scholarly journals De novo activated transcription of newborn coding sequences is inheritable in the plant genome

2020 ◽  
Author(s):  
Takayuki Hata ◽  
Naoto Takada ◽  
Chihiro Hayakawa ◽  
Mei Kazama ◽  
Tomohiro Uchikoba ◽  
...  
Keyword(s):  
Cultured Cells ◽  
De Novo ◽  
Gene Evolution ◽  
Firefly Luciferase ◽  
Plant Genome ◽  
Experimental Scheme ◽  
Transgenic Lines ◽  
Endogenous Genes ◽  
The Individual ◽  
Comparison Of The Results

ABSTRACTThe manner in which newborn genes become transcriptionally activated and fixed in the plant genome is poorly understood. To examine such processes of gene evolution, we performed an artificial evolutionary experiment in Arabidopsis thaliana. As a model of gene-birth events, we introduced a promoterless coding sequence of the firefly luciferase (LUC) gene and established 386 T2-generation transgenic lines. Among them, we determined the individual LUC insertion loci in 76 lines and found that one-third of them were transcribed de novo even in the intergenic or inherently unexpressed regions. In the transcribed lines, transcription-related epigenetic marks were detected across the newly activated transcribed regions. These results agreed with our previous findings in A. thaliana cultured cells under a similar experimental scheme. The comparison of the results of the T2-plant and cultured cell experiments revealed that the de novo-activated transcription caused by local chromatin remodelling was inheritable. During one-generation inheritance, it seems likely that the transcription activities of the LUC inserts trapped by the endogenous genes/transcripts became stronger, while those of de novo transcription in the intergenic/untranscribed regions became weaker. These findings may offer a clue for the elucidation of the mechanism via which newborn genes become transcriptionally activated and fixed in the plant genome.

scholarly journals De novo activated transcription of inserted foreign coding sequences is inheritable in the plant genome

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252674
Author(s):  
Takayuki Hata ◽  
Naoto Takada ◽  
Chihiro Hayakawa ◽  
Mei Kazama ◽  
Tomohiro Uchikoba ◽  
...  
Keyword(s):  
Cultured Cells ◽  
De Novo ◽  
Gene Evolution ◽  
Firefly Luciferase ◽  
Plant Genome ◽  
Coding Sequences ◽  
Experimental Scheme ◽  
Transgenic Lines ◽  
The Individual ◽  
Comparison Of The Results

The manner in which inserted foreign coding sequences become transcriptionally activated and fixed in the plant genome is poorly understood. To examine such processes of gene evolution, we performed an artificial evolutionary experiment in Arabidopsis thaliana. As a model of gene-birth events, we introduced a promoterless coding sequence of the firefly luciferase (LUC) gene and established 386 T2-generation transgenic lines. Among them, we determined the individual LUC insertion loci in 76 lines and found that one-third of them were transcribed de novo even in the intergenic or inherently unexpressed regions. In the transcribed lines, transcription-related chromatin marks were detected across the newly activated transcribed regions. These results agreed with our previous findings in A. thaliana cultured cells under a similar experimental scheme. A comparison of the results of the T2-plant and cultured cell experiments revealed that the de novo-activated transcription concomitant with local chromatin remodelling was inheritable. During one-generation inheritance, it seems likely that the transcription activities of the LUC inserts trapped by the endogenous genes/transcripts became stronger, while those of de novo transcription in the intergenic/untranscribed regions became weaker. These findings may offer a clue for the elucidation of the mechanism by which inserted foreign coding sequences become transcriptionally activated and fixed in the plant genome.

scholarly journals Kozak sequence acts as a negative regulator for de novo transcription initiation of newborn coding sequences in the plant genome

2020 ◽  
Author(s):  
Takayuki Hata ◽  
Soichirou Satoh ◽  
Naoto Takada ◽  
Mitsuhiro Matsuo ◽  
Junichi Obokata
Keyword(s):  
Transcription Initiation ◽  
Cultured Cells ◽  
De Novo ◽  
Gene Evolution ◽  
Firefly Luciferase ◽  
Open Reading Frames ◽  
Negative Regulator ◽  
Plant Genome ◽  
Coding Sequences ◽  
Precise Position

ABSTRACTThe manner in which newborn coding sequences and their transcriptional competency emerge during the process of gene evolution remains unclear. Here, we experimentally simulated eukaryotic gene origination processes by mimicking horizontal gene transfer events in the plant genome. We mapped the precise position of the transcription start sites (TSSs) of hundreds of newly introduced promoterless firefly luciferase (LUC) coding sequences in the genome of Arabidopsis thaliana cultured cells. The systematic characterization of the LUC-TSSs revealed that 80% of them occurred under the influence of endogenous promoters, while the remainder underwent de novo activation in the intergenic regions, starting from pyrimidine-purine dinucleotides. These de novo TSSs obeyed unexpected rules; they predominantly occurred ~100 bp upstream of the LUC inserts and did not overlap with Kozak-containing putative open reading frames (ORFs). These features were the output of the immediate responses to the sequence insertions, rather than a bias in the screening of the LUC gene function. Regarding the wild-type genic TSSs, they appeared to have evolved to lack any ORFs in their vicinities. Therefore, the repulsion by the de novo TSSs of Kozak-containing ORFs described above might be the first selection gate for the occurrence and evolution of TSSs in the plant genome. Based on these results, we characterized the de novo type of TSS identified in the plant genome and discuss its significance in genome evolution.

scholarly journals Kozak sequence acts as a negative regulator for de novo transcription initiation of newborn coding sequences in the plant genome

2021 ◽  
Author(s):  
Takayuki Hata ◽  
Soichirou Satoh ◽  
Naoto Takada ◽  
Mitsuhiro Matsuo ◽  
Junichi Obokata
Keyword(s):  
Transcription Initiation ◽  
Cultured Cells ◽  
De Novo ◽  
Gene Evolution ◽  
Firefly Luciferase ◽  
Open Reading Frames ◽  
Negative Regulator ◽  
Plant Genome ◽  
Coding Sequences ◽  
Precise Position

Abstract The manner in which newborn coding sequences and their transcriptional competency emerge during the process of gene evolution remains unclear. Here, we experimentally simulated eukaryotic gene origination processes by mimicking horizontal gene transfer events in the plant genome. We mapped the precise position of the transcription start sites (TSSs) of hundreds of newly introduced promoterless firefly luciferase (LUC) coding sequences in the genome of Arabidopsis thaliana cultured cells. The systematic characterization of the LUC-TSSs revealed that 80% of them occurred under the influence of endogenous promoters, while the remainder underwent de novo activation in the intergenic regions, starting from pyrimidine-purine dinucleotides. These de novo TSSs obeyed unexpected rules; they predominantly occurred ∼100 bp upstream of the LUC inserts and did not overlap with Kozak-containing putative open reading frames (ORFs). These features were the output of the immediate responses to the sequence insertions, rather than a bias in the screening of the LUC gene function. Regarding the wild-type genic TSSs, they appeared to have evolved to lack any ORFs in their vicinities. Therefore, the repulsion by the de novo TSSs of Kozak-containing ORFs described above might be the first selection gate for the occurrence and evolution of TSSs in the plant genome. Based on these results, we characterized the de novo type of TSS identified in the plant genome and discuss its significance in genome evolution.

scholarly journals Landscape and evolutionary dynamics of terminal-repeat retrotransposons in miniature (TRIMs) in 48 whole plant genomes

2014 ◽  
Author(s):  
Dongying Gao ◽  
Yupeng Li ◽  
Brian Abernathy ◽  
Scott Jackson
Keyword(s):  
Evolutionary Dynamics ◽  
De Novo ◽  
Gene Evolution ◽  
Terminal Repeat ◽  
Plant Genome ◽  
Ltr Retrotransposons ◽  
Functional Roles ◽  
Whole Plant ◽  
Plant Families ◽  
Transposition Mechanism

Terminal-repeat retrotransposons in miniature (TRIMs) are structurally similar to long terminal repeat (LTR) retrotransposons except that they are extremely small and difficult to identify. Thus far, only a few TRIMs have been characterized in the euphyllophytes and the evolutionary and biological impacts and transposition mechanism of TRIMs are poorly understood. In this study, we combined de novo and homology-based methods to annotate TRIMs in 48 plant genome sequences, spanning land plants to algae. We found 156 TRIM families, 146 previously undescribed. Notably, we identified the first TRIMs in a lycophyte and non-vascular plants. The majority of the TRIM families were highly conserved and shared within and between plant families. Even though TRIMs contribute only a small fraction of any plant genome, they are enriched in or near genes and may play important roles in gene evolution. TRIMs were frequently organized into tandem arrays we called TA-TRIMs, another unique feature distinguishing them from LTR retrotransposons. Importantly, we identified putative autonomous retrotransposons that may mobilize specific TRIM elements and detected very recent transpositions of a TRIM in O. sativa. Overall, this comprehensive analysis of TRIMs across the entire plant kingdom provides insight into the evolution and conservation of TRIMs and the functional roles they may play in gene evolution.

scholarly journals Cryptic promoter activation occurs by at least two different mechanisms in the Arabidopsis genome

2020 ◽  
Author(s):  
Hisayuki Kudo ◽  
Mitsuhiro Matsuo ◽  
Soichirou Satoh ◽  
Rei Hachisu ◽  
Masayuki Nakamura ◽  
...  
Keyword(s):  
De Novo ◽  
Core Promoter ◽  
Firefly Luciferase ◽  
Gene Trap ◽  
Plant Genome ◽  
Gene Promoters ◽  
Promoter Activation ◽  
Cryptic Promoter ◽  
Endosymbiotic Gene Transfer ◽  
Reading Frame

ABSTRACTIn gene-trap screening of plant genomes, promoterless reporter constructs are often expressed without trapping of annotated gene promoters. The molecular basis of this phenomenon, which has been interpreted as the trapping of cryptic promoters, is poorly understood. In this study, using Arabidopsis gene-trap lines in which a firefly luciferase (LUC) open reading frame (ORF) was expressed from intergenic regions, we found that cryptic promoter activation occurs by at least two different mechanisms: one is the capturing of pre-existing promoter-like chromatin marked by H3K4me3 and H2A.Z, and the other is the entirely new formation of promoter chromatin near the 5’ end of the inserted LUC ORF. To discriminate between these, we denoted the former mechanism as “cryptic promoter capturing”, and the latter one as “promoter de novo origination”. The latter finding raises a question as to how inserted LUC ORF sequence is involved in this phenomenon. To examine this, we performed a model experiment with chimeric LUC genes in transgenic plants. Using Arabidopsis psaH1 promoter–LUC constructs, we found that the functional core promoter region, where transcription start sites (TSS) occur, cannot simply be determined by the upstream nor core promoter sequences; rather, its positioning proximal to the inserted LUC ORF sequence was more critical. This result suggests that the insertion of the LUC ORF sequence alters the local distribution of the TSS in the plant genome. The possible impact of the two types of cryptic promoter activation mechanisms on plant genome evolution and endosymbiotic gene transfer is discussed.

scholarly journals Combined Luteolin and Indole-3-Carbinol Synergistically Constrains ERα-Positive Breast Cancer by Dual Inhibiting Estrogen Receptor Alpha and Cyclin-Dependent Kinase 4/6 Pathway in Cultured Cells and Xenograft Mice

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2116
Author(s):  
Xiaoyong Wang ◽  
Lijuan Zhang ◽  
Qi Dai ◽  
Hongzong Si ◽  
Longyun Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cell ◽  
Breast Cancer Cells ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
Cyclin Dependent Kinase ◽  
Xenograft Mice ◽  
The Individual

The high concentrations of individual phytochemicals in vitro studies cannot be physiologically achieved in humans. Our solution for this concentration gap between in vitro and human studies is to combine two or more phytochemicals. We screened 12 phytochemicals by pairwise combining two compounds at a low level to select combinations exerting the synergistic inhibitory effect of breast cancer cell proliferation. A novel combination of luteolin at 30 μM (LUT30) and indole-3-carbinol 40 μM (I3C40) identified that this combination (L30I40) synergistically constrains ERα+ breast cancer cell (MCF7 and T47D) proliferation only, but not triple-negative breast cancer cells. At the same time, the individual LUT30 and I3C40 do not have this anti-proliferative effect in ERα+ breast cancer cells. Moreover, this combination L30I40 does not have toxicity on endothelial cells compared to the current commercial drugs. Similarly, the combination of LUT and I3C (LUT10 mg + I3C10 mg/kg/day) (IP injection) synergistically suppresses tumor growth in MCF7 cells-derived xenograft mice, but the individual LUT (10 mg/kg/day) and I3C (20 mg/kg/day) do not show an inhibitory effect. This combination synergistically downregulates two major therapeutic targets ERα and cyclin dependent kinase (CDK) 4/6/retinoblastoma (Rb) pathway, both in cultured cells and xenograft tumors. These results provide a solid foundation that a combination of LUT and I3C may be a practical approach to treat ERα+ breast cancer cells after clinical trials.

scholarly journals Cellular pyrimidine imbalance triggers mitochondrial DNA–dependent innate immunity

2021 ◽  
Author(s):  
Hans-Georg Sprenger ◽  
Thomas MacVicar ◽  
Amir Bahat ◽  
Kai Uwe Fiedler ◽  
Steffen Hermans ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Cultured Cells ◽  
De Novo ◽  
Metabolic Response ◽  
Interferon Response ◽  
Type I ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
De Novo Pyrimidine Synthesis

AbstractCytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remain enigmatic. Here, we show that mtDNA-dependent immune signalling via the cyclic GMP–AMP synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS–STING–TBK1) pathway is under metabolic control and is induced by cellular pyrimidine deficiency. The mitochondrial protease YME1L preserves pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33. Deficiency of YME1L causes inflammation in mouse retinas and in cultured cells. It drives the release of mtDNA and a cGAS–STING–TBK1-dependent inflammatory response, which requires SLC25A33 and is suppressed upon replenishment of cellular pyrimidine pools. Overexpression of SLC25A33 is sufficient to induce immune signalling by mtDNA. Similarly, depletion of cytosolic nucleotides upon inhibition of de novo pyrimidine synthesis triggers mtDNA-dependent immune responses in wild-type cells. Our results thus identify mtDNA release and innate immune signalling as a metabolic response to cellular pyrimidine deficiencies.

scholarly journals Extended haplotype-phasing of long-read de novo genome assemblies using Hi-C

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zev N. Kronenberg ◽  
Arang Rhie ◽  
Sergey Koren ◽  
Gregory T. Concepcion ◽  
Paul Peluso ◽  
...  
Keyword(s):  
Zebra Finch ◽  
Cultured Cells ◽  
De Novo ◽  
Single Cells ◽  
Variant Calling ◽  
Chromatin Interaction ◽  
Extended Haplotype ◽  
Benchmark Datasets ◽  
And Performance ◽  
Genome Assemblies

AbstractHaplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. To date, these assemblies have been best created with complex protocols, such as cultured cells that contain a single-haplotype (haploid) genome, single cells where haplotypes are separated, or co-sequencing of parental genomes in a trio-based approach. These approaches are impractical in most situations. To address this issue, we present FALCON-Phase, a phasing tool that uses ultra-long-range Hi-C chromatin interaction data to extend phase blocks of partially-phased diploid assembles to chromosome or scaffold scale. FALCON-Phase uses the inherent phasing information in Hi-C reads, skipping variant calling, and reduces the computational complexity of phasing. Our method is validated on three benchmark datasets generated as part of the Vertebrate Genomes Project (VGP), including human, cow, and zebra finch, for which high-quality, fully haplotype-resolved assemblies are available using the trio-based approach. FALCON-Phase is accurate without having parental data and performance is better in samples with higher heterozygosity. For cow and zebra finch the accuracy is 97% compared to 80–91% for human. FALCON-Phase is applicable to any draft assembly that contains long primary contigs and phased associate contigs.

High-throughput vectors for efficient gene silencing in plants

2002 ◽  
Vol 29 (10) ◽  
pp. 1217 ◽  
Author(s):  
Chris A. Helliwell ◽  
S. Varsha Wesley ◽  
Anna J. Wielopolska ◽  
Peter M. Waterhouse
Keyword(s):  
Gene Silencing ◽  
Insertional Mutagenesis ◽  
Single Step ◽  
Plant Genome ◽  
Specific Gene ◽  
Single Polymerase Chain Reaction ◽  
Efficient Gene ◽  
Gene Redundancy ◽  
Endogenous Genes ◽  
Rapid Generation

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2853-2859 ◽  
Author(s):  
A. Weng ◽  
T. Magnuson ◽  
U. Storb
Keyword(s):  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Mouse Development ◽  
Partial Methylation ◽  
Distal Chromosome ◽  
Before And After ◽  
Endogenous Genes ◽  
De Novo Methylation

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

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