Control Of Gene Activity Research Articles

Can brain activity transmit transgenerationally?

Memories encoded in the parent's brain should not be able to transfer to the progeny. This assumption, which is compatible with the tenets of modern neuroscience and genetics, is challenged by new insights regarding inheritance of transgenerational epigenetic responses. Here we reflect on new discoveries regarding "molecular memories" in light of older and scandalous work on "Memory transfer" spearheaded by James V. McConnell and Georges Ungar. While the history of this field is filled with controversies, mechanisms for transmission of information across generations are being elucidated in different organisms. Most strikingly, it is now clear that in Caenorhabditis elegans nematodes, somatic responses can control gene activity in descendants via heritable small RNA molecules, and that this type of inheritance is tightly regulated by dedicated machinery. In this perspective we will focus mostly on studies conducted using C. elegans, and examine recent work on the connection between small RNAs in the nervous system and germline. We will discuss the evidence for the inheritance of brain-orchestrated behavior, and its possible significance.

Isolation of Plant Root Nuclei for Single Cell RNA Sequencing.

The characterization of the transcriptional similarities and differences existing between plant cells and cell types is important to better understand the biology of each cell composing the plant, to reveal new molecular mechanisms controlling gene activity, and to ultimately implement meaningful strategies to enhance plant cell biology. To gain a deeper understanding of the regulation of plant gene activity, the individual transcriptome of each plant cell needs to be established. Until recently, single cell approaches were mostly limited to bulk transcriptomic studies on selected cell types. Accessing specific cell types required the development of labor-intensive strategies. Recently, single cell sequencing strategies were successfully applied on isolated Arabidopsis thaliana root protoplasts. However, this strategy relies on the successful isolation of viable protoplasts upon the optimization of the enzymatic cocktails required to digest the cell wall and on the compatibility of fragile plant protoplasts with the use of microfluidic systems to generate single cell transcriptomic libraries. To overcome these difficulties, we present a simple and fast alternative strategy: the isolation and use of plant nuclei to access meaningful transcriptomic information from plant cells. This protocol was specifically developed to enable the use of the plant nuclei with 10× Genomics' Chromium technology partitions technology. Briefly, the plant nuclei are released from the root by chopping into a nuclei isolation buffer before purification by filtration then nuclei sorting. Upon sorting, the nuclei are resuspended in a low divalent ion buffer compatible with the Chromium technology in order to create single nuclei ribonucleic acid-sequencing libraries (sNucRNA-seq). © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis seed sterilization and planting Basic Protocol 2: Nuclei isolation from Arabidopsis roots Basic Protocol 3: Fluorescent-activated nuclei sorting (FANS) purification Support Protocol: Estimation of nuclei density using Countess II automated cell counter Alternate Protocol 1: Proper growth conditions for Medicago truncatula and Sorghum bicolor Alternate Protocol 2: Estimation of nuclei density using sNucRNA-seq technology.

Evolution of Regulated Transcription.

The genomes of all organisms abound with various cis-regulatory elements, which control gene activity. Transcriptional enhancers are a key group of such elements in eukaryotes and are DNA regions that form physical contacts with gene promoters and precisely orchestrate gene expression programs. Here, we follow gradual evolution of this regulatory system and discuss its features in different organisms. In eubacteria, an enhancer-like element is often a single regulatory element, is usually proximal to the core promoter, and is occupied by one or a few activators. Activation of gene expression in archaea is accompanied by the recruitment of an activator to several enhancer-like sites in the upstream promoter region. In eukaryotes, activation of expression is accompanied by the recruitment of activators to multiple enhancers, which may be distant from the core promoter, and the activators act through coactivators. The role of the general DNA architecture in transcription control increases in evolution. As a whole, it can be seen that enhancers of multicellular eukaryotes evolved from the corresponding prototypic enhancer-like regulatory elements with the gradually increasing genome size of organisms.

Molecular Basis of the Function of Transcriptional Enhancers.

Transcriptional enhancers are major genomic elements that control gene activity in eukaryotes. Recent studies provided deeper insight into the temporal and spatial organization of transcription in the nucleus, the role of non-coding RNAs in the process, and the epigenetic control of gene expression. Thus, multiple molecular details of enhancer functioning were revealed. Here, we describe the recent data and models of molecular organization of enhancer-driven transcription.

Estrogen induces dynamic ERα and RING1B recruitment to control gene and enhancer activities in luminal breast cancer.

RING1B, a core Polycomb repressive complex 1 subunit, is a histone H2A ubiquitin ligase essential for development. RING1B is overexpressed in patients with luminal breast cancer (BC) and recruited to actively transcribed genes and enhancers co-occupied by the estrogen receptor α (ERα). Whether ERα-induced transcriptional programs are mediated by RING1B is not understood. We show that prolonged estrogen administration induces transcriptional output and chromatin landscape fluctuations. RING1B loss impairs full estrogen-mediated gene expression and chromatin accessibility for key BC transcription factors. These effects were mediated, in part, by RING1B enzymatic activity and nucleosome binding functions. RING1B is recruited in a cyclic manner to ERα, FOXA1, and GRHL2 cobound sites and regulates estrogen-induced enhancers and ERα recruitment. Last, ChIP exo revealed multiple binding events of these factors at single-nucleotide resolution, including RING1B occupancy approximately 10 base pairs around ERα bound sites. We propose RING1B as a key regulator of the dynamic, liganded-ERα transcriptional regulatory circuit in luminal BC.

Controlling gene activation by enhancers through a drug-inducible topological insulator.

While regulation of gene-enhancer interaction is intensively studied, its application remains limited. Here, we reconstituted arrays of CTCF-binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. In human induced pluripotent stem cells, STITCH inserted between MYC and the enhancer down-regulated MYC. Progressive mutagenesis of STITCH led to a preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. STITCH also altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Finally, STITCH inserted near NEUROG2 impaired the gene activation in differentiating neural progenitor cells. Thus, STITCH should be broadly useful for functional genetic studies.

Engineering light-controllable CAR T cells for cancer immunotherapy.

T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.

Using of bioinformatic software methods for determining PAM sequences by analyzing of CRISPR sites

Abstract Currently, CRISPR/Cas technology is one of the most actively developing in molecular biology as an instrument for genomic DNA editing and controlling gene activity at various levels. For today this technology has a number of limitations that makes it difficult to use. One of the main limitation is the conditions under which the Cas type proteins or their analogues cut DNA: 1) the complementary between sequence of protospacer and guiding RNA; 2 )the special sequences flanking this protospacer on one or both ends. This special sequences usually unique for each enzyme and named PAM sites. However, if the ability to change guiding RNA leads to the emergence of an accurate genomic editing tool, the presence of an immutable PAM site significantly limits the areas of the genome in which editing can be performed. At the moment, more than 3.5 thousand of Cas proteins using different PAM sites have been identified in various bacteria with the help of bioinfomatic tools, but in practice less than ten PAM are used. To be able to use new proteins, it is necessary to obtain their characteristics. In particular the optimal physical and chemical conditions for enzyme, the structure of guiding RNA and PAM-sequence, characteristic for new protein. Currently, several programs have been created to determine PAM sequences for previously unexplored Cas protein analogues, but all of them prone to mistakes and require data confirmation from classical molecular biology. The purpose of this work was to create a new efficient algorithm and software for idetification of PAM sequences characteristic of any microorganism. To test the program, we used CRISPR bacterial cassettes, formed by Cas9 enzyme, as well as current homology search programs for the bacterial genomes. As a result of the study, a modular program was created that allows identifying the sequence of the PAM site based on the analysis of CRISPR cassettes with an efficiency not inferior to world analogues.

An enhancer cluster controls gene activity and topology of the SCN5A-SCN10A locus in vivo

Mutations and variations in and around SCN5A, encoding the major cardiac sodium channel, influence impulse conduction and are associated with a broad spectrum of arrhythmia disorders. Here, we identify an evolutionary conserved regulatory cluster with super enhancer characteristics downstream of SCN5A, which drives localized cardiac expression and contains conduction velocity-associated variants. We use genome editing to create a series of deletions in the mouse genome and show that the enhancer cluster controls the conformation of a >0.5 Mb genomic region harboring multiple interacting gene promoters and enhancers. We find that this cluster and its individual components are selectively required for cardiac Scn5a expression, normal cardiac conduction and normal embryonic development. Our studies reveal physiological roles of an enhancer cluster in the SCN5A-SCN10A locus, show that it controls the chromatin architecture of the locus and Scn5a expression, and suggest that genetic variants affecting its activity may influence cardiac function.

An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing

BackgroundTetracycline (Tet)-regulated expression system has become a widely applied tool to control gene activity. This study aimed to improve the Tet-on system with superior regulatory characteristics.ResultsBy comprehensively comparing factors of transactivators, Tet-responsive elements (TREs), orientations of induced expression cassette, and promoters controlling the transactivator, we developed an optimal Tet-on system with enhanced inducible efficiency and lower leakiness. With the system, we successfully performed effective inducible and reversible expression of microRNA, and presented a more precise and easily reproducible fine-tuning for confirming the target of a miRNA. Finally, the system was applied in CRISPR/Cas9-mediated knockout of nuclear factor of activated T cells-5 (NFAT5), a protective transcription factor in cellular osmoregulation.ConclusionsThis study established an improved Tet-on system for powerful and stringent gene regulation in functional genetic studies.

Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity.

Enhancers are important regulatory elements that can control gene activity across vast genetic distances. However, the underlying nature of this regulation remains obscured because it has been difficult to observe in living cells. Here, we visualize the spatial organization and transcriptional output of the key pluripotency regulator Sox2 and its essential enhancer Sox2 Control Region (SCR) in living embryonic stem cells (ESCs). We find that Sox2 and SCR show no evidence of enhanced spatial proximity and that spatial dynamics of this pair is limited over tens of minutes. Sox2 transcription occurs in short, intermittent bursts in ESCs and, intriguingly, we find this activity demonstrates no association with enhancer proximity, suggesting that direct enhancer-promoter contacts do not drive contemporaneous Sox2 transcription. Our study establishes a framework for interrogation of enhancer function in living cells and supports an unexpected mechanism for enhancer control of Sox2 expression that uncouples transcription from enhancer proximity.

Transcriptional Bursts in a Nonequilibrium Model for Gene Regulation by Supercoiling

We analyze transcriptional bursting within a stochastic nonequilibrium model, which accounts for the coupling between the dynamics of DNA supercoiling and gene transcription. We find a clear signature of bursty transcription when there is a separation between the timescales of transcription initiation and supercoiling dissipation (the latter may either be diffusive or mediated by topological enzymes, such as type I or type II topoisomerases). In multigenic DNA domains, we observe either bursty transcription or transcription waves; the type of behavior can be selected for by controlling gene activity and orientation. In the bursty phase, the statistics of supercoiling fluctuations at the promoter are markedly non-Gaussian.

Analysis of histone ubiquitylation by MSL1/MSL2 proteins in vitro

Histone posttranslational modifications (PTM) control gene activity by targeting chromatin-regulatory proteins. By altering histone charges PTMs could also modulate inter- and intra-nucleosomal interactions, and thus affect chromatin high-order compaction and nucleosome stochastic folding, respectively. However, recently it has been shown that histone H2BK34- ubiquitylation (which is deposited in vivo by MOF-MSL) can destabilize one of the nucleosomal H2A-H2B dimers in symmetrically and (albeit to a lesser extend) asymmetrically modified nucleosomes, and thus promote formation of a hexasome particle. Here we have studied ubiquitylation patterns by purified MSL1/MSL2 using nucleosomes and different histone substrates. We have shown that H2B-ubiquitylation by MSL1/2 depends on substrate configuration. In addition, MSL1/2 efficiently ubiquitylate histone substrates but very poorly modify nucleosomes, which implies a requirement for nucleosome structural alteration for efficient ubiquitylation of H2BK34. Nucleosome modification by MSL1/MSL2 in vitro was analyzed directly using nucleosome gel-mobility shift assay, which suggested that MSL1/2 can deposit two ubiquitin moieties in one nucleosome.

Functional polymorphism within miR-23a∼27a∼24-2 cluster confers clinical outcome of breast cancer in Pakistani cohort.

MicroRNAs (miRNAs) are small regulatory RNA molecules that control gene activity by base pairing with target messenger RNA leading to their cleavage or translational repression. Previous studies show an involvement of miRNAs in various diseases including cancer. Members of the Mir-23a cluster (MIR23A, MIR24-2and MIR27A) are involved in breast cancer (BC). In the present study, miR-23a/24-2/27a cluster was screened for genetic mutation in BC patients. Heterozygous (A/G allele) as well as homozygous (G/G allele) variants were found in mir-27a gene in screened BC patients. RNA structural analysis revealed that the single nucleotide polymorphism (SNP) effects the size of the terminal loop in the precursor miRNA (pre-miRNA). The altered (G allele) hairpin structure observed was two bases longer than the reference (A allele) hairpin.

The histone demethylase UTX/KDM6A in cancer: Progress and puzzles.

The lysine-specific demethylase 6A/UTX (gene name KDM6A) acts as a component of the COMPASS complex to control gene activation. UTX demethylates H3K27me2/3 at genes and enhancers. Deleterious mutations in KDM6A are found in many cancer types, prominently urothelial carcinoma and certain T-cell leukemias. In certain cancers, however, UTX supports oncogenic transcription factors, e.g. steroid hormone receptors in breast and prostate cancer. In fetal development, UTX regulates lineage choice and cell differentiation. Analogously, loss of UTX function in cancer may lead to metaplasia or impede differentiation. Likely because its function is contingent on its interacting transcription factors, the effects of UTX inactivation are not uniform and require detailed investigation in each cancer type. In urothelial carcinoma, in particular, the functional consequences of the frequent mutations in KDM6A and other COMPASS component genes are poorly understood. Nevertheless, UTX inactivation appears to sensitize many cancers to inhibitors of the H3K27 methyltransferase EZH2. Conversely, inhibitors of UTX enzymatic activity may be applicable in cancers with an oncogenic UTX function. Intriguingly, the fact that KDM6A is localized on the X-chromosome, but both copies are expressed, may account for gender-specific differences in cancer susceptibility. In conclusion, despite recent progress, many open questions need to be addressed, most importantly, the detailed mechanisms by which KDM6A inactivation promotes various cancers, but also with which proteins UTX interacts in and apart from the COMPASS complex, and to which extent its catalytic function is required for its tumor-suppressive function.

Buried in PEAT-discovery of a new silencing complex with opposing activities.

Posttranslational histone modifications serve critical roles in gene regulation by determining the functional status of chromatin. Histone‐modifying enzymes often work in large multiprotein complexes. A paper in this issue of The EMBO Journal describes a new chromatin‐modifying complex called PEAT that acts via histone deacetylation. The PEAT complex is involved in heterochromatin formation and gene repression but also appears to have a locus‐specific activating role, possibly through promoting histone acetylation.

Seed genome hypomethylated regions are enriched in transcription factor genes

The precise mechanisms that control gene activity during seed development remain largely unknown. Previously, we showed that several genes essential for seed development, including those encoding storage proteins, fatty acid biosynthesis enzymes, and transcriptional regulators (e.g., ABI3, FUS3) are located within hypomethylated regions of the soybean genome. These hypomethylated regions are similar to the DNA methylation valleys (DMVs), or canyons, found in mammalian cells. Here, we address the question of the extent to which DMVs are present within seed genomes and what role they might play in seed development. We scanned soybean and Arabidopsis seed genomes from postfertilization through dormancy and germination for regions that contain <5% or <0.4% bulk methylation in CG, CHG, and CHH contexts over all developmental stages. We found that DMVs represent extensive portions of seed genomes, range in size from 5-76 kb, are scattered throughout all chromosomes, and are hypomethylated throughout the plant life cycle. Significantly, DMVs are enriched greatly in transcription factor (TF) genes and other developmental genes that play critical roles in seed formation. Many DMV genes are regulated with respect to seed stage, region, and tissue, and contain H3K4me3, H3K27me3, or bivalent marks that fluctuate during development. Our results indicate that DMVs are a unique regulatory feature of both plant and animal genomes, and that a large number of seed genes are regulated in the absence of methylation changes during development, probably by the action of specific TFs and epigenetic events at the chromatin level.

1. A light inducible gene activation system toward controllable cell‐based therapeutics

T cells engineered to express chimeric antigen receptors (CARs) on cell surface can recognize and engage with target cancer cells with redirected specificity. This CAR T cell‐mediated immunotherapy is under intensive investigation as a revolutionary therapy for cancer. However, adverse effects have been reported, including on‐target off‐tumor cytotoxicity, cytokine release syndrome and tumor lysis syndrome. One of the solutions is to engineer on/off‐switchable cells to achieve high‐precision control over their activation. Optogenetics utilizing genetically encoded dimerizers has been applied to remotely activate gene expression. Here, we developed a new blue light‐mediated light‐inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control cell activation, by integrating a LOV2‐based light‐inducible nuclear localization signal with CRY2‐CIB1 dimerization. We demonstrated light‐controllable gene expression and functional modulation in HEK 293T and Jurkat T cell lines. We further applied the LINTAD system in primary human CAR T cells and showed that the light‐stimulated cells possessed a significantly stronger cytotoxicity against target cancer cells than the non‐stimulated ones. We also extended the LINTAD system to activate the expression of the TNF‐related apoptosis‐inducing ligand (TRAIL) in HEK 293T cells, and observed a gain of cytotoxicity upon light stimulation. Therefore, our newly developed LINTAD system can serve as an efficient and general tool to remotely and non‐invasively control gene activation in live cells for therapeutic applications.Support or Funding InformationThis work is supported by grants from NIH HL121365, GM125379 (S.C., Y. Wang), CA204704, CA209629, NSF CBET1360341, DMS1361421 (Y. Wang), and UC San Diego.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential expression profiles of conserved Snail transcription factors in the mouse testis.

Snail transcription factors are key regulators of cellular transitions during embryonic development and tumorigenesis. The closely related SNAI1 and SNAI2 proteins induce epithelial-mesenchymal transitions (EMTs), acting predominantly as transcriptional repressors, while the functions of SNAI3 are unknown. An initial examination of Snai2-deficient mice provided evidence of deficient spermatogenesis. To address the hypothesis that Snail proteins are important for male fertility, this study provides the first comprehensive cellular expression profiles of all three mammalian Snail genes in the post-natal mouse testis. To evaluate Snail transcript expression profiles, droplet digital (dd) PCR and insitu hybridization were employed. Snai1, 2 and 3 transcripts are readily detected at 7, 14, 28days post-partum (dpp) and 7weeks (adult). Unique cellular expression was demonstrated for each by insitu hybridization and immunohistochemistry using Western blot-validated antibodies. SNAI1 and SNAI2 are in the nucleus of the most mature germ cell types at post-natal ages 10, 15 and 26. SNAI3 is only detected from 15 dpp onwards and is localized in the Sertoli cell cytoplasm. In the adult testis, Snai1 and Snai2 transcripts are detected in spermatogonia and spermatocytes, while Snai3 is in both germ and Sertoli cells. SNAI1 protein is evident in nuclei of spermatogonia, spermatocytes, round spermatids and elongated spermatids (Stages IX-XII). SNAI2 is present in the nuclei of spermatogonia and spermatocytes, with a faint signal detected in round spermatids. SNAI3 was detected only in Sertoli cell cytoplasm, as in juvenile testes. Additionally, colocalization of SNAI1 and SNAI2 with previously identified key binding partners, LSD1 and PRC2 complex components, provides strong evidence that these important functional interactions are conserved during spermatogenesis to control gene activity. These distinct expression profiles suggest that each Snail family member has unique functions during spermatogenesis.

The role of epigenetics in renal ageing.

An ability to separate natural ageing processes from processes specific to morbidities is required to understand the heterogeneity of age-related organ dysfunction. Mechanistic insight into how epigenetic factors regulate ageing throughout the life course, linked to a decline in renal function with ageing, is already proving to be of value in the analyses of clinical and epidemiological cohorts. Noncoding RNAs provide epigenetic regulatory circuits within the kidney, which reciprocally interact with DNA methylation processes, histone modification and chromatin. These interactions have been demonstrated to reflect the biological age and function of renal allografts. Epigenetic factors control gene expression and activity in response to environmental perturbations. They also have roles in highly conserved signalling pathways that modulate ageing, including the mTOR and insulin/insulin-like growth factor signalling pathways, and regulation of sirtuin activity. Nutrition, the gut microbiota, inflammation and environmental factors, including psychosocial and lifestyle stresses, provide potential mechanistic links between the epigenetic landscape of ageing and renal dysfunction. Approaches to modify the renal epigenome via nutritional intervention, targeting the methylome or targeting chromatin seem eminently feasible, although caution is merited owing to the potential for intergenerational and transgenerational effects.