Biology subjects

Tong, P.

Publications and source records attributed to Tong, P..

25 records · Page 2Linked to original sources

bioRxiv2021

A systematic analysis of Trypanosoma brucei chromatin factors identifies novel protein interaction networks associated with sites of transcription initiation and termination

Nucleosomes composed of histones are the fundamental units around which DNA is wrapped to form chromatin. Transcriptionally active euchromatin or repressive heterochromatin is regulated in part by the addition or removal of histone post-translational modifications (PTMs) by writer and eraser enzymes, respectively. Nucleosomal PTMs are recognised by a variety of reader proteins which alter gene expression accordingly. The histone tails of the evolutionarily divergent eukaryotic parasite Trypanosoma brucei have atypical sequences and PTMs distinct from those often considered universally conserved. Here we identify 68 predicted readers, writers and erasers of histone acetylation and methylation encoded in the T. brucei genome and, by epitope tagging, systemically localize 63 of them in the parasites bloodstream form. ChIP-seq demonstrated that fifteen candidate proteins associate with regions of RNAPII transcription initiation. Eight other proteins exhibit a distinct distribution with specific peaks at a subset of RNAPII transcription termination regions marked by RNAPIII-transcribed tRNA and snRNA genes. Proteomic analyses identified distinct protein interaction networks comprising known chromatin regulators and novel trypanosome-specific components. Notably, several SET-domain and Bromo-domain protein networks suggest parallels to RNAPII promoter-associated complexes in conventional eukaryotes. Further, we identify likely components of TbSWR1 and TbNuA4 complexes whose enrichment coincides with the SWR1-C exchange substrate H2A.Z at RNAPII transcriptional start regions. The systematic approach employed provides detail of the composition and organization of the chromatin regulatory machinery in Trypanosoma brucei and establishes a route to explore divergence from eukaryotic norms in an evolutionarily ancient but experimentally accessible eukaryote.

molecular biology↗

bioRxiv2020

Inhibition of nonsense-mediated decay rescues functional p53β/γ isoforms in MDM2-amplified cancers

Common mechanisms for p53 loss in cancer include expression of MDM2 or the human papilloma virus (HPV)-encoded E6 protein which both mediate degradation of wild-type (WT) p53 (p53). Here, we show that two alternatively-spliced, functional, truncated isoforms of p53 (p53{beta} and p53{gamma}, containing exons 1-9 of the p53 gene) can be markedly upregulated by pharmacologic or genetic inhibition of nonsense mediated decay (NMD), a regulator of aberrant mRNA stability. These isoforms lack the MDM2 binding domain and hence have reduced susceptibility to MDM2-mediated degradation. In MDM2-overexpressing cells bearing wildtype TP53 gene, NMD blockade increased p53{beta}/{gamma} expression and p53 pathway activation, enhanced radiosensitivity, and inhibited tumor growth. A similar pattern was observed in HPV+ cancer cells and in cancer cells with p53 mutations downstream of exon 9. These results identify a novel therapeutic strategy for restoration of p53 function in tumors rendered p53 deficient through MDM2 overexpression, HPV infection, or certain p53 mutations.

cancer biology↗

bioRxiv2020

Large domains of heterochromatin direct the formation of short mitotic chromosome loops

During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine 9 trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes, such as at centromeres.

cell biology↗

bioRxiv2019

Hap2-Ino80 facilitated transcription promotes de novo establishment of CENP-A chromatin

Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily-conserved histone H3 variant, which directs kinetochore assembly and hence, centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity selected solubilised fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naive centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilises H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.

molecular biology↗

bioRxiv2018

Fitness Landscape of the Fission Yeast Genome

BackgroundNon-protein-coding regions of eukaryotic genomes remain poorly understood. Diversity studies, comparative genomics and biochemical outputs of genomic sites can be indicators of functional elements, but none produce fine-scale genome-wide descriptions of all functional elements.\n\nResultsTowards the generation of a comprehensive description of functional elements in the haploid Schizosaccharomyces pombe genome, we generated transposon mutagenesis libraries to a density of one insertion per 13 nucleotides of the genome. We applied a five-state hidden Markov model (HMM) to characterise insertion-depleted regions at nucleotide-level resolution. HMM-defined functional constraint was consistent with genetic diversity, comparative genomics, gene-expression data and genome annotation.\n\nConclusionsWe infer that transposon insertions lead to fitness consequences in 90% of the genome, including 80% of the non-protein-coding regions, reflecting the presence of numerous non-coding elements in this compact genome that have functional roles. Display of this data in genome browsers provides fine-scale views of structure-function relationships within specific genes.

genomics↗

bioRxiv2018

Inter-species conservation of organisation and function between non-homologous regional centromeres

Despite the conserved essential function of centromeres, centromeric DNA itself is not conserved1-4. The histone-H3 variant, CENP-A, is the epigenetic mark that specifies centromere identity5-8. Paradoxically, CENP-A normally assembles on particular sequences at specific genomic locations. To gain insight into the specification of complex centromeres we took an evolutionary approach, fully assembling genomes and centromeres of related fission yeasts. Centromere domain organization, but not sequence, is conserved between Schizosaccharomyces pombe, S. octosporus and S. cryophilus with a central CENP-ACnp1 domain flanked by heterochromatic outer-repeat regions. Conserved syntenic clusters of tRNA genes and 5S rRNA genes occur across the centromeres of S. octosporus and S. cryophilus, suggesting conserved function. Remarkably, non-homologous centromere central-core sequences from S. octosporus are recognized in S. pombe, resulting in cross-species establishment of CENP-ACnp1 chromatin and functional kinetochores. Therefore, despite the lack of sequence conservation, Schizosaccharomyces centromere DNA possesses intrinsic conserved properties that promote assembly of CENP-A chromatin. Thus, centromere DNA can be recognized and function over unprecedented evolutionary timescales.

genomics↗

bioRxiv2017

Centromeric DNA destabilizes H3 nucleosomes to promote CENP-A deposition during the cell cycle

Active centromeres are defined by the presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and kinetochores are maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A itself promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences. Intrinsic properties of centromeric DNA may favour the assembly of CENP-A rather than H3 nucleosomes. Here we investigate histone dynamics on centromeric DNA. We show that during S-phase histone H3 is deposited as a placeholder at fission yeast centromeres and is subsequently evicted in G2 when we detect deposition of the majority of new CENP-ACnp1. We also find that centromeric DNA has an innate property of driving high rates of turnover of H3 containing nucleosomes resulting in low nucleosome occupancy. When placed at an ectopic chromosomal location in the absence of any CENP-ACnp1 assembly, centromeric DNA retains its ability to impose S-phase deposition and G2 eviction of H3, suggesting that features within this DNA program H3 dynamics. As RNAPII occupancy on this centromere DNA coincides with H3 eviction in G2, we propose a model in which RNAPII-coupled chromatin remodelling promotes replacement of H3 with CENP-ACnp1 nucleosomes.

cell biology↗