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The nuclear compartment is delimited by a specialized expanded sheet of the endoplasmic reticulum (ER) known as the nuclear envelope (NE). Compared to the outer nuclear membrane and the contiguous peripheral ER, the inner nuclear membrane (INM) houses a unique set of transmembrane proteins that serve a staggering range of functions. Many of these functions reflect the exceptional position of INM proteins at the membrane-chromatin interface. Recent research revealed that numerous INM proteins perform crucial roles in chromatin organization, regulation of gene expression, genome stability, and mediation of signaling pathways into the nucleus. Other INM proteins establish mechanical links between chromatin and the cytoskeleton, help NE remodeling, or contribute to the surveillance of NE integrity and homeostasis. As INM proteins continue to gain prominence, we review these advancements and give an overview on the functional versatility of the INM proteome. © 2021, Cold Spring Harbor Perspectives in Biology by Cold Spring Harbor Laboratory Press ISSN:1943-0264

As a BET bromodomain inhibitor, JQ1 has been proven have efficacy against a number of different cancers. In terms of male reproduction, JQ1 may be used as a new type of contraceptive, since JQ1 treatment in male mice could lead to germ cell defects and a decrease of sperm motility, moreover, this effect is reversible. However, the mechanism of JQ1 acting on gene regulation in spermatogenesis remains unclear. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse testes treated with JQ1 or vehicle control to determine the transcriptional regulatory function of JQ1 in spermatogenesis at the single cell resolution. We confirmed that JQ1 treatment could increase the numbers of somatic cells and spermatocytes and decrease the numbers of spermatid cells. Gene Ontology (GO) analysis demonstrated that differentially expressed genes which were down-regulated after JQ1 injection were mainly enriched in ‘‘DNA conformation change” biological process in early developmental germ cells and “spermatid development” biological process in spermatid cells. ATAC-seq data further confirmed that JQ1 injection could change the open state of chromatin. In addition, JQ1 could change the numbers of accessible meiotic DNA double-stranded break sites and the types of transcription factor motif that functioned in pachytene spermatocytes and round spermatids. The multi-omics analysis revealed that JQ1 had the ability to regulate gene transcription by changing chromatin conformation in mouse spermatogenesis, which would potentiate the availability of JQ1 in male contraceptive.

Bidirectional transport of macromolecules occurs through nuclear pores, which were first detected by electron microscopy. Interest in regulation of gene expression, curiosity to understand one of the few remaining uncharacterized translocons of eukaryotic cells, and the hope of ultimate therapeutic relevance have led to a period of intense investigation of these transport pathways. This article outlines the principal features of nucleocytoplasmic transport, highlights less well-understood related issues, and gathers thoughts pertaining to the evolutionary origin of the equipment that accomplishes transport. During the past half century, this field has added a major dimension to the understanding of what it means to be eukaryotic.

Extrachromosomal circular DNA (ecDNA) or double minutes have gained renewed interest since its discovery more than five decades ago, emerging as potent drivers of tumour evolution. This has largely been motivated by recent discovery that the tumour-exclusive ecDNA are highly prevalent in almost all cancers unlike previously thought. EcDNAs contribute to elevated oncogene expression, intratumoural heterogeneity, tumour adaptation and therapy resistance independently of canonical chromosomal alterations. Importantly, ecDNAs play a critical role in patient survival as ecDNA-based oncogene amplification adversely affects clinical outcome to a significantly greater extent than intrachromosomal amplification. Chromothripsis, a major driver of ecDNA biogenesis and gene amplification, is a mutational process characterised by chromosomal shattering and localised complex genome rearrangement. Chemotherapeutic drugs can lead to chromothriptic rearrangements and therapy resistance. In this review, we examine how ecDNAs mediate oncogene overexpression, facilitate accelerated tumour malignancy and enhance rapid adaptation independently of linear chromosomes. We delve into discoveries pertaining to mechanisms of biogenesis, distinctive features of ecDNA, gene regulation and topological interactions with active chromatin. We also discuss the critical role of chromothripsis in engendering ecDNA amplification and evolution. One envisions that insights into ecDNA biology not only hold importance for the cancer genome and tumour evolutionary dynamics, but could also inform prognostication and clinical intervention, particularly for cancers characterised by high oncogene amplification.

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21–encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Higher-order chromatin structures play important roles in regulating multiple biological processes, growth and development, biotic and abiotic stress response. However, little is known about three-dimensional chromatin structures in Paulownia, or about whole-genome chromatin conformational changes that occur in response to Paulownia witches' broom (PaWB) disease. We used high-throughput chromosome conformation capture (Hi-C) to obtain genome-wide profiles of chromatin conformation in healthy and phytoplasma-infected Paulownia fortunei genome. The heatmap results indicated that the strongest interactions between chromosomes were in the telomeres. We confirmed the main structural characteristics, such as A/B compartments, topologically associated domains, and chromatin loops were prominent in Paulownia genome and clearly altered in phytoplasma-infected plants. By combining chromatin Immunoprecipitation sequencing, Hi-C signals, and RNA sequencing data, we inferred that the chromatin structure changed and the modification levels of three histones (H3K4me3/K9ac/K36me3) increased in phytoplasma-infected P. fortunei, which was associated with changes of transcriptional activity. We concluded that epigenetic modifications, transcriptional activity might function in combination to shape chromatin packing in healthy and phytoplasm-infected Paulownia. Finally, 11 genes (such as RPN6, Sec61 subunit alpha), commonly located at specific TAD boundaries, A/B compartment switching, specific loops, and associated with histone marks, were identified and considered as closely related to PaWB stress. Our results provide new insights into the nexus between gene regulation and chromatin conformational alterations in non-model plants upon phytopathogen infection and plant disease resistance. Key words: Paulownia witches' broom; chromatin conformation; histone modification; transcription regulation.

Calvarial bone healing is challenging, especially for individuals with osteoporosis because stem cells from osteoporotic patients are highly prone to adipogenic differentiation. Based on previous findings that chondrogenic induction of adipose-derived stem cells (ASCs) can augment calvarial bone healing, we hypothesized that activating chondroinductive Sox Trio genes (Sox5, Sox6, Sox9) and repressing adipoinductive genes (C/ebp-α, Ppar-γ) in osteoporotic ASCs can reprogram cell differentiation and improve calvarial bone healing after implantation. However, simultaneous gene activation and repression in ASCs is difficult. To tackle this problem, we built a CRISPR-BiD system for bi-directional gene regulation. Specifically, we built a CRISPR-AceTran system that exploited both histone acetylation and transcription activation for synergistic Sox Trio activation. We also developed a CRISPR interference (CRISPRi) system that exploited DNA methylation for repression of adipoinductive genes. We combined CRISPR-AceTran and CRISPRi to form the CRISPR-BiD system, which harnessed three mechanisms (transcription activation, histone acetylation, and DNA methylation). After delivery into osteoporotic rat ASCs, CRISPR-BiD significantly enhanced chondrogenesis and in vitro cartilage formation. Implantation of the engineered osteoporotic ASCs into critical-sized calvarial bone defects significantly improved bone healing in osteoporotic rats. These results implicated the potential of the CRISPR-BiD system for bi-directional regulation of cell fate and regenerative medicine.

Understanding the exquisitely complex nature of the three-dimensional organization of the genome and how it affects gene regulation remains a central question in biology. Recent advances in sequencing- and imaging-based approaches in decoding the three-dimensional chromatin landscape have enabled a systematic characterization of gene regulatory architecture. In this review, we outline how chromatin architecture provides a reference atlas to predict the functional consequences of non-coding variants associated with human traits and disease. High-throughput perturbation assays such as massively parallel reporter assays (MPRA) and CRISPR-based genome engineering in combination with a reference atlas opened an avenue for going beyond observational studies to experimentally validating the regulatory principles of the genome. We conclude by providing a suggested path forward by calling attention to barriers that can be addressed for a more complete understanding of the regulatory landscape of the human brain.

Background Mechanisms governing the diversity of CFTR gene expression throughout the body are complex. Multiple intronic and distal regulatory elements are responsible for regulating differential CFTR expression across tissues. Methods Drawing on published data, 18 high-priority genomic regions were identified and interrogated for CFTR-enhancer function using CRISPR/dCas9-based epigenome editing tools. Each region was evaluated by dCas9p300 and dCas9KRAB for its ability to enhance or repress CFTR expression, respectively. Results Multiple genomic regions were tested for enhancer activity using CRISPR/dCas9 epigenome editing. dCas9p300 mediates a significant increase in CFTR mRNA levels when targeted to the promoter and a region 44 kb upstream of the transcriptional start site in a CFTR-low expressing cell line. Multiple gRNAs targeting the promoter induced a robust increase in CFTR protein levels. In contrast, dCas9KRAB-mediated repression is much more robust with 10 of the 18 evaluated genomic regions inducing CFTR protein knockdown. To evaluate the therapeutic efficacy of modulating CFTR gene regulation, dCas9p300 was used to induce elevated levels of CFTR from the endogenous locus in ΔF508/ΔF508 human bronchial epithelial cells. Ussing chamber studies demonstrated a synergistic increase in ion transport in response to CRISPR-induced expression of ΔF508 CFTR mRNA along with VX809 treatment. Conclusions CRISPR/dCas9-based epigenome-editing provides a previously unexplored tool for interrogating CFTR enhancer function. Here, we demonstrate that therapeutic interventions that increase the expression of CFTR may improve the efficacy of CFTR modulators. A better understanding CFTR regulatory mechanisms could uncover novel therapeutic interventions for the development of cystic fibrosis therapies.

Fibroblasts can be reprogrammed into cardiovascular progenitor cells (CPCs) using transgenic approaches, although the underlying mechanism remains unclear. We determined whether activation of endogenous genes such as Gata4, Nkx2.5, and Tbx5 can rapidly establish autoregulatory loops and initiate CPC generation in adult extracardiac fibroblasts using a CRISPR activation system. The induced fibroblasts (>80%) showed phenotypic changes as indicated by an Nkx2.5 cardiac enhancer reporter. The progenitor characteristics were confirmed by colony formation and expression of cardiovascular genes. Cardiac sphere induction segregated the early and late reprogrammed cells that can generate functional cardiomyocytes and vascular cells in vitro. Therefore, they were termed CRISPR-induced CPCs (ciCPCs). Transcriptomic analysis showed that cell cycle and heart development pathways were important to accelerate CPC formation during the early reprogramming stage. The CRISPR system opened the silenced chromatin locus, thereby allowing transcriptional factors to access their own promoters and eventually forming a positive feedback loop. The regenerative potential of ciCPCs was assessed after implantation in mouse myocardial infarction models. The engrafted ciCPCs differentiated into cardiovascular cells in vivo but also significantly improved contractile function and scar formation. In conclusion, multiplex gene activation was sufficient to drive CPC reprogramming, providing a new cell source for regenerative therapeutics.

Background: MicroRNAs (miRNA) belong to the substantial class of posttranscriptional gene regulators with decisive functions in typical cellular and disease progressions. They are short RNA molecules that are not translated into proteins but bind to the complementary sites of various mRNAs, thus blocking them and leading to translational inhibition. Objective: These miRNA molecules act as signatory molecules or biomarkers for various types of malignancies. Different miRNAs are involved in different cancer-linked pathways depending on the nature, stage, and kind of cancer. The objective of this article is to discuss and review the role and significance of various miRNAs in two of the most prominent cancers; breast and ovarian cancer. Methods: The role of miRNAs in the instigation, propagation, and metastasis of melanoma has been elucidated. Results: This article focuses on the up- and down-regulation of various miRNAs in breast and ovarian cancer, with stress on diversity in their occurrence and specificity in their threshold levels in certain types and stages of cancer, suggesting their potential role as an effective diagnostic and treatment tool for cancer. Conclusion: Considering all the aforementioned information, it can be concluded that miRNAs can act as potential biomarkers for the diagnosis of breast and ovarian cancers.

Plant response to drought stress includes systems for intracellular regulation of gene expression and signaling, as well as inter-tissue and inter-organ signaling, which helps entire plants acquire stress resistance. Plants sense water-deficit conditions both via the stomata of leaves and roots, and transfer water-deficit signals from roots to shoots via inter-organ signaling. ABA is an important phytohormone involved in drought stress response and adaptation, and is synthesized mainly in vascular tissues and guard cells of leaves. In leaves, stress-induced ABA is distributed to various tissues by transporters, which activates stomatal closure and expression of stress-related genes to acquire drought stress resistance. Moreover, stepwise stress response at the whole-plant level is important for proper understanding of the physiological response to drought conditions. Drought stress is sensed by multiple types of sensors as molecular patterns of abiotic stress signals, which are transmitted via separate parallel signaling networks to induce downstream responses, including stomatal closure and synthesis of stress-related proteins and metabolites. Peptide molecules play important roles in the inter-organ signaling of dehydration from roots to shoots, as well as signaling of osmotic changes and reactive oxygen species/Ca2+ . In this review, we have summarized recent advances in research on complex plant drought stress responses, focusing on inter-tissue signaling in leaves and inter-organ signaling from roots to shoots. We have discussed the mechanisms via which drought stress adaptations and resistance are acquired at the whole-plant level, and have proposed the importance of quantitative phenotyping for measuring plant growth under drought conditions.

We profiled the global gene expression landscape across the reproductive lifecycle of Brassica napus. Comparative analysis of this nascent amphidiploid revealed the contribution of each subgenome to plant reproduction. Whole genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive-lethal phenotype. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome-specific transcription factor networks identified distinct transcription factor families enriched in each of the An and Cn subgenome early in seed development. Analysis of laser-microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm, and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm, and seed coat of the developing seed. Transcriptional networks spanning both the An and Cn genomes of the whole B. napus seed can identify valuable targets for seed development research and that-omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses.

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous activation kinetics of a given gene in response to EGF stimulation. Acetylation levels modify these heterogenous patterns and influence both allele activation frequencies and overall expression profile parameters. Our data therefore point to a complex interplay between acetylation equilibria and target gene induction where acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner., Graphical Abstract Graphical AbstractModel showing how acetylation levels driven by the KDAC-KAT equilibrium differentially affects gene activation.

The oxidative stress response is critical for malignant cells. It plays a dual role by helping cancer cells survive and proliferate but also causing apoptosis and apoptosis-- like cell death. The oxidative stress response is characterized by tight regulation of gene expression by a series of transcription factors (OSRts; oxidative stress response transcription factors). In this communication, we review the role of OSRts, notably NRF2 and p53 as well as other transcription factors that modulate the response. We discuss how hierarchal the oxidative stress response is and controls ‘live or die’ signals. This is followed by a discussion on how plant-derived molecules, including polyphenols, which are described both as prooxidants and antioxidants within the cancer cells, have been reported to affect the activities of OSRts. Deriving an example from preliminary data from our group, we discuss how plant-derived molecules might modulate the oxidative stress response by causing structural perturbations in the proteinaceous transcription factors, notably Nrf2 and p53. We look at this information in the light of understanding how plant derived molecules may be used as lead compounds to develop modulators of the oxidative stress response.

Intracellular adhesion molecule 1 (ICAM-1) is one of the most extensively studied inducible cell adhesion molecules which is responsible for several immune functions like T cell activation, extravasation, inflammation, etc. The molecule is constitutively expressed over the cell surface and is regulated up / down in response to inflammatory mediators like cellular stress, proinflammatory cytokines, viral infection. These stimuli modulate the expression of ICAM-1 primarily through regulating the ICAM-1 gene transcription. On account of the presence of various binding sites for NF-κB, AP-1, SP-1, and many other transcription factors, the architecture of the ICAM-1 promoter become complex. Transcription factors in union with other transcription factors, coactivators, and suppressors promote their assembly in a stereospecific manner on ICAM-1 promoter which mediates ICAM-1 regulation in response to different stimuli. Along with transcriptional regulation, epigenetic modifications also play a pivotal role in controlling ICAM-1 expression on different cell types. In this review, we summarize the regulation of ICAM-1 expression both at the transcriptional as well as post-transcriptional level with an emphasis on transcription factors and signaling pathways involved.

Manipulation of gene activity through creation of hypomorphic mutants has been a long-standing tool in examining gene function. Our previous studies have indicated that hypomorphic mutants could be created by inserting cis-regulatory sequences composed of consecutive adenosine nucleotides called poly(A) tracks. Here we use poly(A) tracks to create hypomorphic mutants and functional characterization of membrane, secretory, and endogenous proteins. Insertion of poly(A) tracks into the sequences of interleukin-2 and membrane protein CD20 results in a programmable reduction of mRNA stability and attenuation of protein expression regardless of the presence of a signaling sequence. Likewise, CRISPR-Cas9 targeted insertion of poly(A) tracks into the coding sequence of the endogenous human genes AUF1 and TP53 results in a programmable reduction of targeted protein and mRNA levels. Functional analyses of AUF1-engineered hypomorphs indicate a direct correlation between AUF1 gene levels and the stability of AUF1-regulated mRNAs. Hypomorphs of TP53 affect expression of the target genes differentially depending on the severity of the hypomorphic mutation. Finally, decreases in TP53 protein affect the same cellular pathways in poly(A) track-engineered cells as in cancer cells, indicating these variants’ biological relevance. These results highlight this technology’s power to create predictable, stable hypomorphs in recombinant or endogenous genes in combination with CRISPR-Cas9 engineering tools., Graphical abstract, Powell et al. report an optimized and CRISPR-Cas9 combined method for rapid creation of reduced levels of targeted genes. They show that this method can be used for genes encoding membrane and secretory proteins as well as for endogenous genes when combined with new genome-engineering technologies.

Fam114A1 is a gene closely related to the development of nerve cells, melanocytes, and nerve cells that originate from the neural crest of the embryonic ectoderm. Recent studies showed that Fam114A1 has a role in the occurrence of ankylosing myelitis spondylitis and autoimmune enteritis; still, its cellular function remains poorly understood. In this study, we investigated the effect of Fam114A1 on the biological activity of melanocytes. We found that the expression of Fam114A1 in vitiligo melanocytes (MCV-L, MCV-N, PI3V) was higher than that in normal melanocytes, and the biological function of melanocytes was significantly affected when the Fam114A1 gene was silenced. Inhibition of Fam114A1 increased proliferation, migration, and melanin synthesis proteins, decreased apoptosis, while its overexpression reversed this process. Mechanistically, we discovered that RACK1 is a target protein of Fam114A1 and that RACK1 can be negatively regulated by Fam114A1. Further study showed that Fam114A1 inhibition could not protect melanocytes from apoptosis once the expression of RACK1 protein was silenced. In summary, Fam114A1 is an effective regulatory protein for regulating the function of melanocytes. Inhibition Fam114A1 protects melanocytes from apoptosis through increasing RACK1.

Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the adaptive response that increases the resistance of Escherichia coli cells to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are essentially absent. Whereas genetic deletion of these proteins causes extreme sensitivity to alkylation stress, a temporary lack of expression is tolerated and increases genetic plasticity of the whole population. We demonstrated this by monitoring the dynamics of nascent DNA mismatches during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. We propose that stochastic modulation of DNA repair capacity by the adaptive response creates a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.

The Physcomitrium patens DICER-LIKE1a (PpDCL1a) mRNA encoding the essential Dicer protein for microRNA (miRNA) biogenesis harbors an intronic miRNA (miR1047). An autoregulatory mechanism to control PpDCL1a abundance that is based on competitive processing of the intronic miRNA and proper PpDCL1a mRNA splicing has previously been proposed. If intron splicing occurs first the mRNA can be translated into the functional PpDCL1a protein, whereas the processing of the intronic miRNA catalyzed by PpDCL1a itself, prior to pre-mRNA splicing, generates a truncated transcript unable to produce a functional protein. This proposed autoregulation of DCL1 has not been functionally analyzed in any plant species, and the existence of this autoregulatory control is expected to have a general impact on the overall miRNA biogenesis pathway and the transcriptome that is under miRNA control. We abolished PpDCL1a autoregulatory feedback control by the precise deletion of the MIR1047-containing intron. The generated line displayed hypersensitivity to salt stress and hyposensitivity to the plant hormone ABA, accompanied by the disturbed expression of miRNAs and mRNAs, revealed by transcriptome analyses. The feedback control together with the phenotypic abnormalities and molecular changes in the intron-less line can be rescued by the re-insertion of a modified intron harboring a sequence-unrelated artificial miRNA. Our findings indicate the physiological importance of miR1047-based feedback control of PpDCL1a transcript abundance, which controls the expression of miRNAs, and their cognate target RNAs during salt stress adaptation, and suggests a key role for this autoregulation in the molecular adaptation of land plants to terrestrial habitats.

Translation control by proteins refers to the process by which protein synthesis of specific products is regulated by external trans-acting proteins, often by repression and sometimes autoregulatory on the messenger ribonucleic acid (mRNA) encoding the very protein being synthesized, and also by activation in some systems. Keywords: RNA phage gene regulation; phage T4 gene regulation; ribosomal protein synthesis; translation activation; iron homeostasis; SELEX

Dysregulation of the oncogenic transcription factor HOXA9 is a prominent feature for most aggressive acute myeloid leukemia cases and a strong indicator of poor prognosis in patients. Leukemia subtypes with hallmark overexpression of HOXA9 include those carrying MLL gene rearrangements, NPM1c mutations, and other genetic alternations. A growing body of evidence indicates that HOXA9 dysregulation is both sufficient and necessary for leukemic transformation. The HOXA9 mRNA and protein regulation includes multi-layered controls by transcription factors (such as CDX2/4 and USF2/1), epigenetic factors (such as MLL-Menin-LEDGF, DOT1L, ENL, HBO1, NPM1c-XPO1, and polycomb proteins), microRNAs (such as miR-126 and miR-196b), long non-coding RNAs (such as HOTTIP), three-dimensional chromatin interactions, and post-translational protein modifications. Recently, insights into the dynamic regulation of HOXA9 have led to an advanced understanding of the HOXA9 regulome and provided new cancer therapeutic opportunities, including developing inhibitors targeting DOT1L, Menin, and ENL proteins. This review summarizes recent advances in understanding the molecular mechanisms controlling HOXA9 regulation and the pharmacological approaches that target HOXA9 regulators to treat HOXA9-driven acute myeloid leukemia.

Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes where it acts by engaging its ligand, CD47. SIRPG, which encodes SIRPγ, contains a non-synonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. SIRPG produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of SIRPG transcript isoforms. All of these transcript isoforms produced protein upon transient expression in vitro. However, CRISPR targeting of one of the alternatively spliced exons in SIRPG eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling and had significantly increased levels of cell surface CD47. In primary CD4+ and CD8+ T cells cell surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that SIRPG is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.

The gut-brain axis refers to the bidirectional connection and communication between the gastrointestinal tract and the central nervous system. This paper explores two routes for this communication that have hitherto remained under-examined: epigenetics and redox signaling and their implications for autism spectrum disorder (ASD). The gut microbiota may induce epigenetic changes in the gut and potentially in the brain through their fermentation products. Instead of through other conceptualizations of them acting as neurotransmitters, gut microbial products may act as epigenetic agents, which are supported by the effects of gut bacterial-derived metabolites on gene regulation and expression. In addition to their epigenetic effects, gut bacterial-derived communicative agents can also influence host signaling by contributing to and even substituting host reactive oxygen species (ROS) production. These ROS can act as second messengers and exert oxidative activity on proteins to influence immune, inflammatory, and other signaling processes. ROS and epigenetic mechanisms may have interactive effects as well. ROS, in addition to their role in signaling pathways and cellular redox alterations, also influence redox-sensitive transcription factors, thereby having an effect on gene expression. Specifically, ROS are involved in the activation of transcription factors, chromatin remodeling, and histone/protein deacetylation. These two proposed mechanisms correspond with the recent findings related to ASD, where a cofactor that is shown to be lower in ASD has antioxidative properties, responds to epigenetic modulation, and increases via microbiota interventions. The current evidence reviewed here suggests the need to update models of the gut-brain communication to include these two mechanisms. Such a modeling can also contribute to understanding the unknowns of host metabolism and physiology in ASD and afford potential therapeutic avenues for this as well as other psychiatric and physiological conditions.

Circular RNA is progressively reported to occur in various species including mammals where it is thought to be involved in the post-transcriptional regulation of gene expression, partly via interactions with microRNA. Here, we asked whether the circular topology causes functional differences to linear forms when interacting with short RNA strands in vitro and in human cells. Kinetic studies with human bladder cancer-derived synthetic circular RNA versus linear transcripts, respectively, with short oligoribonucleotides showed similar association rates for both topologies. Conversely, a substantial topology-related difference was measured for the activation entropy and the activation enthalpy of RNA–RNA annealing. This finding strongly indicates a significant difference of the mechanism of RNA–RNA interactions. To investigate whether these characteristics of circular RNA are biologically meaningful we performed transient transfection experiments with a microRNA-regulated expression system for luciferase in bladder cancer-derived cells. We co-transfected linear or circular RNA containing one microRNA binding site for the target-suppressing microRNA mlet7a. Here, the circular isoform showed a strongly increased competition with microRNA function versus linear versions. In summary, this study suggests novel topology-related characteristics of RNA–RNA interactions involving circRNA in vitro and in living cells.

Subcellular localization of transcripts is highly associated with regulation of gene expression, synthesis of protein, and also the development of the human brain cortex. Although many mechanisms are prevalent in the occurrence of neuroinflammation, the mechanisms based on differences in subcellular localization of transcripts have not been explored. To characterize the dynamic profile of nuclear and cytoplasmic transcripts during the progress of haemorrhage-induced neuroinflammation, we isolated nucleo-cytoplasmic RNA fractions of oxyhaemoglobin (oxy-Hb) treated microglia cells and sequenced both fractions. We discovered that cytoplasmic retained genes were the major forces to maintain the neuroinflammatory microenvironment with 10 hub genes and 40 conserved genes were identified. Moreover, antisense RNA Gm44096 and lincRNA Gm47270, which co-expressed with a crowd of inflammatory genes in the cytoplasm, were discovered as regulatory strategies for sustaining the neuroinflammatory microenvironment. Thus, our study provides a new perspective on understanding haemorrhage-induced neuroinflammation and also reveals a mechanism of lncRNA responsible for maintaining the neuroinflammatory microenvironment.

Aryl hydrocarbon receptor (AhR) is a ligand-mediated transcription factor known for regulating response to xenobiotics, including prototypical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the activation of CYP1A1 expression. Upon ligand-binding, AhR translocates to the nucleus, interacts with the AhR nuclear translocator, and binds to xenobiotic response elements (XREs; GCGTG) present in the promoter region of AhR-regulated genes. Recently, we identified a novel tryptophan catabolite, cinnabarinic acid (CA), as an endogenous AhR agonist capable of activating expression of AhR target gene stanniocalcin 2 (stc2). The CA-driven stc2 induction bestowed cytoprotection against hepatotoxicity in an AhR-dependent manner. Interestingly, only CA but not TCDD was able to induce stc2 expression in liver, and CA was unable to upregulate the TCDD responsive cyp1a1 gene. In this report, we identified CA-specific histone H4 lysine 5 acetylation and H3 lysine 79 methylation at the AhR-bound stc2 promoter. Moreover, histone H4 lysine 5 acetylation writer, activating transcription factor 2 (Atf2), and H3 lysine 79 methylation writer, disruptor of telomeric silencing 1-like histone lysine methyltransferase (Dot1l), were interacting with the AhR complex at the stc2 promoter exclusively in response to CA treatment concurrent with the histone epigenetic marks. Suppressing Atf2 and Dot1l expression using RNA interference confirmed their role in stc2 expression. CRISPR/Cas9-assisted replacement of cyp1a1 promoter-encompassing XREs with stc2 promoter XREs resulted in CA-dependent induction of cyp1a1, underlining a fundamental role of quaternary structure of XRE sequence in agonist-specific gene regulation. In conclusion, CA-driven recruitment of specific chromatin regulators to the AhR complex and resulting histone epigenetic modifications may serve as a molecular basis for agonist-specific stc2 regulation by AhR. SIGNIFICANCE STATEMENT: Results reported here provide a mechanistic explanation for the agonist-specific differential gene regulation by identifying interaction of aryl hydrogen receptor with specific chromatin regulators concomitant with unique histone epigenetic marks. This study also demonstrated that the agonist-specific target-gene expression can be transferred with the gene-specific promoter xenobiotic response element-sequence in the context of chromatin architecture.

The cytochrome P450 (Cyp) proteins Cyp1A1 and Cyp1A2 are strongly induced in the mouse liver by the potent environmental toxicant 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), acting through the aryl hydrocarbon receptor (AHR). The induction of Cyp1A1 is localized within the centrilobular regions of the mouse liver at low doses of TCDD, progressing to pan-lobular induction at higher doses. Even without chemical perturbation, metabolic functions and associated genes are basally zonated in the liver lobule along the central-to-portal axis. To investigate the mechanistic basis of spatially restricted gene induction by TCDD, we have developed a multiscale computational model of the mouse liver lobule with single-cell resolution. The spatial location of individual hepatocytes in the model was calibrated from previously published high-resolution images. A systems biology model of the network of biochemical signaling pathways underlying Cyp1A1 and Cyp1A2 induction was then incorporated into each hepatocyte in the model. Model simulations showed that a negative feedback loop formed by binding of the induced Cyp1A2 protein to TCDD, together with cooperative gene induction by the β-catenin/AHR/TCDD transcription factor complex and β-catenin, help produce the spatially localized induction pattern of Cyp1A1. Although endogenous WNT regulates the metabolic zonation of many genes, it was not a driver of zonal Cyp1A1 induction in our model. Conclusion: In this work, we used data-driven computational modeling to identify the mechanistic basis of zonally restricted gene expression induced by the potent and persistent environmental pollutant TCDD. The multiscale model and derived results clarify the mechanisms of dose-dependent hepatic gene induction responses to TCDD. Additionally, this work contributes to our broader understanding of spatial gene regulation along the liver lobule.

Non-healing wounds in Type 2 Diabetes (T2D) patients represent the most common cause of amputation in the US, with an associated 5-year mortality of nearly 50%. Our lab has examined tissue from both T2D murine models and human wounds in order to explore mechanisms contributing to impaired wound healing. Current published data in the field point to macrophage function serving a pivotal role in orchestrating appropriate wound healing. Wound macrophages in mice and patients with T2D are characterized by a persistent inflammatory state; however, the mechanisms that control this persistent inflammatory state are unknown. Current literature demonstrates that gene regulation through histone modifications, DNA modifications, and microRNA can influence macrophage plasticity during wound healing. Further, accumulating studies reveal the importance of cells such as adipocytes, infiltrating immune cells (PMNs and T cells), and keratinocytes secrete factors that may help drive macrophage polarization. This review will examine the role of macrophages in the wound healing process, along with their function and interactions with other cells, and how it is perturbed in T2D. We also explore epigenetic factors that regulate macrophage polarization in wounds, while highlighting the emerging role of other cell types that may influence macrophage phenotype following tissue injury.

Lineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. Here we perform dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis uncovers differential fate commitment dynamics between the two lineages as they exit the stem and progenitor stage. Collectively, these data provide insights into the temporally regulated synergy of the cis- and the trans-regulatory components underlying hematopoietic lineage commitment and differentiation., Lineage differentiation and commitment is driven by transcription regulators and chromatin changes. Here the authors report daily profiling of chromatin accessibility and transcriptome changes during human erythropoiesis, relating these changes to lineage potential between erythropoiesis and megakaryopoieis.

Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

During cellular specification, transcription factors orchestrate cellular decisions through gene regulation. By hijacking these transcriptional networks, human pluripotent stem cells (hPSCs) can be specialized into neurons with different molecular identities for the purposes of regenerative medicine and disease modeling. However, molecular fine tuning cell types to match their in vivo counterparts remains a challenge. Directing cell fates often result in blended or incomplete neuron identities. A better understanding of hPSC to neuron gene regulation is needed. Here, we used single cell RNA sequencing to resolve some of these graded molecular identities during human neurogenesis from hPSCs. Differentiation platforms were established to model neural induction from stem cells, and we characterized these differentiated cell types by 10x single cell RNA sequencing. Using single cell trajectory and co-expression analyses, we identified a co-regulated transcription factor module expressing achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1) and neuronal differentiation 1 (NEUROD1). We then tested the function of these transcription factors in neuron subtype differentiation by gene knockout in a novel human system that reports the expression of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. ASCL1 was identified as a necessary transcription factor for regulating dopaminergic neurotransmitter selection.

Proteasomal activator 28 gamma (PA28γ) upregulates the levels of HBx, a regulatory protein of hepatitis B virus (HBV) to stimulate HBV replication; however, the detailed mechanism remains unknown. Here, we found that PA28γ impaired the ability of seven in absentia homolog 1 (Siah-1) as an E3 ubiquitin ligase of HBx. PA28γ competitively inhibited the binding of Siah-1 to HBx in human hepatoma cells. Accordingly, PA28γ increased the stability of HBx and decreased HBx ubiquitination, abolishing the potential of Siah-1 to downregulate HBx levels. PA28γ also executed its role as an antagonist of Siah-1 during HBV replication, as demonstrated by an in vitro HBV replication system. The present study may provide insights into the mechanisms underlying the regulation of HBV replication.

During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation., Long-range looping of an enhancer to a promoter (E-P looping) is a key feature of gene activation; thus, regulation of E-P looping could serve as an effective strategy to precisely control gene expression. Here the authors propose the Drosophila chromatin insulator antagonist Shep represses expression of genes during neuronal maturation by preventing E-P looping.

Background The Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. The assembly, regulation, and functions of these nuclear condensates remain to be further understood. Results We found that Med15, a subunit in the tail module of the Mediator complex, formed nuclear condensates through a novel mechanism. Nuclear foci of Med15 were detected by both immunostaining of endogenous proteins and live cell imaging. Like Med1 foci and many other biomolecular condensates, Med15 foci were sensitive to 1, 6-Hexanediol and showed rapid recovery during fluorescence recovery after photobleaching. Interestingly, overexpressing DYRK3, a dual-specificity kinase that controls the phase transition of membraneless organelles, appeared to disrupt Med1 foci and Med15 foci. We identified two regions that are required to form Med15 nuclear condensates: the glutamine-rich intrinsically disordered region (IDR) and a short downstream hydrophobic motif. The optodroplet assay revealed that both the IDR and the C-terminal region of Med15 contributed to intracellular phase separation. Conclusions We identified that the Mediator complex subunit Med15 formed nuclear condensates and characterized their features in living cells. Our work suggests that Med15 plays a role in the assembly of transcription coactivator condensates in the nucleus and identifies Med15 regions that contribute to phase separation.

Successful reproduction is dependent on the transfer of male seminal proteins to females upon mating. These proteins arise from secretory tissues in the male reproductive tract, including the prostate and seminal vesicles in mammals and the accessory gland in insects. Although detailed functional studies have provided important insights into the mechanisms by which accessory gland proteins support reproduction, much less is known about the molecular mechanisms that regulate their expression within this tissue. Here we show that the Drosophila HR39 nuclear receptor is required for the proper expression of most genes that encode male accessory gland proteins. Consistent with this role, HR39 mutant males are infertile. In addition, tissue-specific RNAi and genetic rescue experiments indicate that HR39 acts within the accessory glands to regulate gene expression and male fertility. These results provide new directions for characterizing the mammalian orthologs of HR39, the SF-1 and LRH-1 nuclear receptors, both of which are required for glandular secretions and reproduction. In addition, our studies provide a molecular mechanism to explain how the accessory glands can maintain the abundant levels of seminal fluid production required to support fertility.

The aptamer domain of the theophylline riboswitch was randomized to generate a library containing millions of different variants. Dual genetic selection utilizing the cat-upp fusion gene was performed for the library, which successfully led to the identification of a caffeine-specific synthetic riboswitch. When a chloramphenicol-resistance gene was expressed under control of this riboswitch, E. coli cells showed chloramphenicol resistance only in the presence of caffeine. When inserted upstream of the gfpuv or lacZ gene, the caffeine riboswitch induced the expression of green fluorescent protein or β-galactosidase in the presence of caffeine, respectively. When tested with various concentrations of caffeine, the β-galactosidase activity was proportional to the amount of caffeine, clearly indicating the caffeine-dependent gene regulation by the caffeine riboswitch.

DNA methylation alterations are early events during tumourigenesis, affecting genes involved in the crosstalk between cells and surroundings in colorectal cancer (CRC). Among these genes, GRIA4, Glutamate Ionotropic Receptor AMPA Type Subunit 4, displays hypermethylation in the promoter region, and is an early diagnostic biomarker. It is well known that methylation can also affect alternative transcription. The purpose of this study is to evaluate the expression, at transcript and protein level, of GRIA4 main isoforms (the canonical one and a short variant) in 23 CRC and matched normal samples, of which we previously verified the methylation status. We further predicted miRNA/transcript target interactions as a possible post-transcriptional regulation using bioinformatics tools. As expected, downregulation of both variants has been observed in tumours. Interestingly, in contrast to what observed at transcriptional level, the GluR4 protein short isoform displayed higher expression than the canonical one either in normal or tumoural tissues. This may be explained by miRNA specifically targeting the canonical isoform. Our study is the first one that shows the expression of both isoforms in colon tissues. To note, the evident expression of the short isoform suggests a functional role in intestinal cell biology.

BACKGROUND Hepatic stellate cells (HSCs) are the key effector cells mediating the occurrence and development of liver fibrosis, while aerobic glycolysis is an important metabolic characteristic of HSC activation. Transforming growth factor-β1 (TGF-β1) induces aerobic glycolysis and is a driving factor for metabolic reprogramming. The occurrence of glycolysis depends on a high glucose uptake level. Glucose transporter 1 (GLUT1) is the most widely distributed glucose transporter in the body and mainly participates in the regulation of carbohydrate metabolism, thus affecting cell proliferation and growth. However, little is known about the relationship between TGF-β1 and GLUT1 in the process of liver fibrosis and the molecular mechanism underlying the promotion of aerobic glycolysis in HSCs. AIM To investigate the mechanisms of action of GLUT1, TGF-β1 and aerobic glycolysis in the process of HSC activation during liver fibrosis. METHODS Immunohistochemical staining and immunofluorescence assays were used to examine GLUT1 expression in fibrotic liver tissue. A Seahorse extracellular flux (XF) analyzer was used to examine changes in aerobic glycolytic flux, lactate production levels and glucose consumption levels in HSCs upon TGF-β1 stimulation. The mechanism by which TGF-β1 induces GLUT1 protein expression in HSCs was further explored by inhibiting/promoting the TGF-β1/mothers-against-decapentaplegic-homolog 2/3 (Smad2/3) signaling pathway and inhibiting the p38 and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. In addition, GLUT1 expression was silenced to observe changes in the growth and proliferation of HSCs. Finally, a GLUT1 inhibitor was used to verify the in vivo effects of GLUT1 on a mouse model of liver fibrosis. RESULTS GLUT1 protein expression was increased in both mouse and human fibrotic liver tissues. In addition, immunofluorescence staining revealed colocalization of GLUT1 and alpha-smooth muscle actin proteins, indicating that GLUT1 expression was related to the development of liver fibrosis. TGF-β1 caused an increase in aerobic glycolysis in HSCs and induced GLUT1 expression in HSCs by activating the Smad, p38 MAPK and P13K/AKT signaling pathways. The p38 MAPK and Smad pathways synergistically affected the induction of GLUT1 expression. GLUT1 inhibition eliminated the effect of TGF-β1 on HSC proliferation and migration. A GLUT1 inhibitor was administered in a mouse model of liver fibrosis, and GLUT1 inhibition reduced the degree of liver inflammation and liver fibrosis. CONCLUSION TGF-β1 induces GLUT1 expression in HSCs, a process related to liver fibrosis progression. In vitro experiments revealed that TGF-β1-induced GLUT1 expression might be one of the mechanisms mediating the metabolic reprogramming of HSCs. In addition, in vivo experiments also indicated that the GLUT1 protein promotes the occurrence and development of liver fibrosis.

DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1 repeats and promoter methylation of DNA damage response (DDR) and DNA repair (DR) genes (PARP1, ATM, BRCA1, MLH1, XPC, RAD23B, APC, TNFα, DNMT3A, MRE11A, MGMT, CDKN2A, MTHFR) in human peripheral blood mononuclear cells (PBMCs) of healthy donors in response to γ-radiation. Methylation level was correlated with gene expression profile of selected DDR and DR genes (APC, MLH1, PARP1, MRE11A, TNFα, MGMT) to understand their role in gene regulation. Blood samples were collected from 15 random healthy donors, PBMCs were isolated, exposed to 0.1 Gy (low) and 2.0 Gy (high) doses of γ-radiation and proliferated for 48 h and 72 h. Genomic DNA and total RNA were isolated from irradiated PBMCs along with un-irradiated control. Methylation profile was determined from bisulphite converted DNA and amplified by methylation sensitive high resolution melting (MS-HRM) method. Total RNA was converted to cDNA and relative expression was analysed using real time quantitative-PCR. Our results revealed that at 0.1 Gy, MRE11A and TNFα showed significant (P < 0.05) increase in methylation at 72 h. At 2.0 Gy, significant increase (P < 0.05) in methylation profile was observed at LINE1, MRE11A, PARP1, BRCA1, DNMT3A and RAD23B at 48 h and 72 h. PARP1 showed significant positive correlation of methylation status with gene expression. In conclusion, low and high doses of γ-radiation have significant influence on DNA methylation status of LINE1, DDR and DR genes suggesting their potential role as epigenetic signatures in human PBMCs, which can be further explored in human populations.

Messenger RNA (mRNA) can be transported and targeted to different subcellular compartments and locally translated. Local translation is an evolutionally conserved mechanism that in mammals, provides an important tool to exquisitely regulate the subcellular proteome in different cell types, including neurons. Local translation in axons is involved in processes such as neuronal development, function, plasticity, and diseases. Here, we summarize the current progress on axonal mRNA transport and translation. We focus on the regulatory mechanisms governing how mRNAs are transported to axons and how they are locally translated in axons. We discuss the roles of axonally synthesized proteins, which either function locally in axons, or are retrogradely trafficked back to soma to achieve neuron-wide gene regulation. We also examine local translation in neurological diseases. Finally, we give a critical perspective on the remaining questions that could be answered to uncover the fundamental rules governing local translation, and discuss how this could lead to new therapeutic targets for neurological diseases.

RNA viral vectors that replicate without DNA intermediates are attractive platforms for manipulation of cells for biomedical and veterinary applications because they have minimal risk of chromosomal integration. Vesicular stomatitis virus (VSV) vectors are among the most well-studied RNA viral vectors due to their low pathogenicity to humans and ability to express transgenes at high levels for weeks to months. However, their applications have been mostly limited to oncolytic and vaccine vectors due to their cytopathogenicity. We discovered two mutations in the VSV vector that synergistically confer improved stability in mouse embryonic stem cells (ESCs) with markedly lower cytopathic effects. We also demonstrated chemical regulation of transgene expression through embedded riboswitches. The ESCs infected with the mutant vector were shown to maintain pluripotency. This new vector sets the stage for precise regulation of gene expression in ESCs to produce a variety of differentiated cells without chromosomal alteration.

Increasing evidence supports the existence of cross-kingdom gene regulation. However, the therapeutic potential of stress-specific plant miRNAs and their role in UV-related pathologies in human tissue remain largely unexplored. The aim of this study was to investigate the therapeutic potential and mechanisms of action of stress-induced miRNA cocktails (SI-WmiRs) from Einkorn wheat (Triticum monococcum L.) on human keratinocyte (HaCaT) cells exposed to a high dose of UV-B radiation. We used a biofactory approach and irradiated wheatgrass with UV-C for 240 min to obtain the specific SI-WmiRs that wheat produces to recover from UV stress. We followed the plant with molecular and biochemical analyses and extracted our SI-WmiRs at the most appropriate time (0 h and 6 h after UV-C application). Then, we applied the SI-WmiR cocktail to HaCaT cells exposed to high-dose of UV-B radiation. Our results show that UV-B radiation induced lipid peroxidation and DNA damage, as demonstrated by increased malondialdehyde (MDA) levels and changes in the RAPD band profile, respectively. UV stress also impaired IL6/JAK2/STAT3 signalling and activated the inflammatory mediators IL6 and TNF-α in HaCaT cells, leading to significant induction of apoptotic cell death. We found that SI-WmiR transfection prevents lipid peroxidation and oxidative stress-related DNA damage by increasing antioxidant (CuZn-SOD, Mn-SOD) and DNA repair (EXO1, SMUG1 and XRCC3) gene expression. In addition, SI-WmiRs regulated IL6/JAK2/STAT3 signalling by reducing JAK2 and STAT3 gene expression and phosphorylated protein levels compared to the control treatments. Moreover, SI-WmiRs inhibited pro-apoptotic BAX, Caspase 3 and Caspase 8 gene expression and protein levels to prevent apoptosis of UV-stressed HaCaT cells. Our results demonstrate that stress-induced wheat miRNAs produced using a biofactory approach have strong potential as a novel and effective alternative therapy for UV stress-related skin damage.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a topologically associated domain (TAD) in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB domain and CNC homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here, we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (∼8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.

Extracellular vesicles (EVs) transfer functional molecules between cells. CD63 is a widely recognized EV marker that contributes to EV secretion from cells. However, the regulation of its expression remains largely unknown. Ferritin is a cellular iron storage protein that can also be secreted by the exosome pathway, and serum ferritin levels classically reflect body iron stores. Iron metabolism–associated proteins such as ferritin are intricately regulated by cellular iron levels via the iron responsive element-iron regulatory protein (IRE-IRP) system. Herein, we present a novel mechanism demonstrating that the expression of the EV-associated protein CD63 is under the regulation of the IRE-IRP system. We discovered a canonical IRE in the 5′ untranslated region of CD63 messenger RNA that is responsible for regulating its expression in response to increased iron. Cellular iron loading caused a marked increase in CD63 expression and the secretion of CD63+ EVs from cells, which were shown to contain ferritin-H and ferritin-L. Our results demonstrate that under iron loading, intracellular ferritin is transferred via nuclear receptor coactivator 4 (NCOA4) to CD63+ EVs that are then secreted. Such iron-regulated secretion of the major iron storage protein ferritin via CD63+ EVs, is significant for understanding the local cell-to-cell exchange of ferritin and iron.

Brain homeostasis depends on the existence of the blood-brain barrier (BBB). Despite decades of research, the factors and signalling pathways for modulating and maintaining BBB integrity are not fully elucidated. Here, we characterize the expression and function of the multifunctional receptor, sortilin, in the cells of the BBB, in vivo and in vitro. We show that sortilin acts as an important regulatory protein of the BBB's tightness. In rats lacking sortilin, the BBB was leaky, which correlated well with relocated distribution of the localisation of zonula occludens-1, VE-cadherin and β-catenin junctional proteins. Furthermore, the absence of sortilin in brain endothelial cells resulted in decreased phosphorylation of Akt signalling protein and increased the level of phospho-ERK1/2. As a putative result of MAPK/ERK pathways activity, the junctions between the brain endothelial cells were disintegrated and the integrity of the BBB became compromised. The identified barrier differences between wild type and Sort1-/- brain endothelial cells can pave the way for a better understanding of sortilin's role in the healthy and diseased BBB.

Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5' untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.

GTPases are a large superfamily of evolutionarily conserved proteins involved in a variety of fundamental cellular processes. The developmentally regulated GTP-binding protein (DRG) subfamily of GTPases consists of two highly conserved paralogs, DRG1 and DRG2, both of which have been implicated in the regulation of cell proliferation, translation and microtubules. Furthermore, DRG1 and 2 proteins both have a conserved binding partner, DRG family regulatory protein 1 and 2 (DFRP1 and DFRP2), respectively, that prevents them from being degraded. Similar to DRGs, the DFRP proteins have also been studied in the context of cell growth control and translation. Despite these proteins having been implicated in several fundamental cellular processes they remain relatively poorly characterized, however. In this review, we provide an overview of the structural biology and biochemistry of DRG GTPases and discuss current understanding of DRGs and DFRPs in normal physiology, as well as their emerging roles in diseases such as cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03961-0.

Histones constitute the chief protein component of DNA. They help to maintain chromatin structure and regulate gene expression. The long double-stranded DNA molecule winds around histone octamers to form nucleosomes which serve the purpose of compacting DNA within the confines of the nuclear membrane. There are five major types of histones, namely H1/H5, H2, H3 and H4. H3.3 is a subtype of H3 histone and can be encoded either by the H3F3A or H3F3B genes independently. Amino acids such as lysine and arginine found in the histone tails are sites of post-translational modifications (PTMs) such as methylation and acetylation. These PTMs in histones are involved in the regulation of gene expression by chromatin remodelling and by controlling DNA methylation patterns. Mutations in histone genes can affect sites of PTMs causing changes in local and global DNA methylation status. These effects are directly linked to neoplastic transformation by altered gene expression. Recurrent H3.3 histone mutations are increasingly identified in several malignancies and developmental disorders. The following review attempts to shed light on the diseases associated with H3.3 histone mutations.