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

Unusually for a eukaryote, genes transcribed by RNA polymerase II (pol II) in Trypanosoma brucei are arranged in polycistronic transcription units. With one exception, no pol II promoter motifs have been identified, and how transcription is initiated remains an enigma. T. brucei has four histone variants: H2AZ, H2BV, H3V, and H4V. Using chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) to examine the genome-wide distribution of chromatin components, we show that histones H4K10ac, H2AZ, H2BV, and the bromodomain factor BDF3 are enriched up to 300-fold at probable pol II transcription start sites (TSSs). We also show that nucleosomes containing H2AZ and H2BV are less stable than canonical nucleosomes. Our analysis also identifies >60 unexpected TSS candidates and reveals the presence of long guanine runs at probable TSSs. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable pol II transcription termination sites. Our findings suggest that histone modifications and histone variants play crucial roles in transcription initiation and termination in trypanosomes and that destabilization of nucleosomes by histone variants is an evolutionarily ancient and general mechanism of transcription initiation, demonstrated in an organism in which general pol II transcription factors have been elusive.

Telomerase-independent proliferation is influenced by cell type in Saccharomyces cerevisiae.

Yeast strains harboring mutations in genes required for telomerase function (TLC1 and the EST genes) exhibit progressive shortening of telomeric DNA and replicative senescence. A minority of cells withstands loss of telomerase through RAD52-dependent amplification of telomeric and subtelomeric sequences; such survivors are now capable of long-term propagation with telomeres maintained by recombination rather than by telomerase. Here we report that simultaneous expression in haploid cells of both MATa and MATalpha information suppresses the senescence of telomerase-deficient mutants, with suppression occurring via the RAD52-dependent survivor pathway(s). Such suppression can be mimicked by deletion of SIR1-SIR4, genes that function in transcriptional silencing of several loci including the silent mating-type loci. Furthermore, telomerase-defective diploid strains that express only MATa or MATalpha information senesce at a faster rate than telomerase-defective diploids that are heterozygous at the MAT locus. This suggests that the RAD52-dependent pathway(s) for telomere maintenance respond to changes in the levels of recombination, a process regulated in part by the hierarchy of gene control that includes MAT regulation. We propose that cell-type-specific regulation of recombination at human telomeres may similarly contribute to the tissue-specific patterns of disease found in telomerase-deficient tumors.

Selective di- or trimethylation of histone H3 lysine 76 by two DOT1 homologs is important for cell cycle regulation in Trypanosoma brucei.

DOT1 is an evolutionarily conserved histone H3 lysine 79 (H3K79) methyltransferase. K79 methylation is associated with transcriptional activation, meiotic checkpoint control, and DNA double-strand break (DSB) responses. Trypanosoma brucei has two homologs, DOT1A and DOT1B, which are responsible for dimethylation and trimethylation of H3K76, respectively (K76 in T. brucei is synonymous to K79 in other organisms). K76 dimethylation is only detectable during mitosis, whereas trimethylation occurs throughout the cell cycle. Deletion of DOT1B resulted in dimethylation of K76 throughout the cell cycle and caused subtle defects in cell cycle regulation and impaired differentiation. RNAi-mediated depletion of DOT1A appears to disrupt a mitotic checkpoint, resulting in premature progression through mitosis without DNA replication, generating a high proportion of cells with a haploid DNA content, an unprecedented state for trypanosomes. We propose that DOT1A and DOT1B influence the trypanosome cell cycle by regulating the degree of H3K76 methylation.

Histone H2AZ dimerizes with a novel variant H2B and is enriched at repetitive DNA in Trypanosoma brucei.

H2AZ is a widely conserved histone variant that is implicated in protecting euchromatin from the spread of heterochromatin. H2AZ is incorporated into nucleosomes as a heterodimer with H2B, by the SWR1 ATP-dependent chromatin-remodeling complex. We have identified a homolog of H2AZ in the protozoan parasite Trypanosoma brucei, along with a novel variant of histone H2B (H2BV) that shares approximately 38% sequence identity with major H2B. Both H2AZ and H2BV are essential for viability. H2AZ localizes within the nucleus in a pattern that is distinct from canonical H2A and is largely absent from sites of transcription visualized by incorporation of 5-bromo-UTP (BrUTP). H2AZ and H2BV colocalize throughout the cell cycle and exhibit nearly identical genomic distribution patterns, as assessed by chromatin immunoprecipitation. H2AZ co-immunoprecipitates with H2BV but not with histones H2B or H2A nor with the variant H3V. These data strongly suggest that H2AZ and H2BV function together within a single nucleosome, marking the first time an H2AZ has been shown to associate with a non-canonical histone H2B.

A variant histone H3 is enriched at telomeres in Trypanosoma brucei.

Variant histones play critical roles in transcriptional activation and repression, DNA repair and chromosome segregation. We have identified HTV, a single-copy gene in Trypanosoma brucei encoding a variant form of histone H3 (H3V). H3V is present at discrete nuclear foci that shift over the course of the cell cycle and associate with the mitotic spindle, a pattern of localization reminiscent of that described previously for both mini-chromosomes and telomeres. By combining fluorescence in situ hybridization with indirect immunofluorescence, we confirmed that the H3V foci overlap with a 177-bp repetitive sequence element found predominantly in mini-chromosomes, as well as with the TTAGGG repeats that compose telomeres. Chromatin immunoprecipitation studies, however, reveal that only the telomeric repeat DNA is substantially enriched with H3V. HTV is not essential for viability, mini-chromosome segregation, telomere maintenance or transcriptional silencing at the telomere-proximal expression sites from which bloodstream-form T. brucei controls antigenic variation. We propose that H3V represents a novel class of histone H3 variant, a finding that has evolutionary implications.