEGs (Tp53, STAT3, Jun, etc.), DEmiRs (miR-124, let-7b, etc.), TFs (NF-kB, etc.), and lncRNAs (HOTAIR, etc.) were introduced as potential factors in the pathogenesis of IgAN.ConclusionIntegration of two different expression datasets and construction of a multilayer regulatory network not only provided a deeper insight into the pathogenesis of IgAN, but also introduced several key molecules as potential therapeutic target/non-invasive biomarkers.

Jasmonate- and abscisic acid-activated AaGSW1-AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua.

SummaryArtemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual‐responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA‐induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA‐AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA‐responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1‐AaTCP15/AaORA regulatory module to integrate JA and ABA‐mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1‐AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants.

Recent insights into the regulatory networks of NLRP3 inflammasome activation.

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a fascinating cellular machinery endowed with the capacity for rapid proteolytic processing of the pro-inflammatory cytokine IL-1β and the cell death effector gasdermin D (GSDMD). Although its activity is essential to fight infection and support tissue homeostasis, the inflammasome complex, which consists of the danger sensor NLRP3, the adaptor apoptosis-associated speck-like protein containing a CARD (ASC; also known as PYCARD), caspase-1 and probably other regulatory proteins, also bears considerable potential for detrimental inflammation, as observed in human conditions such as gout, heart attack, stroke and Alzheimer's disease. Thus, multi-layered regulatory networks are required to ensure the fine balance between rapid responsiveness versus erroneous activation (sufficient and temporally restricted versus excessive and chronic activity) of the inflammasome. These involve multiple activation, secretion and cell death pathways, as well as modulation of the subcellular localization of NLRP3, and its structure and activity, owing to post-translational modification by other cellular proteins. Here, we discuss the exciting progress that has recently been made in deciphering the regulation of the NLRP3 inflammasome. Additionally, we highlight open questions and describe areas of research that warrant further exploration to obtain a more comprehensive molecular and cellular understanding of the NLRP3 inflammasome.

Identification of rice genotypes for seedling stage multiple abiotic stress tolerance

Under changing climatic conditions, crops are expected to experience combination of abiotic stresses rather than challenged by a single abiotic stress and hence, breeding for specific abiotic stress tolerance is unavailing. Exposure of crops to abiotic stress during seedling stage leads to poor crop establishment due to early death of seedlings. Hence, tolerance to multiple abiotic stress at seedling stage is imperative for breeding crops. Therefore, there is a need to identify donors for multiple abiotic stress tolerance in rice. Our study aimed at identifying rice genotypes which has multiple abiotic stress tolerance particularly for salinity, drought and heat stress, the most common abiotic stresses under tropical condition. We hypothesis that since abiotic stress tolerance mechanism involves multi-layered regulatory networks, identifying genotypes conferring tolerance through overlapping mechanisms across different abiotic stress might be a multiple abiotic stress tolerant genotype. An important adaptive mechanism for stress tolerance is high root to shoot ratio and this physiological trait has been used as a criterion for tolerance. Twelve rice genotypes were grown under induced water deficit, salinity and high temperature conditions and seedling growth parameters were recorded. Root shoot ratio on length basis was derived from the recorded parameters and the data was normalized by Z distribution and plotted using scatter plot across stresses. KR15058 and KR15007 were identified as the seedling stage multiple abiotic stress tolerant lines by scatter plot analysis. These lines are promising source for the crop improvement programme for developing a multiple stress tolerant genotype with superior agronomical background.

Long noncoding RNA lnc-NAP sponges mmu-miR-139-5p to modulate Nanog functions in mouse ESCs and embryos

ABSTRACT The pluripotency of embryonic stem cells (ESCs) is controlled by a multilayer regulatory network, of which the key factors include core pluripotency genes Oct4, Sox2 and Nanog, and multiple microRNAs (miRNAs). Recently, long noncoding RNAs (lncRNAs) have been discovered as a class of new regulators for ESCs, and some lncRNAs could function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we identify mmu-miR-139-5p as a new regulator for Nanog by targeting Nanog 3′ untranslated region (UTR) to repress Nanog expression in mouse ESCs and embryos. Such regulation could be released by an ESC-specifically expressed ceRNA named lnc-NAP. The expression of lnc-NAP is activated by OCT4, SOX2, as well as NANOG through promoter binding. Downregulation of lnc-NAP reduces Nanog abundance, which leads to decreased pluripotency of mouse ESCs and embryonic lethality. These results reveal lnc-NAP as a new regulator for Nanog in mouse ESCs, and uncover a feed-forward regulatory loop of Nanog through the participation of lnc-NAP.

Multilayered Control of Protein Turnover by TORC1 and Atg1

The target of rapamycin complex 1 (TORC1) is a master regulator of cell homeostasis, which promotes anabolic reactions and synchronously inhibits catabolic processes such as autophagy-mediated protein degradation. Its prime autophagy target is Atg13, a subunit of the Atg1 kinase complex that acts as the gatekeeper of canonical autophagy. To study whether the activities of TORC1 and Atg1 are coupled through additional, more intricate control mechanisms than simply this linear pathway, we analyzed the epistatic relationship between TORC1 and Atg1 by using quantitative phosphoproteomics. Our invivo data, combined with targeted invitro TORC1 and Atg1 kinase assays, not only uncover numerous TORC1 and Atg1 effectors, but also suggest distinct bi-directional regulatory feedback loops and characterize Atg29 as a commonly regulated downstream target of both TORC1 and Atg1. Thus, an exquisitely multilayered regulatory network appears to coordinate TORC1 and Atg1 activities to robustly tune autophagy in response to nutritional cues.

Gene Coexpression Networks Reveal Key Drivers of Flavonoid Variation in Eleven Tea Cultivars (Camellia sinensis).

Following the recent completion of the draft genome sequence of the tea plant, high-throughput decoding of gene function, especially for those involved in complex secondary metabolic pathways, has become a major challenge. Here, we profiled the metabolome and transcriptome of 11 tea cultivars, and then illustrated a weighted gene coexpression network analysis (WGCNA)-based system biological strategy to interpret metabolomic flux, predict gene functions, and mine key regulators involved in the flavonoid biosynthesis pathway. We constructed a multilayered regulatory network, which integrated the gene coexpression relationship with the microRNA target and promoter cis-regulatory element information. This allowed us to reveal new uncharacterized TFs (e.g., MADSs, WRKYs, and SBPs) and microRNAs (including 17 conserved and 15 novel microRNAs) that are potentially implicated in different steps of the catechin biosynthesis. Furthermore, we applied metabolic-signature-based association method to capture additional key regulators involved in catechin pathway. This provides important clues for the functional characterization of five SCPL1A acyltransferase family members, which might be implicated in the production balance of anthocyanins, galloylated catechins, and proanthocyanins. Application of an "omics"-based system biology strategy should facilitate germplasm utilization and provide valuable resources for tea quality improvement.

Physiological and biochemical aspects of the functioning of Arg-X proteolytic system in the cell nuclei of plants

Plants are structural and biochemical systems in which at each moment of ontogenesis are established inherent only to this plant, the ratio of growth, biorhythmic and morphogenetic processes. The conjugacy of circadian rhythm and the activity of chromatin-modifying enzymes is becoming increasingly important. In this aspect, histones play an important role, since they are able to fine-tune transcriptional programs through a series of accurately organized post-translational modifications that combinatorially regulate chromatin function. It is proved that almost all histones are proteolytically modified. It was shown that differences in the exhibition of activity Arg-X proteolysis between spring and winter seedlings are observed mainly in the fractions of core histones at the level of chromatin tightly bound. The preferential localization of Arg-X protease activity was found in the histone (H3 + H4) block as spring and winter varieties. In addition, activity Arg-X proteolysis at the level of nuclear matrix correlated with the growth characteristics of spring and winter seedlings in coleoptiles and roots growing by cell stretching. Results can be viewed as an illustration of a multi-layered regulatory proteinase network involved in proteolytic processing of nuclear proteins, taking part in circadian spatial and temporal structural changes of chromatin states.

Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function.

Porphyromonas gingivalis, a keystone pathogen of chronic periodontitis, uses ferric uptake regulator homolog (PgFur) to regulate production of virulence factors. This study aimed to characterize PgFur protein in regard to its structure-function relationship. We experimentally identified the 5′ mRNA sequence encoding the 171-amino-acid-long PgFur protein in the A7436 strain and examined this PgFur version as a full-length protein. PgFur protein did not bind to the canonical Escherichia coli Fur box, but the wild-type phenotype of the mutant Δpgfur strain was restored partially when expression of the ecfur gene was induced from the native pgfur promoter. The full-length PgFur protein contained one zinc atom per protein monomer, but did not bind iron, manganese, or heme. Single cysteine substitutions of CXXC motifs resulted in phenotypes similar to the mutant Δpgfur strain. The modified proteins were produced in E. coli at significantly lower levels, were highly unstable, and did not bind zinc. The pgfur gene was expressed at the highest levels in bacteria cultured for 24 h in the absence of iron and heme or at higher levels in bacteria cultured for 10 h in the presence of protoporphyrin IX source. No influence of high availability of Fe2+, Zn2+, or Mn2+ on pgfur gene expression was observed. Two chromosomal mutant strains producing protein lacking 4 (pgfurΔ4aa) or 13 (pgfurΔ13aa) C-terminal amino acid residues were examined in regard to importance of the C-terminal lysine-rich region. The pgfurΔ13aa strain showed a phenotype typical for the mutant Δpgfur strain, but both the wild-type PgFur protein and its truncated version bound zinc with similar ability. The Δpgfur mutant strain produced higher amounts of HmuY protein compared with the wild-type strain, suggesting compromised regulation of its expression. Potential PgFur ligands, Fe2+, Mn2+, Zn2+, PPIX, or serum components, did not influence HmuY production in the Δpgfur mutant strain. The mutant pgfurΔ4aa and pgfurΔ13aa strains exhibited affected HmuY protein production. PgFur, regardless of the presence of the C-terminal lysine-rich region, bound to the hmu operon promoter. Our data suggest that cooperation of PgFur with partners/cofactors and/or protein/DNA modifications would be required to accomplish its role played in an in vivo multilayer regulatory network.

SignaLink: Multilayered Regulatory Networks.

Biological networks are graphs used to represent the inner workings of a biological system. Networks describe the relationships of the elements of biological systems using edges and nodes. However, the resulting representation of the system can sometimes be too simplistic to usefully model reality. By combining several different interaction types within one larger multilayered biological network, tools such as SignaLink provide a more nuanced view than those relying on single-layer networks (where edges only describe one kind of interaction). Multilayered networks display connections between multiple networks (i.e., protein-protein interactions and their transcriptional and posttranscriptional regulators), each one of them describing a specific set of connections. Multilayered networks also allow us to depict cross talk between cellular systems, which is a more realistic way of describing molecular interactions. They can be used to collate networks from different sources into one multilayered structure, which makes them useful as an analytic tool as well.

Non-coding RNAs demystify constitutive heterochromatin as essential modulator of epigenotype

Heterochromatin, ever since its discovery in 1928 as a distinct cytological entity showing differential condensation cycle than euchromatin, has remained enigmatic because of the general paucity of typical protein-coding genes and highly variable quantities in different genomes. While the significance of developmentally regulated facultative heterochromatin regions has been relatively better understood and appreciated, the repetitive and transposon sequence rich but protein-coding gene poor constitutive heterochromatin has continued to be a puzzle and ideas about its requirement for biological systems have varied from ‘junk’ or ‘selfish’ to very significant. Studies in recent decades on the diverse non-coding RNAs (ncRNAs) in eukaryotes have revealed them as essential parts of multi-layered regulatory networks. Interestingly, a significant contribution to the cellular ncRNAs pool seems to be derived from the constitutive heterochromatic regions and thus, as was suggested by several classical genetic studies, the constitutive heterochromatin can indeed have significant to subtle effects on activities of numerous other genes and, therefore, have far-reaching evolutionary consequences under natural conditions. This review briefly discusses, using examples mostly from Drosophila, the general organization of constitutive heterochromatin and how these regions can affect the spatial organization of chromatin in nucleus and how the diverse ncRNAs derived from such regions directly or indirectly modulate activities of many genes on other chromosomes. Interestingly, the diversity of small and large phenotypic effects that were empirically ascribed in classical studies to be associated with constitutive heterochromatin can now be understood through ncRNA metabolism. The functional and evolutionary significance of the heterochromatin is much more than it merely being a condensed and repressive state as a mechanism to keep the transposons etc. silent. The enigma of heterochromatin is to be viewed in light of the fact that biological systems are products of chance and necessity and, therefore, do not always follow the human reductionist logic.