Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome

Abstract

In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties were originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays.

Figure 1:. Mapping the 5-hmU and chromatin accessibility landscape in B. minutum.

(A) Proportions of human and B. minutum gDNA in 5-hmU Methylated DNA immunoprecipitation sequencing (MeDIP-seq) and control libraries. A mixture of human and dinoflagellate gDNA was used as input to MeDIP-seq experiments, and the fraction of reads that map to each genome is shown. The 5-hmU MeDIP-seq library is enriched for dinoflagellate reads confirming the specificity of 5-hmU pull down. (B) Proportion of multimapping reads in 5-hmU MeDIP-seq and control libraries. The 5-hmU MeDIP-seq library exhibits a higher fraction of multimapping reads, suggesting that 5-hmU is enriched over repetitive elements. (C) Metaprofiles of 5-hmU and control libraries signal over B. minutum transcription start sites/gene starts. (D) Basepair-resolution chemical mapping of 5-hmU does not reveal a sequence motif associated with the modification in B. minutum. (E) Fragment length distribution of B. minutum ATAC-seq datasets. Shown are uniquely mapping reads alone as well as all reads that can be mapped. (F) Proportion of multimapping reads in B. minutum ATAC-seq datasets as well as a control genomic DNA (gDNA) library. (G) Metaprofiles of ATAC-seq signal over B. minutum transcription start sites/gene starts as well as the gDNA control. (H) Distribution of ATAC-seq regions of enrichment relative to annotated genomic features. (I) Differential accessibility analysis for the 27 °C and 34 °C conditions.

Figure 2:. Inverse correlation between 5-hmU and chromatin accessibility and association with dinoTADs boundaries in the B. minutum genome.

(A-B) Representative snapshots of the distribution of 5-hmU enrichment and decreased chromatin accessibility relative to dinoTAD boundaries. (C) Depletion of ATAC-seq signal around dinoTAD boundaries. (D) Enrichment of MeDIP-seq signal around dinoTAD boundaries. (E) Increased 5-hmU chemical mapping conversion rate around dinoTAD boundaries. (F-G) ATAC-seq and MeDIP-seq are generally anti-correlated (calculated for 5-kbp bins over the whole genome) (H) ATAC-seq and MeDIP-seq are specifically strongly anti-correlated around dinoTAD boundaries.

Figure 3:. Association of 5-hmU and chromatin accessibility with repetitive elements in the B. minutum genome.

(A-C) Distribution of all repeats, individual repeat families, and some DNA elements around dinoTAD boundaries. (D) ATAC-seq enrichment/depletion over repetitive elements. (E) MeDIP-seq enrichment/depletion over repetitive elements. (F-G) V-plot around positioned nucleosomes in S. cerevisiae (for comparison) and de novo identified putative positioned nucleosomes in B. minutum. (H) Enrichment/depletion of positioned nucleosomes over repetitive elements.