F the panelfortuitous transcription initiation of these silent VSGs, thereby maintaining mono-allelic expression of the active VSG. The more poorly positioned nucleosomes SCR7 web covering the VSG arrays may represent an open chromatin structure more amenable to DNA recombination events, or may be due to a repressive chromatin structure resulting in decreased MNase cleavage.TbORC1 and DNA replication originsIt had previously been shown that ORC1, a component of the origin recognition complex, binds to numerous regions in the T. brucei genome, many of which act as origins of DNA replication [74]. It has also previously been shown in S. cerevisiae that ORC1, apart from its role in DNAreplication, is also a component of silencing complexes assembled at silencing elements such as the E-element of the Mat alpha silent mating-type locus. Consequently, we were interested in establishing whether the T. brucei ORC1 was similarly involved in a specialized nucleosomal organization which could be implicated in gene silencing. We mapped ORC1 sites identified in T. brucei 927 [74] to the equivalent sequences in the genome of T. brucei 427. The ORC1 binding sequences originally identified by Tiengwe and colleagues [74] ranged from 65 bp to 3 kb. This mapping was therefore at a low resolution, below that of single nucleosomes. Nevertheless, we utilized this dataset to investigate the nucleosomal organization in the vicinity of assigned ORC1 binding sites.Maree et al. Epigenetics Chromatin (2017) 10:Page 14 ofWe first investigated the DNA region surrounding ORC1 sites in the core region of chromosomes (994 sites), which contain the constitutively expressed housekeeping genes. Here, very little nucleosomal organization relative to the center of the assigned DNA replication origin was discernible (Fig. 5c). We next investigated the subtelomeric regions (119 sites), which are defined as regions adjacent to telomeric ends. These contain the silent VSG PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26778282 gene and pseudogene arrays, expression site-associated genes (ESAGs), and the highly repetitive retrotransposon hotspot proteins (RHS) gene family. Here, a very clear nucleosomal organization was evident (Fig. 5d). The differences in organization of nucleosomes within the coding and flanking regions of the silent VSG arrays are consistent with the presence of specialized bordering silencing complexes, as also suggested by McCulloch and colleagues [72]. Intriguingly, of the 15 high confidence (false discovery rate (FDR) <0.05; [74]) ORC1 binding sites identified in the subtelomeric region of chromosome 9, 14 were present in the silent VSG array. This result strongly suggests that ORC1 binding sites, and presumably ORC1 itself are involved in the demarcation of specialized chromatin domains associated with silencing of the VSG gene and pseudogene arrays, as is the case in S. cerevisiae silent mating-type loci [73]. This suggests that the role of ORC1 in arranging surrounding chromatin structure and repressing regional transcription is evolutionarily ancient, and was likely present in LECA.Discussion Trypanosoma brucei regulates the expression of most of its protein-coding genes at the posttranscriptional level. However, the T. brucei genome encodes homologs for putative chromatin writers, readers and erasers, as well as chromatin remodeling enzymes and histone variants [3?]. It has been shown that specific histone variants demarcate the borders of PTUs [42]. In addition, various epigenetic players were shown to be im.