The modified base J is the target for a novelDNA-binding protein in kinetoplastid protozoans

Cross, Mike; Kieft, Rudo; Sabatini, Robert; Wilm, Matthias; Kort, Martin de; Marel, Gijs A. van der; Boom, Jacques H. van; Leeuwen, Fred van; Borst, Piet

doi:10.1093/emboj/18.22.6573

Cited by 68 publications

(71 citation statements)

References 37 publications

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“…As most transcriptional factors bind the major groove of DNA, the idea that base J can induce steric hindrance is highly plausible. The ability of base J to recruit specific proteins leading to repressed chromatin is unlikely, since the only base J-containing DNA binding protein identified in trypanosome nuclear extracts is the thymidine hydroxylase involved in base J synthesis, JBP1 (12). Gel shift analysis of extracts from the JBP1KO cell line failed to indicate any other base J-containing DNA binding activity.…”

Section: Discussionmentioning

confidence: 99%

Epigenetic Regulation of Transcription and Virulence in Trypanosoma cruzi by O-Linked Thymine Glucosylation of DNA

Ekanayake

Minning

Weatherly

et al. 2011

Molecular and Cellular Biology

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Unlike other eukaryotes, the protein-coding genes of Trypanosoma cruzi are arranged in large polycistronic gene clusters transcribed by polymerase II (Pol II). Thus, it is thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. Here, we show that the glucosylated thymine DNA base (␤-D-glucosyl-hydroxymethyluracil or base J) is present within sequences flanking the polycistronic units (PTUs) in T. cruzi. The loss of base J at sites of transcription initiation, via deletion of the two enzymes that regulate base J synthesis (JBP1 and JBP2), correlates with an increased rate of Pol II transcription and subsequent genome-wide increase in gene expression. The affected genes include virulence genes, and the resulting parasites are defective in host cell invasion and egress. These studies indicate that base J is an epigenetic factor regulating Pol II transcription initiation in kinetoplastids and provides the first biological role of the only hypermodified DNA base in eukaryotes.Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is the major cause of cardiac disease in South and Central America (1). The parasite has a complex life cycle with two hosts and four developmental stages. Epimastigotes develop in the hindgut of the triatomine insect vector and differentiate into metacyclic forms. Infective metacyclic forms enter the vertebrate host, invade the host cell, and differentiate to form amastigotes. Trypomastigotes released from the infected cell are able to reinvade a wide variety of host cells. Success of the parasite throughout the life cycle is ensured by the regulated expression of surface proteins such as mucin and trans-sialidase, which allow differential adherence and evasion of the host immune responses (2). Members of the surface glycoprotein gene family colocalize with a novel hypermodified DNA base, ␤-D-glucosyl-hydroxymethyluracil or base J, suggesting an epigenetic mechanism of regulating T. cruzi pathogenesis (3).In base J, the thymine base exhibits O-linked glucosylation in telomeric DNA of all kinetoplastid flagellates and some closely related unicellular flagellates, but base J is not present in the genomes of other protozoa or metazoa (3, 4). Base J was initially discovered on the basis of its distinct presence within the 19 silent telomeric variant surface glycoprotein (VSG) expression sites (ES) of Trypanosoma brucei but absence from the single transcribed ES, suggesting its role in the regulation of telomeric VSG gene expression (3, 35). Recent genomewide analysis revealed that base J is also present throughout the T. brucei genome, enriched at regions flanking polymerase II (Pol II) polycistronic transcription units (PTUs) (9). PTUs are large gene clusters that are cotranscribed by Pol II to yield polycistronic pre-mRNAs that are then processed into mature mRNAs by trans-splicing and polyadenylation (7). The localization of base J at PTU-flanking regions suggests a role for the modified base in regulating Pol II transcription initi...

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Section: Discussionmentioning

confidence: 99%

Epigenetic Regulation of Transcription and Virulence in Trypanosoma cruzi by O-Linked Thymine Glucosylation of DNA

Ekanayake

Minning

Weatherly

et al. 2011

Molecular and Cellular Biology

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“…These proteins could lead to gene silencing and/or suppression of DNA recombination, both of which are involved in the mechanism of antigenic variation. Our recent discovery of J-binding proteins (JBPs) in kinetoplastids that specifically bind J-containing DNA is compatible with this idea (10). By understanding how JBP specifically recognizes and binds the unique modified base in DNA could represent a first step in elucidating the function and mechanism of J action.…”

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confidence: 94%

Recognition of Base J in Duplex DNA by J-binding Protein

Sabatini¹,

Meeuwenoord²,

Boom³

et al. 2002

Journal of Biological Chemistry

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␤-D-Glucosylhydroxymethyluracil, also called base J, is an unusual modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously found a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia. We have now characterized the binding properties of recombinant JBP from Crithidia using synthetic J-DNA substrates that contain the glycosylated base in various DNA sequences. We find that JBP recognizes base J only when presented in double-stranded DNA but not in singlestranded DNA or in an RNA:DNA duplex. It also fails to interact with free glucose or free base J. JBP is unable to recognize nonmodified DNA or intermediates of J synthesis, suggesting that JBP is not directly involved in J biosynthesis. JBP binds J-DNA with high affinity (K d ‫؍‬ 40 -140 nM) but requires at least 5 bp flanking the glycosylated base for optimal binding. The nature of the flanking sequence affects binding because J in a telomeric sequence binds JBP with higher affinity than J in another sequence known to contain J in trypanosome DNA. We conclude that JBP is a structure-specific DNAbinding protein. The significance of these results in relation to the biological role and mechanism of action of J modification in kinetoplastids is discussed.In the DNA of kinetoplastid flagellates, a fraction of thymine is replaced by the modified base ␤-D-glucosylhydroxymethyluracil, called J (1-3). In all kinetoplastids, J is abundantly present in telomeric repeats (3). In the parasite Trypanosoma brucei, J is also found in the telomeric variant surface glycoprotein (VSG) 1 gene expression sites involved in antigenic variation (4, 5). The presence of J in inactive telomeric VSG gene expression sites but not in the active site suggests that J may be involved in the transcriptional repression of VSG gene expression sites and thus antigenic variation (3-9).It has been suggested that J is involved in long term transcriptional repression and that J could also suppress unwanted recombination between repetitive sequences in the genome (3,8,9). Whether this is true and whether J is the cause or the consequence of this silencing remain to be determined. However, consistent with both ideas, the protrusion of the sugar group of the major groove of DNA, at specific locations, could allow the recognition and binding of proteins that would mediate J function. These proteins could lead to gene silencing and/or suppression of DNA recombination, both of which are involved in the mechanism of antigenic variation. Our recent discovery of J-binding proteins (JBPs) in kinetoplastids that specifically bind J-containing DNA is compatible with this idea (10). By understanding how JBP specifically recognizes and binds the unique modified base in DNA could represent a first step in elucidating the function and mechanism of J action.In this report, we use recombinant JBP to study the interaction between JBP and J-DNA. We utilize DNA ...

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“…Although both JBP1 and JBP2 stimulate de novo thymidine hydroxylation in vivo, the ability of JBP1 to bind J-DNA is thought to play a role in J propagation/maintenance (9,14,19). Deletion of either JBP1 or JBP2 from the bloodstream form T. brucei results in a 20-and 8-fold reduction in the levels of base J, respectively (10,14,20). The simultaneous deletion of both JBP1 and JBP2 yields a cell line that is unable to synthesize base J unless cells are fed hmU (14).…”

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Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

Bullard

Rosa-Spiegler

Liu

et al. 2014

Journal of Biological Chemistry

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The modified base J is the target for a novelDNA-binding protein in kinetoplastid protozoans

Cited by 68 publications

References 37 publications

Epigenetic Regulation of Transcription and Virulence in Trypanosoma cruzi by O-Linked Thymine Glucosylation of DNA

Epigenetic Regulation of Transcription and Virulence in Trypanosoma cruzi by O-Linked Thymine Glucosylation of DNA

Recognition of Base J in Duplex DNA by J-binding Protein

Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

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