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

Abstract

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.

Figure 1.

Histone modifications and gene organization in T. brucei. (A) T. brucei contains highly divergent histone sequences. Shown, as examples, are the N-terminal histone H4 sequences of Tetrahymena thermophilia, Homo sapiens, and T. brucei. Known sites of acetylation (blue) and methylation (red) are indicated. (B) Divergent SSRs contain pol II TSSs. Convergent SSRs contain pol II TTSs. Orange boxes represent ORFs and green arrows indicate the direction of transcription.

Figure 2.

Histone H4K10ac is enriched at probable pol II TSSs. (A) For a representative region of chromosome 10, the relative number of tags for histone H4K10ac (black) and histone H4 (gray) are calculated for a window size of 100 bp. Orange boxes represent ORFs and green arrows indicate direction of transcription. (B) H4K10ac-rich regions at non-SSRs are often located next to tRNA genes (blue boxes).

Figure 3.

Genome-wide distribution of histone H4K10ac. Relative number of tags for H4K10ac in BF (chr 1–11) and PF (chr 7 and 11). The green asterisk marks the previously identified actin promoter (Ben Amar et al. 1991). The red asterisks mark life cycle-specific peaks. Window size, 100 bp.

Figure 4.

Histone variants H2AZ and H2BV and BDF3 are enriched at probable pol II TSSs. (A) For representative regions of chromosome 10, the relative number of tags for H2AZ (red), H2BV (yellow), and H4K10ac (black) are overlaid. Window size, 100 bp. Orange boxes represent ORFs and green arrows indicate direction of transcription. (B) For a representative region of chromosome 10, the relative number of tags for H4K10ac (black) and BDF3 (magenta) are calculated for a window size of 100 bp and 1000 bp, respectively. The number of tags from the BDF3 ChIP was normalized based on the number of tags from an H4 ChIP (not shown), to eliminate artifacts attributable to repetitive sequences. For unnormalized BDF3 data, see Supplemental Figure S4. Orange boxes represent ORFs. (C) Chromatin was prepared from cell lines containing Ty1-tagged H2A or H2AZ and equal amounts were washed with buffers containing increasing levels of NaCl. Chromatin was treated with micrococcal nuclease to generate mononucleosomes and immunoprecipitated with monoclonal antibody BB2. Coimmunoprecipitated histones were separated by SDS-PAGE and immunoblotted (top and middle panels) or stained with amido black (bottom panel), to determine the levels of coimmunoprecipitated histones in the H2A-IP and H2AZ-IP. (D) A representative IF colocalization analysis of BDF3-HA (red), DAPI (blue), and H4K10ac (top panel, green) or H4 (bottom panel, green). Bar, 2 μm.

Figure 5.

Histone variants H3V and H4V are enriched at probable pol II TTSs. For a representative region of chromosome 10, the relative number of tags for H3V (first panel, green) and histone H4V (second panel, blue) ChIPs were calculated for a window size of 2000 bp and normalized based on the number of tags from an H4 ChIP, to eliminate artifacts attributable to repetitive sequences. For unnormalized data, see Supplemental Figure S5. (Third panel) The relative number of tags for H4K10ac (black) is calculated for a window size of 100 bp. (Fourth panel) Overlay of H4K10ac, H3V, and H4V data. Orange boxes represent ORFs, vertical gray boxes mark pol II TTSs, and green arrows indicate direction of transcription.

Figure 6.

5′ ends of probable pol II TSSs are marked by runs of guanine. (A) Divergent SSRs contain two peaks of high GC richness (red). The left peak indicates high levels of C in the top strand and the right peak indicates high levels of G in the top strand (blue). Orange boxes represent ORFs and green arrows indicate direction of transcription. Numbers on x-axis indicate nucleotide position on chr 10. (B) Average density of stretches of G (n ≥ 9) from 128 TSSs (dark blue, right axis). Density shown is the mean over 38 putative TSSs at non-SSRs and 90 TSSs at divergent SSRs using a 1000-bp moving average (densities were statistically indistinguishable between the two types of TSSs; data not shown). For comparison, the mean shapes of H2AZ (red) and BDF3 (magenta) enrichment are shown at non-SSR TSSs (left axis, see also Supplemental Fig. S6). BDF3 data were scaled to have the same maximum as H2AZ data. TSSs centers were determined based on Gaussian fits to the H2AZ data. (C) Model indicating how regions upstream of probable pol II TSSs are enriched for guanine. Sequence and peak size are not drawn to scale.

Figure 7.

Model to illustrate a possible cascade of events leading to pol II transcription initiation. Histone H3K4me3 enrichment at TSSs could provide a binding site for HAT2, which contains a chromodomain and is responsible for H4K10 acetylation. H4K10ac is strongly enriched at probable pol II TSSs and might serve as a binding site for BDF3. The latter is enriched at the 5′ end of probable pol II TSSs, where it may play a role in recruiting pol II transcription factors or the chromatin remodeling complex involved in H2AZ and H2BV incorporation. These two histone variants are enriched at probable pol II TSSs and form less stable nucleosomes that could facilitate binding of the transcriptional machinery. The G tract at the 5′ end of probable pol II TSSs could block antisense transcription and/or provide binding sites for transcription factors. H3V and H4V variants are enriched at probable pol II TTSs, which often contain tRNA or other genes transcribed by RNA pol III.