Construction and Evaluation of Self-Directing Expression System Using Regulatory Elements of Cry Gene of Bacillus Thuringiensis

An expression system based on the cry gene regulatory elements was constructed. The Terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1 was cloned in pSG1151 plasmid downstream to gfpmut1. The promoter region of the cry gene was amplified to give three different reading frames. The Promoter region of cry gene was cloned in pSG1151T plasmid upstream to gfpmut1. The expression of GFP under the promoter/terminator expression system was evaluated by checking the expression of gfpmut1 under the same promoter. The GFP content of pSG1151 and three constructs; pDSA1, pDSA2 and pDSA3 were compared by fluorescence spectroscopy. The fluorescent intensity of pSG1151 and pDSA1 were compared at time interval of 6 hours upto 72 hours. Both the samples showed detectable fluorescence that increased with time up to 12 hours, but the increase in the fluorescence of pDSA1 was 3 times higher as compared to pSG1151. A cold peptidase gene was cloned under the control of the cry promoter. The transformed E.coli DH5α colonies were patched on skim milk agar plates and the clones of pSG1151CP and pDSA1CP were compared on the basis of zone of clearance. The zone of clearance of pDSA1CP was much higher as compared to that of pSG1151CP. The cell-free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the specific activity of the extracellular protease. At 72 hours the protease activity in pDSA1CP was 2.7 fold higher compared to that of wild Bacillus sp. S1DI 10.

Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

Precise control of gene expression underpins normal development. This relies on mechanisms that enable communication between gene promoters and other regulatory elements. In embryonic stem cells (ESCs), the CDK-Mediator (CDK-MED) complex has been reported to physically link gene regulatory elements to enable gene expression and also prime genes for induction during differentiation. Here we discover that CDK-MED contributes little to 3D genome organisation in ESCs, but has a specific and essential role in controlling interactions between inactive gene regulatory elements bound by Polycomb repressive complexes (PRCs). These interactions are established by the canonical PRC1 (cPRC1) complex but rely on CDK-MED, which facilitates binding of cPRC1 to its target sites. Importantly, through separation of function experiments, we reveal that this collaboration between CDK-MED and cPRC1 in creating long-range interactions does not function to prime genes for induction during differentiation. Instead, we discover that priming relies on an interaction-independent mechanism whereby the CDK module supports core Mediator engagement with gene promoters to enable gene activation.

The RNA chaperone StpA enables fast RNA refolding by destabilization of mutually exclusive base pairs within competing secondary structure elements

In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.

GABAergic Gene Regulatory Elements Used in Adeno-Associated Viral Vectors

Several neurological and psychiatric disorders have been associated with impairments in GABAergic inhibitory neurons in the brain. Thus, in the current era of accelerated development of molecular medicine and biologically-based drugs, there is a need to identify gene regulatory sequences that can be utilized for selectively manipulating the expression of nucleic acids and proteins in GABAergic neurons. This is particularly important for the use of viral vectors in gene therapy. In this Mini Review, we discuss the use of various gene regulatory elements for targeting GABAergic neurons, with an emphasis on adeno-associated viral vectors, the most widely used class of viral vectors for treating brain diseases.

Transcriptional Regulation of RUNX1: An Informatics Analysis

The RUNX1/AML1 gene encodes a developmental transcription factor that is an important regulator of haematopoiesis in vertebrates. Genetic disruptions to the RUNX1 gene are frequently associated with acute myeloid leukaemia. Gene regulatory elements (REs), such as enhancers located in non-coding DNA, are likely to be important for Runx1 transcription. Non-coding elements that modulate Runx1 expression have been investigated over several decades, but how and when these REs function remains poorly understood. Here we used bioinformatic methods and functional data to characterise the regulatory landscape of vertebrate Runx1. We identified REs that are conserved between human and mouse, many of which produce enhancer RNAs in diverse tissues. Genome-wide association studies detected single nucleotide polymorphisms in REs, some of which correlate with gene expression quantitative trait loci in tissues in which the RE is active. Our analyses also suggest that REs can be variant in haematological malignancies. In summary, our analysis identifies features of the RUNX1 regulatory landscape that are likely to be important for the regulation of this gene in normal and malignant haematopoiesis.

A Reassessment of Copy Number Variations in Congenital Heart Defects: Picturing the Whole Genome

Copy number variations (CNVs) can modulate phenotypes by affecting protein-coding sequences directly or through interference of gene expression. Recent studies in cancer and limb defects pinpointed the relevance of non-coding gene regulatory elements such as long non-coding RNAs (lncRNAs) and topologically associated domain (TAD)-related gene-enhancer interactions. The contribution of such non-coding elements is largely unexplored in congenital heart defects (CHD). We performed a retrospective analysis of CNVs reported in a cohort of 270 CHD patients. We reviewed the diagnostic yield of pathogenic CNVs, and performed a comprehensive reassessment of 138 CNVs of unknown significance (CNV-US), evaluating protein-coding genes, lncRNA genes, and potential interferences with TAD-related gene-enhancer interactions. Fifty-two of the 138 CNV-US may relate to CHD, revealing three candidate CHD regions, 19 candidate CHD genes, 80 lncRNA genes of interest, and six potentially CHD-related TAD interferences. Our study thus indicates a potential relevance of non-coding gene regulatory elements in CNV-related CHD pathogenesis. Shortcomings in our current knowledge on genomic variation call for continuous reporting of CNV-US in international databases, careful patient counseling, and additional functional studies to confirm these preliminary findings.

Gene-regulatory elements may change the amount, timing, or location of gene expression, cis-Regulation therapy platforms might become a gene therapy to treat many genetic diseases

Changes in gene expression levels above or below a particular threshold may have a dramatic impact on phenotypes, leading to a wide spectrum of human illnesses. Gene-regulatory elements, also known as cis-regulatory elements (CREs), may change the amount, timing, or location (cell/tissue type) of gene expression, whereas mutations in a gene's coding sequence may result in lower or higher gene expression levels resulting in protein loss or gain. Loss-of-function mutations in both genes produce recessive human illness, while haploinsufficient mutations in 65 genes are also known to be deleterious due to function gain, according to the ClinVar1 and ClinGen3 databases. CREs are promoters living near to a gene's transcription start site and switching it on at predefined times, places, and levels. Other distal CREs, like enhancers and silencers, are temporal and tissue-specific control promoters. Enhancers activate promoters, commonly referred to as "promoters," whereas silencers turn them off. Insulators also restrict promiscuous interactions between enhancers and gene promoters. Systematic genomic approaches can help understand the cis-regulatory circuitry of gene expression by highly detecting and functionally defining these CREs. This includes the new use of CRISPR–CRISPR-associated protein 9 (CRISPR–Cas9) and other editing approaches to discover CREs. Cis-Regulation therapy (CRT) provides many promises to heal human ailments. CRT may be used to upregulate or downregulate disease-causing genes due to lower or higher levels of expression, and it may also be used to precisely adjust the expression of genes that assist in alleviating disease features. CRT may employ proteins that generate epigenetic modifications like methylation, histone modification, or gene expression regulation looping. Weighing CRT's advantages and downsides against alternative treatment methods is crucial. CRT platforms might become a practical technique to treat many genetic diseases that now lack treatment alternatives if academics, patient communities, clinicians, regulators and industry work together.

Methods of massive parallel reporter assays for investigation of enhancers

The correct deployment of genetic programs for development and differentiation relies on finely coordinated regulation of specific gene sets. Genomic regulatory elements play an exceptional role in this process. There are few types of gene regulatory elements, including promoters, enhancers, insulators and silencers. Alterations of gene regulatory elements may cause various pathologies, including cancer, congenital disorders and autoimmune diseases. The development of high-throughput genomic assays has made it possible to significantly accelerate the accumulation of information about the characteristic epigenetic properties of regulatory elements. In combination with high-throughput studies focused on the genome-wide distribution of epigenetic marks, regulatory proteins and the spatial structure of chromatin, this significantly expands the understanding of the principles of epigenetic regulation of genes and allows potential regulatory elements to be searched for in silico. However, common experimental approaches used to study the local characteristics of chromatin have a number of technical limitations that may reduce the reliability of computational identification of genomic regulatory sequences. Taking into account the variability of the functions of epigenetic determinants and complex multicomponent regulation of genomic elements activity, their functional verification is often required. A plethora of methods have been developed to study the functional role of regulatory elements on the genome scale. Common experimental approaches for in silico identification of regulatory elements and their inherent technical limitations will be described. The present review is focused on original high-throughput methods of enhancer activity reporter analysis that are currently used to validate predicted regulatory elements and to perform de novo searches. The methods described allow assessing the functional role of the nucleotide sequence of a regulatory element, to determine its exact boundaries and to assess the influence of the local state of chromatin on the activity of enhancers and gene expression. These approaches have contributed substantially to the understanding of the fundamental principles of gene regulation.

The transcriptional landscape of a rewritten bacterial genome reveals control elements and genome design principles

Sequence rewriting enables low-cost genome synthesis and the design of biological systems with orthogonal genetic codes. The error-free, robust rewriting of nucleotide sequences can be achieved with a complete annotation of gene regulatory elements. Here, we compare transcription in Caulobacter crescentus to transcription from plasmid-borne segments of the synthesized genome of C. ethensis 2.0. This rewritten derivative contains an extensive amount of supposedly neutral mutations, including 123’562 synonymous codon changes. The transcriptional landscape refines 60 promoter annotations, exposes 18 termination elements and links extensive transcription throughout the synthesized genome to the unintentional introduction of sigma factor binding motifs. We reveal translational regulation for 20 CDS and uncover an essential translational regulatory element for the expression of ribosomal protein RplS. The annotation of gene regulatory elements allowed us to formulate design principles that improve design schemes for synthesized DNA, en route to a bright future of iteration-free programming of biological systems.

Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function

Lung cancer is the leading cause of cancer deaths worldwide and remains one of the most incurable. Tyrosine kinase receptors, such as the epidermal growth factor receptor (EGFR), are often aberrantly activated and drive tumor growth. Monotherapy with tyrosine kinase inhibitors to deactivate EGFR has shown initial efficacy, but their benefits tend to decline over time. EGFR acts as a transcriptional factor promoting the expression of co-oncogenic drivers, which, in turn, interact with canonical EGFR mutations to induce therapeutic relapse. This study reports that sortilin, a crucial regulator of cytoplasmic EGFR, attenuates its transducing function. Genome-wide chromatin binding revealed that sortilin interacts with gene regulatory elements occupied by EGFR. These results suggest a model, in which sortilin exhibits potential tumor suppressor-like activity by concurrently binding to regulatory elements of cMYC. Sortilin expression in lung adenocarcinoma may be predictive of the efficacy of anti-EGFR strategies.