Transcription terminator-mediated enhancement in transgene expression in maize: preponderance of the AUGAAU motif overlapping with poly(A) signals

AbstractThe selection of transcription terminators (TTs) for pairing with high expressing constitutive promoters in chimeric constructs is crucial to deliver optimal transgene expression in plants. In this study, the use of the native combinations of four polyubiquitin gene promoters and corresponding TTs resulted in up to >3-fold increase in transgene expression in maize. Of the eight polyubiquitin promoter and TT regulatory elements utilized, seven were novel and identified from the polyubiquitin genes of Brachypodium distachyon, Setaria italica, and Zea mays. Furthermore, gene expression driven by the Cassava mosaic virus promoter was studied by pairing the promoter with distinct TTs derived from the high expressing genes of Arabidopsis. Of the three TTs studied, the polyubiquitin10 gene TT produced the highest transgene expression in maize. Polyadenylation patterns and mRNA abundance from eight distinct TTs were analyzed using 3’-RACE and next-generation sequencing. The results exhibited one to three unique polyadenylation sites in the TTs. The poly(A) site patterns for the StPinII TT were consistent when the same TT was deployed in chimeric constructs irrespective of the reporter gene and promoter used. Distal to the poly(A) sites, putative polyadenylation signals were identified in the near-upstream regions of the TTs based on previously reported mutagenesis and bioinformatics studies in rice and Arabidopsis. The putative polyadenylation signals were 9 to 11 nucleotides in length. Six of the eight TTs contained the putative polyadenylation signals that were overlaps of either canonical AAUAAA or AAUAAA-like polyadenylation signals and AUGAAU, a top-ranking-hexamer of rice and Arabidopsis gene near-upstream regions. Three of the polyubiquitin gene TTs contained the identical 9-nucleotide overlap, AUGAAUAAG, underscoring the functional significance of such overlaps in mRNA 3’ end processing. In addition to identifying new combinations of regulatory elements for high constitutive trait gene expression in maize, this study demonstrated the importance of TTs for optimizing gene expression in plants. Learning from this study could be applied to other dicotyledonous and monocotyledonous plant species for transgene expression.

Download Full-text

The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽

10.1182/blood.v85.2.319.319 ◽

1995 ◽

Vol 85 (2) ◽

pp. 319-329 ◽

Cited By ~ 66

Author(s):

S Dziennis ◽

RA Van Etten ◽

HL Pahl ◽

DL Morris ◽

TL Rothstein ◽

...

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Transgene Expression ◽

Heterologous Gene Expression ◽

Myeloid Cells ◽

Reporter Genes ◽

Regulatory Elements ◽

Specific Expression ◽

High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

Download Full-text

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

10.31219/osf.io/xc5a2 ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Gene Expression ◽

Genetic Diseases ◽

Regulatory Elements ◽

Tissue Type ◽

Gene Promoters ◽

Expression Levels ◽

Cis Regulation ◽

Gene Regulatory Elements ◽

Gene Regulatory ◽

Gene Expression Levels

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.

Download Full-text

The Role of Histone Demethylases and DNA Methyltransferases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients

Blood ◽

10.1182/blood.v128.22.3921.3921 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3921-3921

Author(s):

Katerina Rejlova ◽

Alena Musilova ◽

Martina Slamova ◽

Karel Fiser ◽

Karolina Skvarova Kramarzova ◽

...

Keyword(s):

Gene Expression ◽

Hox Genes ◽

Fold Increase ◽

Dna Methyltransferases ◽

Histone Mark ◽

Gene Promoters ◽

Histone Demethylases ◽

Atra Treatment ◽

Hox Gene

Abstract Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous results showed that HOX gene expression differs among genetically characterized subtypes of pediatric acute myeloid leukemia (AML). Specifically, PML-RARa positive AML patients have overall lowest HOX gene expression which positively correlates with expression of histone 3 lysine 27 (H3K27) demethylases - JMJD3 and UTX and negatively with the expression of DNA methyltransferases - DNMT3a and DNMT3b. Interestingly, JMJD3 was already shown to be a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). From these findings we postulated a hypothesis that reduced levels of HOX genes in PML-RARa positive AML are a consequence of suppressed expression of histone demethylases resulting in increased H3K27 methylation and/or of elevated levels of DNMTs leading to de novoDNA methylation. We studied the role of histone demethylases and DNMTs in the regulation of HOX gene expression and the effect of treatment in PML-RARa positive cell lines (NB4 and ATRA-resistant clones NB4-LR2 and NB4-MR2). We treated NB4 cell line by all-trans retinoic acid (ATRA; 1uM), which was described to release the differentiation block caused by the presence of PML-RARa and to degrade the fusion protein. We observed that expression of particular HOX genes (HOXA1, HOXA3, HOXA4, HOXA5, HOXA7, HOXB4, HOXB6) measured by qPCR was significantly increased after ATRA treatment. While the level of JMJD3 was significantly increased upon ATRA treatment as well, the expression of UTX did not change. Furthermore, we detected significantly reduced expression of DNMT3b gene. To exclude a non-specific effect of ATRA, independent of PML-RARa, we used resistant clones LR2 and MR2 bearing mutations in retinoic acid-binding domain. HOX gene expression together with JMJD3, UTX and DNMT3b expression did not change upon ATRA treatment. These results confirm the PML-RARa-dependent regulation of HOX genes. To test the role of JMJD3 in the HOX gene expression regulation, we cultured NB4 cells with a specific inhibitor of histone demethylases, GSK-J4 (1 uM, 10 uM), in the presence of ATRA. The co-treatment caused significant decrease in the expression of studied HOX genes (HOXA1, HOXA3, HOXA5, HOXA7, HOXA10, HOXB4, HOXB6) in comparison to ATRA alone which supports the role of JMJD3 in the transcription regulation. Further, we performed chromatin immunoprecipitation (ChIP) to investigate if the changes of HOX gene expression upon ATRA and GSK-J4 treatment would correspond with changes of histone code on HOX gene promoter regions. ATRA treatment caused reduction of repressive histone mark (H3K27me3) on particular HOX gene promoters (HOXA1, HOXA3, HOXA5, HOXA7), by contrast, combinational treatment of ATRA and GSK-J4 reversed this effect. Accordingly, we detected that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2. Next we were interested if JMJD3 inhibition would interfere with the differentiation effect of ATRA. As shown previously, ATRA treatment alone caused differentiation of NB4 cell line whereas the combination with GSK-J4 did not reduce the effect. Interestingly, in addition to differentiation it led cells to apoptosis. Combination of drugs (ATRA - 1uM, GSK-J4 - 1, 2, 5uM) increased significantly the percentage of dead cells in comparison to ATRA or GSK treatment alone (GSK-J4 alone vs in combination with ATRA, 1uM - 1.8 fold, 2uM - 2.2 fold, 5 uM - 2.3 fold increase). Next we measured apoptosis in resistant clones LR2 and MR2. In both cases the highest concentration used of GSK-J4 (5uM) in combination with ATRA caused significant increase of dead cells as well (LR2 - 2.1 fold, MR2 - 2.0 fold increase). Our results indicate that JMJD3 is responsible for the regulation of HOX gene expression in PML-RARa positive leukemia since changes of HOX gene expression correspond with histone modifications on the regions of HOX gene promoters. We assume that DNA methylation driven by DNMT3b can also participate in this process. Moreover, our findings demonstrate potential therapeutic implications of GSK-J4 inhibitor in combination with ATRA in patients with acute promyelocytic leukemia who are not responsive to ATRA monotherapy. Supported by P304/12/2214 and GAUK 196616 Disclosures No relevant conflicts of interest to declare.

Download Full-text

Plant Soft Rot Development and Regulation from the Viewpoint of Transcriptomic Profiling

Plants ◽

10.3390/plants9091176 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1176

Author(s):

Ivan Tsers ◽

Vladimir Gorshkov ◽

Natalia Gogoleva ◽

Olga Parfirova ◽

Olga Petrova ◽

...

Keyword(s):

Gene Expression ◽

Plant Cell Wall ◽

Soft Rot ◽

Regulatory Elements ◽

Plant Responses ◽

Gene Promoters ◽

Rna Seq ◽

Induced Plant Responses ◽

Regulatory Systems ◽

Genome Level

Soft rot caused by Pectobacterium species is a devastating plant disease poorly characterized in terms of host plant responses. In this study, changes in the transcriptome of tobacco plants after infection with Pectobacterium atrosepticum (Pba) were analyzed using RNA-Seq. To draw a comprehensive and nontrivially itemized picture of physiological events in Pba-infected plants and to reveal novel potential molecular “players” in plant–Pba interactions, an original functional gene classification was performed. The classifications present in various databases were merged, enriched by “missed” genes, and divided into subcategories. Particular changes in plant cell wall-related processes, perturbations in hormonal and other regulatory systems, and alterations in primary, secondary, and redox metabolism were elucidated in terms of gene expression. Special attention was paid to the prediction of transcription factors (TFs) involved in the disease’s development. Herewith, gene expression was analyzed within the predicted TF regulons assembled at the whole-genome level based on the presence of particular cis-regulatory elements (CREs) in gene promoters. Several TFs, whose regulons were enriched by differentially expressed genes, were considered to be potential master regulators of Pba-induced plant responses. Differential regulation of genes belonging to a particular multigene family and encoding cognate proteins was explained by the presence/absence of the particular CRE in gene promoters.

Download Full-text

Hepatic gene expression patterns in thyroid hormone-treated hypothyroid rats

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310291 ◽

2003 ◽

Vol 31 (2) ◽

pp. 291-303 ◽

Cited By ~ 42

Author(s):

JM Weitzel ◽

S Hamann ◽

M Jauk ◽

M Lacey ◽

A Filbry ◽

...

Keyword(s):

Gene Expression ◽

Thyroid Hormone ◽

Expression Patterns ◽

Regulatory Elements ◽

Activation Mechanism ◽

Gene Expression Patterns ◽

Peroxisome Proliferator ◽

Gene Promoters ◽

Protein Levels

Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. We have performed DNA microarray experiments using hepatic RNA from hypothyroid and T3-treated hypothyroid rats in order to characterize T3-induced gene expression patterns after various time points (6, 24 and 48 h after the administration of the hormone). Sixty-two of 4608 different genes displayed a reproducible T3-response, and cluster analysis divided these differentially regulated genes into six expression patterns. Thirty-six genes were not significantly regulated within the first 24 h. Transient transfection experiments of eight late-induced gene promoters failed to detect a thyroid hormone response element within their regulatory elements, suggesting an indirect activation mechanism(s). In search for an intermediate factor of T3 action, we examined whether various rather ubiquitous transcription factors, peroxisome proliferator-activated receptors (PPARs) and coactivators of the PPARgamma coactivator 1 family (PGC-1) are regulated by T3. Only PPARgamma and PERC/PGC-1beta exhibit a significant T3-response within the first 6 h after treatment, identifying these factors as candidate components for mediating the late-induced expression pattern. Regulation of early-induced genes within the first 6 h after administration of T3 on transcript levels correlates with altered protein levels after 24 and 48 h in vivo.

Download Full-text

BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome

10.1101/2020.11.13.381251 ◽

2020 ◽

Author(s):

Nadezda A. Fursova ◽

Anne H. Turberfield ◽

Neil P. Blackledge ◽

Emma L. Findlater ◽

Anna Lastuvkova ◽

...

Keyword(s):

Gene Expression ◽

Histone Modifications ◽

Transcription Initiation ◽

Target Genes ◽

Regulatory Elements ◽

Polycomb Group ◽

Gene Promoters ◽

Histone H2a ◽

Gene Regulatory Elements ◽

Gene Regulatory

AbstractHistone-modifying systems play fundamental roles in gene regulation and the development of multicellular organisms. Histone modifications that are enriched at gene regulatory elements have been heavily studied, but the function of modifications that are found more broadly throughout the genome remains poorly understood. This is exemplified by histone H2A mono-ubiquitylation (H2AK119ub1) which is enriched at Polycomb-repressed gene promoters, but also covers the genome at lower levels. Here, using inducible genetic perturbations and quantitative genomics, we discover that the BAP1 deubiquitylase plays an essential role in constraining H2AK119ub1 throughout the genome. Removal of BAP1 leads to pervasive accumulation of H2AK119ub1, which causes widespread reductions in gene expression. We show that elevated H2AK119ub1 represses gene expression by counteracting transcription initiation from gene regulatory elements, causing reductions in transcription-associated histone modifications. Furthermore, failure to constrain pervasive H2AK119ub1 compromises Polycomb complex occupancy at a subset of Polycomb target genes leading to their derepression, therefore explaining the original genetic characterisation of BAP1 as a Polycomb group gene. Together, these observations reveal that the transcriptional potential of the genome can be modulated by regulating the levels of a pervasive histone modification, without the need for elaborate gene-specific targeting mechanisms.

Download Full-text

Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of RNAi Mediated Repression.

Blood ◽

10.1182/blood.v106.11.3627.3627 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3627-3627

Author(s):

Elliot M. Epner ◽

Jin Wang ◽

Jing Huang

Keyword(s):

Gene Expression ◽

Gene Silencing ◽

Globin Gene ◽

K562 Cells ◽

Regulatory Elements ◽

Gene Promoters ◽

Globin Genes ◽

Phenotypic Analysis ◽

Pol Ii ◽

Globin Gene Expression

Abstract The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18. The recent demonstration of the involvement of Pol II in the synthesis of miRNA’s prompted us to investigate the role of miRNA’s in gene silencing in this system. A small miRNA was identified at the intergenic enhancer region. ChIP assays showed the binding of two components of the RISC (Dicer and Ago2) at the chicken globin regulatory elements. These results are consistent with the involvement of RISC and miRNA’s in gene silencing in this system.

Download Full-text

The mouse muscle creatine kinase promoter faithfully drives reporter gene expression in transgenicXenopus laevis

Physiological Genomics ◽

10.1152/physiolgenomics.00148.2003 ◽

2004 ◽

Vol 18 (1) ◽

pp. 79-86 ◽

Cited By ~ 11

Author(s):

Wayland Lim ◽

Eric S. Neff ◽

J. David Furlow

Keyword(s):

Gene Expression ◽

Creatine Kinase ◽

Transgene Expression ◽

Muscle Development ◽

Fluorescent Protein ◽

Regulatory Sequences ◽

Gene Promoters ◽

Muscle Creatine Kinase ◽

Mouse Muscle ◽

Muscle Creatine

Developing Xenopus laevis experience two periods of muscle differentiation, once during embryogenesis and again at metamorphosis. During metamorphosis, thyroid hormone induces both muscle growth in the limbs and muscle death in the tail. In mammals, the muscle creatine kinase (MCK) gene is activated during the differentiation from myoblasts to myocytes and has served as both a marker for muscle development and to drive transgene expression in transgenic mice. Transcriptional control elements are generally highly conserved throughout evolution, potentially allowing mouse promoter use in transgenic X. laevis. This paper compares endogenous X. laevis MCK gene expression and the mouse MCK (mMCK) promoter driving a green fluorescent protein reporter in transgenic X. laevis. The mMCK promoter demonstrated strong skeletal muscle-specific transgene expression in both the juvenile tadpole and adult frog. Therefore, our results clearly demonstrate the functional conservation of regulatory sequences in vertebrate muscle gene promoters and illustrate the utility of using X. laevis transgenesis for detailed comparative study of mammalian promoter activity in vivo.

Download Full-text

Three-Dimensional Genome Organization in Breast and Gynecological Cancers: How Chromatin Folding Influences Tumorigenic Transcriptional Programs

Cells ◽

10.3390/cells11010075 ◽

2021 ◽

Vol 11 (1) ◽

pp. 75

Author(s):

Stephanie I. Nuñez-Olvera ◽

Jonathan Puente-Rivera ◽

Rosalio Ramos-Payán ◽

Carlos Pérez-Plasencia ◽

Yarely M. Salinas-Vera ◽

...

Keyword(s):

Gene Expression ◽

Protein Function ◽

Three Dimensional ◽

Gene Mutations ◽

Regulatory Elements ◽

Specific Gene ◽

Gene Promoters ◽

Nucleotide Polymorphisms ◽

Non Coding Rnas ◽

Insulator Elements

A growing body of research on the transcriptome and cancer genome has demonstrated that many gynecological tumor-specific gene mutations are located in cis-regulatory elements. Through chromosomal looping, cis-regulatory elements interact which each other to control gene expression by bringing distant regulatory elements, such as enhancers and insulators, into close proximity with promoters. It is well known that chromatin connections may be disrupted in cancer cells, promoting transcriptional dysregulation and the expression of abnormal tumor suppressor genes and oncogenes. In this review, we examine the roles of alterations in 3D chromatin interactions. This includes changes in CTCF protein function, cancer-risk single nucleotide polymorphisms, viral integration, and hormonal response as part of the mechanisms that lead to the acquisition of enhancers or super-enhancers. The translocation of existing enhancers, as well as enhancer loss or acquisition of insulator elements that interact with gene promoters, is also revised. Remarkably, similar processes that modify 3D chromatin contacts in gene promoters may also influence the expression of non-coding RNAs, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), which have emerged as key regulators of gene expression in a variety of cancers, including gynecological malignancies.

Download Full-text

The global and promoter-centric 3D genome organization temporally resolved during a circadian cycle

10.1101/2020.07.23.217992 ◽

2020 ◽

Author(s):

Masami Ando-Kuri ◽

Rodrigo G. Arzate-Mejía ◽

Jorg Morf ◽

Jonathan Cairns ◽

Cesar A. Poot-Hernández ◽

...

Keyword(s):

Gene Expression ◽

Mouse Liver ◽

Regulatory Elements ◽

Open Chromatin ◽

Chromatin Conformation ◽

Gene Promoters ◽

Circadian Gene ◽

Circadian Genes ◽

Physiological Importance ◽

3D Genome

SummaryCircadian gene expression is essential for organisms to adjust cellular responses and anticipate daily changes in the environment. In addition to its physiological importance, the clock circuit represents an ideal, temporally resolved, system to study transcription regulation. Here, we analysed changes in spatial mouse liver chromatin conformation using genome-wide and promoter-capture Hi-C alongside daily oscillations in gene transcription in mouse liver. We found circadian topologically associated domains switched assignments to the transcriptionally active, open chromatin compartment and the inactive compartment at different hours of the day while their boundaries stably maintain their structure over time. Individual circadian gene promoters displayed maximal chromatin contacts at times of peak transcriptional output and the expression of circadian genes and contacted transcribed regulatory elements, or other circadian genes, was phase-coherent. Anchor sites of promoter chromatin loops were enriched in binding sites for liver nuclear receptors and transcription factors, some exclusively present in either rhythmic or stable contacts. The circadian 3D chromatin maps provided here identify the scales of chromatin conformation that parallel oscillatory gene expression and protein factors specifically associated with circadian or stable chromatin configurations.

Download Full-text