scholarly journals Faculty Opinions recommendation of Sequential in cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster.

2021 ◽  
Author(s):  
Peter Verrijzer
Keyword(s):  
Hoxd Cluster ◽  
In Cis

scholarly journals Sequential in-cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster

2021 ◽  
Author(s):  
Rita Lhopitallier ◽  
Leonardo Beccari ◽  
Lucille Lopez-Delisle ◽  
Benedicte Mascrez ◽  
Jozsef Zakany ◽  
...  
Keyword(s):  
Target Genes ◽  
Gene Clusters ◽  
Ctcf Binding ◽  
Allelic Series ◽  
Hox Cluster ◽  
Hoxd Cluster ◽  
Insulator Elements ◽  
In Cis ◽  
Selection Of

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in-cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the mis-regulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, it seems that the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions and some doing both, whereas all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites amongst paralogous Hox clusters or between various vertebrates.

scholarly journals Sequential in cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster

2021 ◽  
Author(s):  
Ana Rita Amândio ◽  
Leonardo Beccari ◽  
Lucille Lopez-Delisle ◽  
Bénédicte Mascrez ◽  
Jozsef Zakany ◽  
...  
Keyword(s):  
Target Genes ◽  
Gene Clusters ◽  
Ctcf Binding ◽  
Allelic Series ◽  
Hoxd Cluster ◽  
Insulator Elements ◽  
In Cis ◽  
Regulatory Landscapes ◽  
Selection Of

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the misregulation of Hoxd genes in specific structures. Altogether, while most enhancer–promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer–promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites among paralogous Hox clusters or between various vertebrates.

An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4034-4034
Author(s):  
Lingxia Chen ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
Rita Sarkar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hek293 Cells ◽  
Light Chains ◽  
Trans Splicing ◽  
In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

scholarly journals Polydatin Attenuates Cisplatin-Induced Acute Kidney Injury by Inhibiting Ferroptosis

2022 ◽  
Vol 2022 ◽  
pp. 1-14
Author(s):  
Lu Zhou ◽  
Peng Yu ◽  
Ting-ting Wang ◽  
Yi-wei Du ◽  
Yang Chen ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Kidney Injury ◽  
Inhibitory Effect ◽  
Ros Generation ◽  
Molecular Events ◽  
Mda Content ◽  
Effect Of Pd ◽  
In Cis

Cisplatin is widely used in the treatment of solid tumors, but its application is greatly limited due to its nephrotoxicity; thus, there is still no effective medicine for the treatment of cisplatin-induced acute kidney injury (Cis-AKI). We previously identified that polydatin (PD) exerts nephroprotective effects by antioxidative stress in AKI models. Recent evidence suggests that oxidative stress-induced molecular events overlap with the process of ferroptosis and that there are common molecular targets, such as glutathione (GSH) depletion and lipid peroxidation. Nevertheless, whether the nephroprotective effect of PD is related to anti-ferroptosis remains unclear. In this study, the inhibitory effect of PD on ferroptosis was observed in both cisplatin-treated HK-2 cells (20 μM) in vitro and a Cis-AKI mouse model (20 mg/kg, intraperitoneally) in vivo, characterized by the reversion of excessive intracellular free iron accumulation and reactive oxygen species (ROS) generation, a decrease in malondialdehyde (MDA) content and GSH depletion, and an increase in glutathione peroxidase-4 (GPx4) activity. Remarkably, PD dose-dependently alleviated cell death induced by the system Xc− inhibitor erastin (10 μM), and the effect of the 40 μM dose of PD was more obvious than that of ferrostatin-1 (1 μM) and deferoxamine (DFO, 100 μM), classical ferroptosis inhibitors. Our results provide insight into nephroprotection with PD in Cis-AKI by inhibiting ferroptosis via maintenance of the system Xc−-GSH-GPx4 axis and iron metabolism.

Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae

1988 ◽  
Vol 8 (5) ◽  
pp. 2184-2194
Author(s):  
M Snyder ◽  
R J Sapolsky ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Conditions ◽  
Proper Function ◽  
Dna Fragments ◽  
Transcriptional Interference ◽  
Autonomously Replicating Sequence ◽  
Transcript Stability ◽  
Colony Color ◽  
In Cis

Transcription directed into a Saccharomyces cerevisiae autonomously replicating sequence (ARS) causes high-frequency loss of minichromosomes. Conditionally stable artificial yeast chromosomes were constructed that contain an inducible GAL promoter upstream of ARS1. Under growth conditions in which the promoter was inactive, these chromosomes were mitotically stable; however, when the GAL promoter was induced, the chromosomes became extremely unstable as a result of transcriptional impairment of ARS function. This interference by the GAL promoter occurred only in cis but can occur from either side of ARS1. Transcriptional interference of ARS function can be monitored readily by using a visual colony-color assay (P. Hieter, C. Mann, M. Snyder, and R.W. Davis, Cell 40:381-392, 1985), which was further developed as a sensitive in vivo assay for sequences which rescue ARS from transcription. DNA fragments from the 3' ends of genes, inserted downstream of the GAL promoter, protected ARS function from transcriptional interference. This assay is expected to be independent of both RNA transcript stability and processing. Philippsen et al. have shown that transcription into a yeast centromere inhibits CEN function in vivo (L. Panzeri, I. Groth-Clausen, J. Shepard, A. Stotz, and P. Philippsen, Chromosomes Today 8:46-58, 1984). We identified two 200- to 300-base-pair DNA fragments flanking CEN4 that rescued ARS1 from transcription. Both of these fragments protected ARS from transcription when inserted in either orientation. The 3' ends of stable transcripts are encoded by fragments that protected the ARS from transcription, suggesting that the protection was achieved by transcription termination. It is suggested that protection of elements important for the replication and segregation of eucaryotic chromosomes from transcription is necessary for their proper function in vivo.

scholarly journals Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis

2005 ◽  
Vol 201 (10) ◽  
pp. 1627-1635 ◽  
Author(s):  
Yok-Ai Que ◽  
Jacques-Antoine Haefliger ◽  
Lionel Piroth ◽  
Patrice François ◽  
Eleonora Widmer ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Domain ◽  
Disease Evolution ◽  
Cell Internalization ◽  
Fibrinogen Binding ◽  
In Trans ◽  
Fibronectin Binding ◽  
In Cis

The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.

scholarly journals iRAPs curb antisense transcription in E. coli

2019 ◽  
Vol 47 (20) ◽  
pp. 10894-10905 ◽  
Author(s):  
Andrés Magán ◽  
Fabian Amman ◽  
Fatinah El-Isa ◽  
Natascha Hartl ◽  
Ilya Shamovsky ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Antisense Transcription ◽  
Rna Aptamers ◽  
Antisense Strand ◽  
Genomic Context ◽  
Reporter System ◽  
E Coli ◽  
Polymerase Binding ◽  
In Cis

Abstract RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli.

scholarly journals Functional interaction between transcriptional elements in the long terminal repeat of reticuloendotheliosis virus: cooperative DNA binding of promoter- and enhancer-specific factors.

1988 ◽  
Vol 8 (12) ◽  
pp. 5232-5244 ◽  
Author(s):  
A Hirano ◽  
T Wong
Keyword(s):  
Long Terminal Repeat ◽  
Repeated Sequence ◽  
Terminal Repeat ◽  
Coding Region ◽  
Protein Coding ◽  
Cell Extracts ◽  
Cellular Factors ◽  
In Cis ◽  
Transcriptional Elements

Transcription from reticuloenodotheliosis virus strain T (REV-T), an avian retrovirus unrelated to avian leukosis and sarcoma viruses, is modulated by sequences in at least five functional domains. A promoter containing a TATA and multiple CCAAT motifs in U3 of the long terminal repeat was absolutely required for transcription. Transcriptional efficiency was greatly augmented by an enhancer immediately upstream, which contained a 22-base-pair repeated sequence. Transcription was further influenced by a negative-acting domain in the 5' region of U3 and two downstream domains in the transcribed non-protein-coding region. One of these latter domains contained a consensus enhancer core sequence and positively affected transcription in both mammalian and avian cells; the other acted negatively in a dog cell line. Transcription from REV-T in vivo required cellular factors which could be competed for specifically by the promoter or enhancer domain. The downstream domains competed with reporter genes containing these domains, but not directly with the U3 sequences. The promoter, enhancer, and the positive-acting downstream domains formed multiple complexes with distinct classes of cellular factors in both avian and mammalian cell extracts. Binding of factors to the promoter and enhancer domains was cooperative when these domains were joined in cis.

scholarly journals Capturing the onset of PRC2-mediated repressive domain formation

2018 ◽  
Author(s):  
Ozgur Oksuz ◽  
Varun Narendra ◽  
Chul-Hwan Lee ◽  
Nicolas Descostes ◽  
Gary LeRoy ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Long Range ◽  
De Novo ◽  
Histone H3 ◽  
Domain Formation ◽  
Polycomb Repressive Complex 2 ◽  
Nucleation Sites ◽  
In Cis ◽  
Step Mechanism

SummaryPolycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting “nucleation sites”, creating H3K27me3-forming polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.

scholarly journals Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Tatiana Kent ◽  
Pedro A Mateos-Gomez ◽  
Agnel Sfeir ◽  
Richard T Pomerantz
Keyword(s):  
Terminal Deoxynucleotidyl Transferase ◽  
Transferase Activity ◽  
End Joining ◽  
Switching Mechanism ◽  
Terminal Transferase ◽  
Alternative End Joining ◽  
Nucleotide Insertions ◽  
In Cis

DNA polymerase θ (Polθ) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Polθ transfers nucleotides to the 3’ terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Polθ template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Polθ terminal transferase activity is most efficient on DNA containing 3’ overhangs, is facilitated by an insertion loop and conserved residues that hold the 3’ primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Polθ to generate genetic diversity during alt-EJ and characterizes Polθ as among the most proficient terminal transferases known.

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