scholarly journals HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex

2021 ◽  
Author(s):  
Matthias S. Leisegang ◽  
Jasleen Kaur Bains ◽  
Sandra Seredinski ◽  
James A. Oo ◽  
Nina M. Krause ◽  
...  
Keyword(s):  
Loss Of Function ◽  
Cellular Functions ◽  
Eph Receptor ◽  
Triplex Formation ◽  
Expression Control ◽  
Gene Expression Control ◽  
Biophysical Studies ◽  
Oxygen Sensitive ◽  
Hoogsteen Base Pairing

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduced the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 was down-regulated in pulmonary hypertension and loss-of-function approaches not only resulted in gene de-repression but also enhanced angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

scholarly journals Synthetic gene regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae

2019 ◽  
Author(s):  
Robin A. Sorg ◽  
Clement Gallay ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Regulatory Networks ◽  
Expression Patterns ◽  
Combinatorial Libraries ◽  
Regulatory Elements ◽  
Expression Control ◽  
Gene Expression Control

AbstractStreptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND and IMPLY gates. Finally, we demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

scholarly journals TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome

2019 ◽  
Vol 30 (12) ◽  
pp. 2338-2353 ◽  
Author(s):  
Lina L. Kampf ◽  
Ronen Schneider ◽  
Lea Gerstner ◽  
Roland Thünauer ◽  
Mengmeng Chen ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Slit Diaphragm ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Steroid Resistant ◽  
Steroid Resistant Nephrotic Syndrome ◽  
Whole Exome ◽  
Immortalized Cell

BackgroundMutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology.MethodsWe used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). We analyzed the functional significance of an SRNS-associated gene in vitro and in podocyte-like Drosophila nephrocytes.ResultsWe identified hemizygous missense mutations in the gene TBC1D8B in five families with nephrotic syndrome. Coimmunoprecipitation assays indicated interactions between TBC1D8B and active forms of RAB11. Silencing TBC1D8B in HEK293T cells increased basal autophagy and exocytosis, two cellular functions that are independently regulated by RAB11. This suggests that TBC1D8B plays a regulatory role by inhibiting endogenous RAB11. Coimmunoprecipitation assays showed TBC1D8B also interacts with the slit diaphragm protein nephrin, and colocalizes with it in immortalized cell lines. Overexpressed murine Tbc1d8b with patient-derived mutations had lower affinity for endogenous RAB11 and nephrin compared with wild-type Tbc1d8b protein. Knockdown of Tbc1d8b in Drosophila impaired function of the podocyte-like nephrocytes, and caused mistrafficking of Sns, the Drosophila ortholog of nephrin. Expression of Rab11 RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane, whereas RAB11 overexpression revealed a partial phenotypic overlap to Tbc1d8b loss of function.ConclusionsNovel mutations in TBC1D8B are monogenic causes of SRNS. This gene inhibits RAB11. Our findings suggest that RAB11-dependent vesicular nephrin trafficking plays a role in the pathogenesis of nephrotic syndrome.

Modifying the Epigenome as a Therapeutic Strategy in Myelodysplasia

Hematology ◽  
2007 ◽  
Vol 2007 (1) ◽  
pp. 405-411 ◽  
Author(s):  
Guillermo Garcia-Manero
Keyword(s):  
Dna Methylation ◽  
Clinical Information ◽  
Histone Code ◽  
Epigenetic Therapy ◽  
Epigenetic Alterations ◽  
Expression Control ◽  
Gene Expression Control ◽  
Clinical Interest

AbstractThe term epigenetics refers to a number of biochemical modifications of chromatin that, without altering the primary sequence of DNA, have a role in genomic regulation and in particular gene expression control. These modifications can occur at the DNA level (i.e., DNA methylation), and affect the chromatin protein scaffold (i.e., histone code modifications), among several others. The study of these modifications is a very active area of research both at the basic and clinical levels. Clinical interest in these epigenetic alterations stems mainly from two observations. First, detection of specific epigenetic alterations could be used to develop cancer biomarkers (e.g., for the early detection or prognostication of cancer). Second, most epigenetic alterations are reversible both in vitro and in vivo, leading the way to the development of new anticancer therapies. This review focuses on the current clinical information regarding different forms of epigenetic therapy in patients with myelodysplastic syndromes (MDS). Basic aspects of DNA methylation or histone code alterations are not covered in detail in this review.

scholarly journals DivergentIn vitroMIC Characteristics and underlying isogenic mutations in host-specializedPseudomonas aeruginosa

2017 ◽  
Author(s):  
Xuan Qin ◽  
Alexander L Greninger ◽  
Chuan Zhou ◽  
Amanda Adler ◽  
Shuhua Yuan ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agents ◽  
Drug Susceptibility ◽  
Resistance Rate ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Dna Mismatch ◽  
Compare And Contrast

AbstractClinical isolates ofPseudomonas aeruginosa (Pa)from patients with cystic fibrosis (CF) are known to differ from those associated with infections of non-CF hosts in colony morphology, drug susceptibility patterns, and genomic hypermutability. AlthoughPaisolates from CF have long been recognized for their overall higher resistance rate calculated generally by reduced “percent susceptible”, this study takes the approach to compare and contrast Etest MIC distributions between two distinct cohorts of clinical strains (n=224 from 56 CF patients and n=130 from 68 non-CF patients respectively) isolated in 2013. Logarithmic transformed MIC (logMIC) values of 11 antimicrobial agents were compared between the two groups. CF isolates tended to produce heterogeneous and widely dispersed MICs compared to non-CF isolates. By applying a test for equality of variances, we were able to confirm that the MICs generated from CF isolates against 9 out of the 11 agents were significantly more dispersed than those from non-CF (p<0.02-<0.001). Quantile-quantiles plots indicated little agreement between the two cohorts of isolates. Based on whole genome sequencing of 19 representative CFPaisolates, divergent gain- or loss-of-function mutations in efflux and porin genes and their regulators between isogenic or intra-clonal associates were evident. Not one, not a few, but the net effect all adaptive mutational changes in the genomes of CFPa, both shared and unshared between isogenic strains, are responsible for the divergent heteroresistance patterns. Moreover, the isogenic variations are suggestive of a bacterial syntrophic lifestyle when “lockedȍ inside a host focal airway environment over prolonged periods.Significance statementBacterial heteroresistance is associated with niche specialized organisms interacting with host species for prolonged period of time, medically characterized by “chronic focal infections”. A prime example is found inPseudomonas aeruginosaisogenic/non-homogeneous isolates from patient airways with cystic fibrosis. The development of pseudomonal polarizing MICsin vitroto many actively used antimicrobial agents among isogenic isolates and “Eagle-type” heteroresistance patterns are common and characteristic. Widespread isogenic gene lesions were evident for defects in drug transporters, DNA mismatch repair, and many other structural or cellular functions—a result of pseudomonal symbiotic response to host selection. Co-isolation of extremely susceptible and resistant isogenicPastrains suggests intra-airway evolution of a multicellular syntrophic bacterial lifestyle, which has laboratory interpretation and clinical treatment implications.

scholarly journals Molecular and Phenotypic Analysis of 25 Recessive, Homozygous-Viable Alleles at the Mouse agouti Locus

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 659-674
Author(s):  
Rosalynn J Miltenberger ◽  
Kazumasa Wakamatsu ◽  
Shosuke Ito ◽  
Richard P Woychik ◽  
Liane B Russell ◽  
...  
Keyword(s):  
Loss Of Function ◽  
Wild Type ◽  
Allelic Series ◽  
Phenotypic Analysis ◽  
Protein Coding ◽  
Agouti Locus ◽  
Expression Control ◽  
Gene Expression Control ◽  
Specific Locus

Abstract Agouti is a paracrine-acting, transient antagonist of melanocortin 1 receptors that specifies the subapical band of yellow on otherwise black hairs of the wild-type coat. To better understand both agouti structure/function and the germline damage caused by chemicals and radiation, an allelic series of 25 recessive, homozygous-viable agouti mutations generated in specific-locus tests were characterized. Visual inspection of fur, augmented by quantifiable chemical analysis of hair melanins, suggested four phenotypic categories (mild, moderate, umbrous-like, severe) for the 18 hypomorphs and a single category for the 7 amorphs (null). Molecular analysis indicated protein-coding alterations in 8 hypomorphs and 6 amorphs, with mild-moderate phenotypes correlating with signal peptide or basic domain mutations, and more devastating phenotypes resulting from C-terminal lesions. Ten hypomorphs and one null demonstrated wild-type coding potential, suggesting that they contain mutations elsewhere in the ≥125-kb agouti locus that either reduce the level or alter the temporal/spatial distribution of agouti transcripts. Beyond the notable contributions to the field of mouse germ cell mutagenesis, analysis of this allelic series illustrates that complete abrogation of agouti function in vivo occurs most often through protein-coding lesions, whereas partial loss of function occurs slightly more frequently at the level of gene expression control.

scholarly journals Noise-reducing optogenetic negative-feedback gene circuits in human cells

2019 ◽  
Vol 47 (14) ◽  
pp. 7703-7714 ◽  
Author(s):  
Michael Tyler Guinn ◽  
Gábor Balázsi
Keyword(s):  
Gene Expression ◽  
Noise Reduction ◽  
Negative Feedback ◽  
Mammalian Cells ◽  
Cellular Proliferation ◽  
Protein Domain ◽  
Gene Circuits ◽  
Expression Control ◽  
Gene Expression Control

Abstract Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology and other biomedical fields of research.

Embryonic mesodermal defects in alpha 5 integrin-deficient mice

Development ◽  
1993 ◽  
Vol 119 (4) ◽  
pp. 1093-1105 ◽  
Author(s):  
J.T. Yang ◽  
H. Rayburn ◽  
R.O. Hynes
Keyword(s):  
Embryonic Stem ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Fibronectin Receptor ◽  
Beta 1 Integrin ◽  
Integrin Gene ◽  
Posterior Trunk ◽  
Mutant Cells ◽  
Deficient Mice

A loss of function mutation of the murine alpha 5 integrin gene generated by gene targeting in embryonic stem cells is a recessive embryonic lethal. The mutant embryos start to show observable defects by day 9 of gestation and die around day 10–11. The alpha 5-null embryos have pronounced defects in posterior trunk and yolk sac mesodermal structures, suggesting a role for alpha 5 beta 1 integrin in mesoderm formation, movement or function. However, the embryos progress significantly further than embryos null for fibronectin, for which alpha 5 beta 1 integrin is a receptor, suggesting the involvement of other fibronectin receptors. In vitro studies on cells derived from the alpha 5-null embryos confirm that the alpha 5 beta 1 integrin is not expressed on mutant cells and show that the mutant cells are able to assemble fibronectin matrix, form focal contacts, and migrate on fibronectin despite the complete absence of the alpha 5 beta 1 fibronectin receptor integrin. All these functions have previously been thought to involve or require alpha 5 beta 1. The results presented show that these cellular functions involving fibronectin can proceed using other receptors.

scholarly journals Epigenetic Control of Circadian Clock Operation during Development

2012 ◽  
Vol 2012 ◽  
pp. 1-8
Author(s):  
Chengwei Li ◽  
Changxia Gong ◽  
Shuang Yu ◽  
Jianguo Wu ◽  
Xiaodong Li
Keyword(s):  
Circadian Clock ◽  
Cell Types ◽  
Circadian Oscillation ◽  
Chromatin Modifications ◽  
Circadian Gene ◽  
Cellular Functions ◽  
Future Studies ◽  
Expression Control ◽  
Gene Expression Control ◽  
Genomic Scale

The molecular players of circadian clock oscillation have been identified and extensively characterized. The epigenetic mechanisms behind the circadian gene expression control has also been recently studied, although there are still details to be illucidated. In this review, we briefly summarize the current understanding of the mammalian clock. We also provide evidence for the lack of circadian oscillation in particular cell types. As the circadian clock has intimate interaction with the various cellular functions in different type of cells, it must have plasticity and specicity in its operation within different epigenetic environments. The lack of circadian oscillation in certain cells provide an unique opportunity to study the required epigenetic environment in the cell that permit circadian oscillation and to idenfify key influencing factors for proper clock function. How epigenetic mechansims, including DNA methylaiton and chromatin modifications, participate in control of clock oscillation still awaits future studies at the genomic scale.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

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