Transcription-independent TFIIIC-bound sites cluster near heterochromatin boundaries within lamina-associated domains in C. elegans

Abstract

BACKGROUND: Chromatin organization is central to precise control of gene expression. In vertebrates, the insulator protein CTCF plays a central role in organizing chromatin into topologically associated domains (TADs). In nematode C. elegans, however, a CTCF homolog is absent, and pervasive TAD structures are limited to the dosage-compensated sex chromosome, leaving the 25 principle of C. elegans chromatin organization unclear. Transcription Factor III C (TFIIIC) is a basal transcription factor complex for RNA Polymerase III (Pol III), also implicated in chromatin organization. TFIIIC binding without Pol III co-occupancy, referred to as extra-TFIIIC binding, has been implicated in insulating active and inactive chromatin domains in yeasts, flies, and mammalian cells. Whether extra-TFIIIC sites are present and contribute to chromatin organization in C. elegans remain unknown. 30 RESULTS: We identified, in C. elegans embryos, 504 TFIIIC-bound sites absent of Pol III and TATAbinding protein co-occupancy characteristic of extra-TFIIIC sites. Extra-TFIIIC sites constituted half of all identified TFIIIC binding sites in the genome. Unlike Pol III-associated TFIIIC sites predominantly localized in the sex chromosome, extra-TFIIIC sites were highly over-represented within autosomes. Extra-TFIIIC sites formed dense clusters in cis. The autosomal regions enriched for extra-TFIIIC site 35 clusters presented a high level of heterochromatin-associated histone H3K9 trimethylation (H3K9me3). Furthermore, extra-TFIIIC site clusters were embedded in the lamina-associated domains. Despite the heterochromatin environment of extra-TFIIIC sites, the individual clusters of extra-TFIIIC sites were devoid of and resided near the individual H3K9me3-marked regions. CONCLUSION: Clusters of extra-TFIIIC sites were pervasive near the outer boundaries of H3K9me3-40 marked regions in C. elegans. Given the reported activity of extra-TFIIIC sites in heterochromatin insulation in yeasts, our observation raised the possibility that TFIIIC may also demarcate heterochromatin in C. elegans. 3 BACKGROUND 45 Eukaryotic genomes are organized into domains of various chromatin features including actively transcribed regions, transcription factor-bound regions, and transcriptionally repressed regions [1-4].Demarcation of chromatin domains is central to precise control and memory of gene expression patterns. Several proteins have been proposed to have activity in demarcating chromatin domains by acting as a physical boundary [5,6], generating nucleosome depleted regions [7], mediating long-range 50 chromatin interactions [8,9], or tethering chromatin to the nuclear periphery [10]. Despite intense studies [11][12][13][14][15], how chromatin domains are demarcated remains poorly understood.The genome of nematode Carnorhabditis elegans has served as a model to study chromatin organization [3,[16][17][18]. The highest level of chromatin organization in C. elegans is the chromatin feature that distinguishes between the X chromosome and the autosomes. The X chrom...