scholarly journals Genetic Interaction Between Site-Specific Epigenetic Marks and Roles of H4v in Transcription Termination in Trypanosoma brucei

2021 ◽  
Vol 9 ◽  
Author(s):  
Hee-Sook Kim
Keyword(s):  
Trypanosoma Brucei ◽  
Cell Cycle Progression ◽  
Genetic Interaction ◽  
Transcription Termination ◽  
Genome Structure ◽  
Histone Variants ◽  
Mrna Levels ◽  
Messenger Rnas ◽  
Compensatory Mechanisms ◽  
Polycistronic Transcription

In Trypanosoma brucei, genes are assembled in polycistronic transcription units (PTUs). Boundaries of PTUs are designated transcription start sites and transcription termination sites (TTSs). Messenger RNAs are generated by trans-splicing and polyadenylation of precursor RNAs, and regulatory information in the 3′ un-translated region (UTR), rather than promoter activity/sequence-specific transcription factors, controls mRNA levels. Given this peculiar genome structure, special strategies must be utilized to control transcription in T. brucei. TTSs are deposition sites for three non-essential chromatin factors—two of non-canonical histone variants (H3v and H4v) and a DNA modification (base J, which is a hydroxyl-glucosyl dT). This association generated the hypothesis that these three chromatin marks define a transcription termination site in T. brucei. Using a panel of null mutants lacking H3v, H4v, and base J, here I show that H4v is a major sign for transcription termination at TTSs. While having a secondary function at TTSs, H3v is important for monoallelic transcription of telomeric antigen genes. The simultaneous absence of both histone variants leads to proliferation and replication defects, which are exacerbated by the J absence, accompanied by accumulation of sub-G1 population. Thus, I propose that the coordinated actions of H3v, H4v, and J provide compensatory mechanisms for each other in chromatin organization, transcription, replication, and cell-cycle progression.

scholarly journals Site-specific epigenetic marks in Trypanosoma brucei transcription termination, antigenic variation, and proliferation

2021 ◽  
Author(s):  
Hee-Sook Kim
Keyword(s):  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Transcription Termination ◽  
Cell Cycle Control ◽  
Histone Variants ◽  
Transcription Start ◽  
Compensatory Mechanisms ◽  
Polycistronic Transcription ◽  
Secondary Function ◽  
Transcription And Replication

In Trypanosoma brucei, genes assemble into polycistronic transcription units (PTUs). Transcription termination sites (TTSs) hold deposition sites for three non-essential chromatin factors, histone variants (H3v and H4v) and a DNA modification (base J, a hydroxyl-glucosyl dT). Here, I found that H4v is a major sign for transcription termination at TTSs and readthrough transcription machineries progress until they encounter the next bidirectional transcription start site. While having a secondary function at TTSs, H3v is important for monoallelic transcription of telomeric antigen genes. The simultaneous absence of both histone variants leads to proliferation and replication defects, which are exacerbated by the J deficiency, accompanied by accumulation of sub-G1 population. Base J likely contributes to DNA replication and cell-cycle control. I propose that the coordinated actions of H3v, H4v and J function in concert for cellular fate determination and provide compensatory mechanisms for each other in chromatin organization, transcription, and replication.

scholarly journals Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

2019 ◽  
Author(s):  
Rudo Kieft ◽  
Yang Zhang ◽  
Alexandre P. Marand ◽  
Jose Dagoberto Moran ◽  
Robert Bridger ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Binding Protein ◽  
Transcription Termination ◽  
Regulatory Protein ◽  
Gene Clusters ◽  
Pol Ii ◽  
Polycistronic Transcription ◽  
Dna Base ◽  
Multimeric Complex

AbstractBase J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3’-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for host immune evasion. Our results provide the first direct mechanistic link between base J and regulation of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating polycistronic transcription in trypanosomatids.Author SummaryTrypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including divergent organisms that utilize polycistronic transcription and novel epigenetic marks such as base J and H3.V. The studies also provide the first direct mechanistic link between J modification of DNA at termination sites and regulated Pol II termination and gene expression in kinetoplastids.

scholarly journals Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei

2009 ◽  
Vol 23 (9) ◽  
pp. 1063-1076 ◽  
Author(s):  
T. N. Siegel ◽  
D. R. Hekstra ◽  
L. E. Kemp ◽  
L. M. Figueiredo ◽  
J. E. Lowell ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Histone Variants ◽  
Polycistronic Transcription

scholarly journals Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

1989 ◽  
Vol 9 (5) ◽  
pp. 1940-1945 ◽  
Author(s):  
B Y Tseng ◽  
C E Prussak ◽  
M T Almazan
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Mrna Levels ◽  
Proliferating Cells ◽  
Restriction Point ◽  
Serum Stimulation ◽  
G1 Cells

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

Cloning, Heterologous Expression and Kinetic Analysis of Glycerol Kinase (TbGLK1) from Trypanosoma brucei

2000 ◽  
Vol 381 (11) ◽  
pp. 1071-1077 ◽  
Author(s):  
K. Steinborn ◽  
A. Szallies ◽  
D. Mecke ◽  
M. Duszenko
Keyword(s):  
Heterologous Expression ◽  
Kinetic Analysis ◽  
Trypanosoma Brucei ◽  
Blot Analysis ◽  
Spatial Separation ◽  
Single Copy ◽  
Glycerol Kinase ◽  
Mrna Levels ◽  
Atp Production ◽  
C Terminus

Abstract We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.

Posttranscriptional regulation of cytochrome c expression during the developmental cycle of Trypanosoma brucei

1988 ◽  
Vol 8 (11) ◽  
pp. 4625-4633
Author(s):  
A F Torri ◽  
S L Hajduk
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Trypanosoma Brucei ◽  
Posttranscriptional Regulation ◽  
Mitochondrial Protein ◽  
Developmental Cycle ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Partial Cdna ◽  
Steady State Levels

We examined the expression of a nucleus-encoded mitochondrial protein, cytochrome c, during the life cycle of Trypanosoma brucei. The bloodstream forms of T. brucei, the long slender and short stumpy trypanosomes, have inactive mitochondria with no detectable cytochrome-mediated respiration. The insect form of T. brucei, the procyclic trypanosomes, has fully functional mitochondria. Cytochrome c is spectrally undetectable in the bloodstream forms of trypanosomes, but during differentiation to the procyclic form, spectrally detected holo-cytochrome c accumulates rapidly. We have purified T. brucei cytochrome c and raised antibodies that react to both holo- and apo-cytochrome c. In addition, we isolated a partial cDNA to trypanosome cytochrome c. An examination of protein expression and steady-state mRNA levels in T. brucei indicated that bloodstream trypanosomes did not express cytochrome c but maintained significant steady-state levels of cytochrome c mRNA. The results suggest that in T. brucei, cytochrome c is developmentally regulated by a posttranscriptional mechanism which prevents either translation or accumulation of cytochrome c in the bloodstream trypanosomes.

Abstract 19285: Notch1 Haploinsufficiency Increases Risk of Congenital Heart Defects in the Setting of Maternal Diabetes by an Epigenetic Mechanism

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Madhumita Basu ◽  
Kevin Bosse ◽  
Vidu Garg
Keyword(s):  
Signaling Pathway ◽  
Congenital Heart ◽  
Genetic Interaction ◽  
Heart Defects ◽  
Maternal Diabetes ◽  
Epigenetic Mechanism ◽  
Luciferase Reporter ◽  
Environment Interaction ◽  
Mrna Levels ◽  
Dependent Manner

Congenital heart disease (CHD) is the most common type of birth defect. Epidemiologic studies have shown the importance of genetic and environmental factors in the multifactorial etiology of CHD. Maternal diabetes mellitus (DM) is one of the non-genetic risk factors that predisposes to CHD predominantly cardiac septation and cardiac outflow tract defects. DM is known to be associated with endothelial cell dysfunction and we recently demonstrated a genetic interaction between endothelial nitric oxide synthase and Notch1, which encodes a receptor that functions in an important cardiac developmental signaling pathway. We hypothesized that maternal DM in the setting of Notch1 heterozygosity of the developing embryo will predispose to CHD. Notch1 +/- embryos (E13.5) exposed to maternal DM demonstrated an increased incidence (86%) of ventricular septal defects compared to wildtype littermates (22%) (Table). Gene expression studies in non-diabetic wildtype, diabetic wildtype and Notch1 +/- embryos showed DM was associated with decreased Notch1 mRNA levels and upregulation in Jarid2, a histone H3K9 demethylase known to regulate Notch1. In H9C2 and endocardial-derived cells and chick embryos, we showed that hyperglycemia led to decreased expression of Notch1 and its downstream targets in a dose-dependent manner. Similarly, Jarid2 mRNA levels increased with high glucose. Furthermore, we found reduced luciferase reporter activity in cells transfected with a constitutively active Notch1 intracellular domain with hyperglycemia. Studies demonstrating the relative enrichment of Jarid2 on Notch1 locus with hyperglycemia by ChIP-qPCR will also be presented. Our findings reveal that maternal DM disrupts cardiac development by deregulating the Notch1 signaling pathway and suggest that this gene-environment interaction is mediated by an epigenetic mechanism involving Jarid2 providing the first mechanistic insights for this association.

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