A tandem repeat array in IG-DMR is essential for imprinting of paternal allele at the Dlk1–Dio3 domain during embryonic development

In a previous study we isolated a partial cDNA with a tandem repeat of 48 bp, which allowed us to map a novel human mucin gene named MUC4to chromosome 3q29. Here we report the organization and sequence of the 5´-region and its junction with the tandem repeat array of MUC4. Analysis of three overlapping genomic clones allowed us to obtain a partial restriction map of MUC4 and to locate the complete 48 bp tandem repeat domain on a PstI/EcoRI genomic fragment that exhibits a very large variation in number of tandem repeats (7–19 kb). cDNA clonal extension allowed us to obtain the entire 5´ coding region of MUC4. Exon 1 consists of a 5´ untranslated region and an 82 bp fragment encoding the signal peptide. This latter shows a high degree of similarity to the signal peptide of another apomucin, ASGP-1. Exon 2 is extremely large and contains a unique sequence that is followed by the whole tandem repeat domain. It encodes only one cysteine residue, making MUC4 different from mucin genes belonging to the 11p15.5 family. Moreover, an intron downstream from the tandem repeat array consists mainly of a 15 bp tandem repeat that exhibits a polymorphism in having a variable number of tandem repeats.

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Immunization with MUC1/X protein enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target cancer cells

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.10072 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 10072-10072

Author(s):

D. B. Rubinstein ◽

R. Ziv ◽

M. Karmely ◽

O. Leitner ◽

D. Wreschner

Keyword(s):

Cell Surface ◽

Tandem Repeat ◽

Transmembrane Protein ◽

High Titer ◽

Type I ◽

X Protein ◽

Α Subunit ◽

Covalent Interaction ◽

Repeat Array ◽

Tandem Repeat Array

10072 Background: MUC1, a glycoprotein highly expressed in epithelial malignancies including breast, prostate, and ovarian, and on the malignant cells of multiple myeloma has generated considerable interest as a tumor marker and target for tumor killing. The most intensively studied MUC1 protein is a type I transmembrane protein (MUC1/TM) which is proteolytically cleaved soon after synthesis into α and β subunits which bind in a strong non-covalent interaction. Almost all antibodies generated to date against MUC1 recognize epitopes within the highly immunogenic tandem-repeat-array. A major shortcoming in use of such antibodies is the fact that the tandem-repeat-array-containing part of MUC1 is shed from the cell surface into the circulation. Soluble, shed MUC1 sequesters circulating anti-tandem-repeat-array antibodies, limiting their ability to reach targeted MUC1-expressing cells. Antibodies to MUC1 epitopes tethered to the cell surface would likely be more effective therapeutic agents. Despite efforts in recent years, such antibodies have remained elusive; generation of anti-cell antibodies requires characterization of cell-bound epitopes. The junction of the MUC1 α-subunit binding the membrane-tethered β-subunit provides such an epitope. Methods: By use of a novel protocol, entailing immunization with MUC1/TM cDNA and boosting with MUC1/X protein, a MUC1 isoform lacking the tandem-repeat-array, we generated monoclonal antibodies designated DMC209 which recognize the MUC1 α/β junction. Results: DMC209 is exquisitely unique for the target site; all amino acid mutations which abrogate MUC1 cleavage also abrogate DMC209 binding. Additionally, DMC209 binds the MUC1 α/β junction on cell-tethered tandem-repeat-array-containing MUC1 (MUC1/TM) on breast and ovarian cancer, and on myeloma cells. Conclusion: DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly, and as an immunotoxin conjugate. Moreover, the novel high-titer immunization procedure used in generating DMC209 can be used to generate anti-MUC1 α/β junction antibodies acting as ligand and, analogously to herceptin, may have direct cytotoxic activity. No significant financial relationships to disclose.

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Generation of antibodies that target and kill MUC1 expressing cancer cells via a novel cell-tethered MUC1 α/β junction epitope

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.3034 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 3034-3034

Author(s):

M. Karmely ◽

D. B. Rubinstein ◽

D. H. Wreschner

Keyword(s):

Cancer Cells ◽

Tandem Repeat ◽

Plasma Cells ◽

Cell Killing ◽

Ovarian Cancer Cells ◽

Malignant Cells ◽

X Protein ◽

Repeat Array ◽

A Chain ◽

Tandem Repeat Array

3034 Background: MUC1 protein has generated considerable interest as a target for tumor killing. However, a serious shortcoming of anti-MUC1 antibodies generated to date is that they recognize epitopes within the strongly immunogenic tandem-repeat-array of the MUC1 a- chain which is released from the cell into the circulation. Soluble shed MUC1 a-chain sequesters anti-tandem-repeat-array antibodies, severely limiting their ability to reach MUC1-expressing malignant cells. Rather than target freely circulating MUC1 a-chain, we identified the junction of the MUC1 a-subunit to cell-membrane-bound MUC1 β-subunit as a preferable cell-tethered MUC1 epitope; since the site is not shed, antibodies recognizing it are more effective in targeting MUC1 expressing cells. Methods: To circumvent the immunogenicity of the a-chain tandem repeat array we formulated a novel antibody generating protocol utilizing immunization with both MUC1 cDNA and with the alternatively-spliced MUC1/X protein isoform from which the tandem repeat array is deleted. After immunization and hybridoma formation, anti- MUC1 a/β junction antibodies were selected. Results: DMC209 monoclonal antibodies uniquely specific for the MUC1 a/β junction were generated. The antibodies specifically bind the MUC1 a/β junction on full-length MUC1 expressed by breast and ovarian cancer cells, and on MUC1-positive malignant plasma cells of multiple myeloma. To demonstrate that anti-MUC1 a/β junction antibodies kill malignant cells, immunotoxin conjugates were formed with anti-MUC1 a/β junction polyclonal antibodies generated in our cDNA/protein immunization protocol and PE38, a powerful pseudomonas exotoxin. The antibody-exotoxin conjugates were potently cytocidal to MUC1-expressing malignant cells. Significantly, cell killing was abrogated by addition of soluble MUC1/X protein, highlighting that the cell-killing immunotoxin gains cell entry via the MUC1 a/β junction. Conclusions: The MUC1 a/β junction has been identified as an important cell-tethered MUC1 epitope against which highly specific antibodies can be generated capable of killing MUC1-expressing cells. These studies point to effective anti-MUC1-based immuno-therapeutic strategies. No significant financial relationships to disclose.

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Efficient production and transmission of CRISPR/Cas9-mediated mutant alleles at the IG-DMR via generation of mosaic mice using a modified 2CC method

Scientific Reports ◽

10.1038/s41598-019-56676-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Satoshi Hara ◽

Miho Terao ◽

Akari Muramatsu ◽

Shuji Takada

Keyword(s):

Imprinted Genes ◽

Efficient Production ◽

Genomic Locus ◽

Aberrant Expression ◽

Repeat Array ◽

Homology Directed Repair ◽

Functional Regions ◽

Important Approach ◽

Imprinting Control Region ◽

Tandem Repeat Array

AbstractGeneration of mutant imprinting control region (ICR) mice using genome editing is an important approach for elucidating ICR functions. IG-DMR is an ICR in the Dlk1-Dio3 imprinted domain that contains functional regions—in both parental alleles—that are essential for embryonic development. One drawback of this approach is that embryonic lethality can occur from aberrant expression of the imprinted genes if IG-DMR gets mutated in either the paternal or maternal allele. To overcome this problem, we generated mosaic mice that contained cells with modified IG-DMR alleles and wild-type cells using the 2CC method that allowed for microinjection of the CRISPR/Cas9 constructs into a blastomere of 2-cell embryos. This method improved the birth rate of the founder pups relative to that obtained using the standard protocol. We also successfully produced mosaic mice in which the tandem repeat array sequence in the IG-DMR had been replaced by homology directed repair. Additionally, paternal transmission of the replaced allele caused aberrant expression of the imprinted genes due to hypomethylation of the IG-DMR, indicating that the replaced allele recapitulated our deletion model. Our results indicate that this method is useful for the generation of mutant mice in which a genomic locus essential for normal development has been genetically edited.

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Tandem Repeat Hypothesis in Imprinting: Deletion of a Conserved Direct Repeat Element Upstream of H19 Has No Effect on Imprinting in the Igf2-H19 Region

Molecular and Cellular Biology ◽

10.1128/mcb.24.13.5650-5656.2004 ◽

2004 ◽

Vol 24 (13) ◽

pp. 5650-5656 ◽

Cited By ~ 28

Author(s):

Annabelle Lewis ◽

Kohzoh Mitsuya ◽

Miguel Constancia ◽

Wolf Reik

Keyword(s):

Tandem Repeat ◽

Tandem Repeats ◽

Direct Repeat ◽

Regulatory Elements ◽

Repeat Element ◽

Allelic Expression ◽

Differentially Methylated Region ◽

Paternal Allele ◽

Imprinted Genes ◽

Allele Specific

ABSTRACT Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct repeat element adjacent to a DMR is responsible for establishing paternal allele-specific methylation at the DMR and therefore allelic expression of the Rasgrf1 transcript. We identified a prominent and conserved direct tandem repeat 1 kb upstream of the H19 DMR and proposed that it played a similar role in imprinted regulation of H19. To test our hypothesis, we generated mice harboring a 1.7-kb targeted deletion of the direct repeat element and analyzed fetal growth, allelic expression, and methylation within the Igf2-H19 region. Surprisingly the deletion had no effect on imprinting. These results together with deletions of other repeats close to imprinted genes suggest that direct repeats may not be important for the targeting of methylation at the majority of imprinted loci and that the Rasgrf1 locus may be an exception to this rule.

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Microhomologies Are Associated with Tandem Duplications and Structural Variation in Plant Mitochondrial Genomes

Genome Biology and Evolution ◽

10.1093/gbe/evaa172 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1965-1974

Author(s):

Hanhan Xia ◽

Wei Zhao ◽

Yong Shi ◽

Xiao-Ru Wang ◽

Baosheng Wang

Keyword(s):

Tandem Repeat ◽

Structural Variation ◽

Tandem Repeats ◽

Repeat Unit ◽

Mitochondrial Genomes ◽

Repeat Array ◽

Mitochondrial Genome Evolution ◽

Tandem Duplications ◽

Short Tandem

Abstract Short tandem repeats (STRs) contribute to structural variation in plant mitochondrial genomes, but the mechanisms underlying their formation and expansion are unclear. In this study, we detected high polymorphism in the nad7-1 region of the Pinus tabuliformis mitogenome caused by the rapid accumulation of STRs and rearrangements over a few million years ago. The STRs in nad7-1 have a 7-bp microhomology (TAG7) flanking the repeat array. We then scanned the mitogenomes of 136 seed plants to understand the role of microhomology in the formation of STR and mitogenome evolution. A total of 13,170 STRs were identified, and almost half of them were associated with microhomologies. A substantial amount (1,197) of microhomologies was long enough to mediate structural variation, and the length of microhomology is positively correlated with the length of tandem repeat unit. These results suggest that microhomology may be involved in the formation of tandem repeat via microhomology-mediated pathway, and the formation of longer duplicates required greater length of microhomology. We examined the abundance of these 1,197 microhomologies, and found 75% of them were enriched in the plant mitogenomes. Further analyses of the 400 prevalent microhomologies revealed that 175 of them showed differential enrichment between angiosperms and gymnosperms and 186 differed between angiosperms and conifers, indicating lineage-specific usage and expansion of microhomologies. Our study sheds light on the sources of structural variation in plant mitochondrial genomes and highlights the importance of microhomology in mitochondrial genome evolution.

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A Fob1 independent role for the Smc5/6 complex in the rDNA that modulates lifespan

10.1101/2020.01.24.917203 ◽

2020 ◽

Author(s):

Sarah Moradi-Fard ◽

Aditya Mojumdar ◽

Megan Chan ◽

Troy A. A. Harkness ◽

Jennifer A. Cobb

Keyword(s):

Saccharomyces Cerevisiae ◽

Replication Fork ◽

Repetitive Element ◽

Transcriptional Silencing ◽

List Type ◽

Replicative Lifespan ◽

Repeat Array ◽

Rdna Array ◽

Nucleolar Organization ◽

Tandem Repeat Array

SUMMARYThe ribosomal DNA (rDNA) in Saccharomyces cerevisiae is in one tandem repeat array on Chromosome XII. Two spacer regions within each repetitive element, called non-transcribed spacer 1 (NTS1) and NTS2, are important in nucleolar organization. Smc5/6 localizes to both NTS1 and NTS2 and is involved in the regulation of Sir2 and Cohibin binding at NTS1, whereas Fob1 and Sir2 are required for optimal binding of the complex to NTS1 and NTS2, respectively. We demonstrate that Smc5/6 functions in chromatin silencing at NTS1 independently of its role in homologous recombination (HR) when forks pause at the replication fork barrier (RFB). In contrast, when the complex does not localize to the rDNA in nse3-1 mutants, the shortened lifespan correlates with NTS2 homeostasis independently of FOB1 status. Our data identify the importance of Smc5/6 integrity in NTS2 transcriptional silencing and repeat tethering, which in turn underscores rDNA stability and replicative lifespan.HighlightsSmc5/6 is important for transcriptional silencing in the rDNA.Smc5/6 tethers the rDNA array to the periphery.Transcriptional silencing of ncRNA at NTS1 and NTS2 is differentially regulated.Smc5/6 has a role in rDNA maintenance independent of HR processing at the RFB.Fob1-independent disruption of Smc5/6 at NTS2 leads to lifespan reduction.

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An ancient satellite repeat controls gene expression and embryonic development in Aedes aegypti through a highly conserved piRNA

10.1101/2020.01.15.907428 ◽

2020 ◽

Cited By ~ 3

Author(s):

Rebecca Halbach ◽

Pascal Miesen ◽

Joep Joosten ◽

Ezgi Taşköprü ◽

Bas Pennings ◽

...

Keyword(s):

Gene Expression ◽

Embryonic Development ◽

Aedes Aegypti ◽

Gene Silencing ◽

Tandem Repeat ◽

Satellite Repeat ◽

Central Function ◽

Satellite Repeats ◽

In Trans ◽

Regulatory Potential

AbstractTandem repeat elements such as the highly diverse class of satellite repeats occupy large parts of eukaryotic chromosomes. Most occur at (peri)centromeric and (sub)telomeric regions and have been implicated in chromosome organization, stabilization, and segregation1. Others are located more dispersed throughout the genome, but their functions remained largely enigmatic. Satellite repeats in euchromatic regions were hypothesized to regulate gene expression in cis by modulation of the local heterochromatin, or in trans via repeat-derived transcripts2,3. Yet, due to a lack of experimental models, gene regulatory potential of satellite repeats remains largely unexplored. Here we show that, in the vector mosquito Aedes aegypti, a satellite repeat promotes sequence-specific gene silencing via the expression of two abundant PIWI-interacting RNAs (piRNAs). Strikingly, whereas satellite repeats and piRNA sequences generally evolve extremely fast4-6, this locus was conserved for approximately 200 million years, suggesting a central function in mosquito biology. Tandem repeat-derived piRNA production commenced shortly after egg-laying and inactivation of the most abundant of the two piRNAs in early embryos resulted in an arrest of embryonic development. Transcriptional profiling in these embryos revealed the failure to degrade maternally provided transcripts that are normally cleared during maternal-to-zygotic transition. Our results reveal a novel mechanism in which satellite repeats regulate global gene expression in trans via piRNA-mediated gene silencing, which is fundamental to embryonic development. These findings highlight the regulatory potential of this enigmatic class of repeats.

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Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold

Scientific Reports ◽

10.1038/s41598-019-49905-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Albert Perez-Riba ◽

Elizabeth Komives ◽

Ewan R. G. Main ◽

Laura S. Itzhaki

Keyword(s):

Tandem Repeat ◽

Modular Architecture ◽

Tetratricopeptide Repeats ◽

Repeat Protein ◽

Repeat Array ◽

Functional Capabilities ◽

Repeat Proteins ◽

Weakly Coupled ◽

Tandem Repeat Proteins ◽

Modular Platform

Abstract The simple topology and modular architecture of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats makes them straightforward to dissect and redesign. Repeat-protein stability can be manipulated in a predictable way using site-specific mutations. Here we explore a different type of modification - loop insertion - that will enable a simple route to functionalisation of this versatile scaffold. We previously showed that a single loop insertion has a dramatically different effect on stability depending on its location in the repeat array. Here we dissect this effect by a combination of multiple and alternated loop insertions to understand the origins of the context-dependent loss in stability. We find that the scaffold is remarkably robust in that its overall structure is maintained. However, adjacent repeats are now only weakly coupled, and consequently the increase in solvent protection, and thus stability, with increasing repeat number that defines the tandem-repeat protein class is lost. Our results also provide us with a rulebook with which we can apply these principles to the design of artificial repeat proteins with precisely tuned folding landscapes and functional capabilities, thereby paving the way for their exploitation as a versatile and truly modular platform in synthetic biology.

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Folding cooperativity and allosteric function in the tandem-repeat protein class

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2017.0188 ◽

2018 ◽

Vol 373 (1749) ◽

pp. 20170188 ◽

Cited By ~ 5

Author(s):

Albert Perez-Riba ◽

Marie Synakewicz ◽

Laura S. Itzhaki

Keyword(s):

Binding Site ◽

Tandem Repeat ◽

Structural Changes ◽

Tetratricopeptide Repeats ◽

Repeat Protein ◽

Repeat Array ◽

Repeat Proteins ◽

Bacterial Quorum Sensing ◽

Tandem Repeat Proteins ◽

Bacterial Quorum

The term allostery was originally developed to describe structural changes in one binding site induced by the interaction of a partner molecule with a distant binding site, and it has been studied in depth in the field of enzymology. Here, we discuss the concept of action at a distance in relation to the folding and function of the solenoid class of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats. Distantly located repeats fold cooperatively, even though only nearest-neighbour interactions exist in these proteins. A number of repeat-protein scaffolds have been reported to display allosteric effects, transferred through the repeat array, that enable them to direct the activity of the multi-subunit enzymes within which they reside. We also highlight a recently identified group of tandem-repeat proteins, the RRPNN subclass of TPRs, recent crystal structures of which indicate that they function as allosteric switches to modulate multiple bacterial quorum-sensing mechanisms. We believe that the folding cooperativity of tandem-repeat proteins and the biophysical mechanisms that transform them into allosteric switches are intimately intertwined. This opinion piece aims to combine our understanding of the two areas and develop ideas on their common underlying principles. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.

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