JBP2, a SWI2/SNF2-like protein, regulates de novo telomeric DNA glycosylation in bloodstream form Trypanosoma brucei

Kieft, Rudo; Brand, Verena; Ekanayake, Dilrukshi K.; Sweeney, Kate; DiPaolo, Courtney; Reznikoff, William S.; Sabatini, Robert

doi:10.1016/j.molbiopara.2007.06.010

Cited by 28 publications

(35 citation statements)

References 15 publications

(45 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous work has identified two distinct thymidine hydroxylases involved in base J biosynthesis, JBP1 and JBP2 (8,33,36). Deletion of JBP1 (base J binding protein 1) and JBP2 in T. brucei resulted in 20-and 8-fold reduction in base J levels, respectively (8,11,23). Deletion of both enzymes resulted in a trypanosome cell line unable to synthesize base J (J null) (8).…”

mentioning

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

Self Cite

View full text Add to dashboard Cite

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...

show abstract

mentioning

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…SC-805) (1:1000 dilution), anti-histone H3 (Abcam, catalog no. ab8580) (1:2500 dilution) or anti-JBP2 antibody, as described previously (19). Bound antibodies were detected by a secondary goat anti-rabbit antibody conjugated to HRP and visualized by ECL.…”

Section: Preparation Of Recombinant Jgt-mentioning

confidence: 99%

“…JBP2 does not bind the modified base directly, but is able to bind chromatin in a base J-independent manner, presumably via the C-terminal SWI2/SNF2 domain (9). 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).…”

mentioning

confidence: 99%

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…Both of them contain a N-terminal thymine hydroxylase (TH) domain [12], but only JBP1 can bind to J in DNA via a particular J-binding domain in its C-terminal half [13]. On the other hand, JBP2 contains a SWI2/SNF2 domain homologous to ATPase/DNA helicases, which has been thought to be important for its activity [14]. Recently, the JBP proteins have been grouped together with mammalian TET proteins into the new TET/JBP subfamily of Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenases [15].…”

Section: Introductionmentioning

confidence: 99%

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

Liu

Cliffe

et al. 2014

J. Am. Soc. Mass Spectrom.

Self Cite

View full text Add to dashboard Cite

β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: First, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (β-D-glucosyl-5-hydroxymethyl-2′-deoxycytidine, 5-gHmdC), for the accurate detection of β-D-glucosyl-5-hydroxymethyl-2′-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis, i.e. 5-hydroxymethyl-2′-deoxyuridine (5-HmdU). We found that base J was not detectable in the JBP-null cells while it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the future.

show abstract

JBP2, a SWI2/SNF2-like protein, regulates de novo telomeric DNA glycosylation in bloodstream form Trypanosoma brucei

Cited by 28 publications

References 15 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

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

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

Contact Info

Product

Resources

About