[
International Worm Meeting,
2021]
DNA is an antiparallel double helix; its transcription requires separating the two strands. This creates supercoils, underwound and overwound duplex DNA, which accumulate behind and in front of the transcription bubble, respectively. Overwound supercoils are detrimental to helix opening and transcription bubble formation, as they increase the energy necessary to separate strands. In vivo, these structures are resolved by the action of topoisomerases which release supercoils from DNA. Conversely, the action of condensins, molecular machines creating loops in chromatin, has been shown in vitro to increase supercoiling. The interplay between supercoiling, transcription and condensin action could be a powerful regulator of gene expression. To understand this interplay, we mapped supercoiling genome-wide in vivo in L3 larvae using biotinylated 4,5,8-trimethylpsoralen (bTMP). bTMP preferentially intercalates in negatively supercoiled DNA and is crosslinked to it using UV illumination. Using biotin pulldown, we can then enrich for negatively supercoiled DNA before high-throughput sequencing. Our results show highly reproducible supercoiling profiles. At chromosome scale, we did not observe a significant difference in supercoiling between chromosomes. In contrast, telomeric thirds of autosomes (perinuclear heterochromatic B domains) show a lower bTMP enrichment than at central domains (euchromatic A domains), in agreement with higher transcription levels in euchromatin. At the gene level, bTMP enrichment shows two peaks, one 5' of the transcription start site (TSS) and one at the transcription end site (TES). This suggests the accumulation of negative supercoils in the promoter region of genes and at the end of the transcription units. In agreement with a transcriptional cause, the bTMP enrichment profile along the genes correlates with gene expression levels. In hermaphrodite animals, a variant of condensin I called condensin IDC, is part of the dosage compensation complex which downregulates gene expression of X-linked genes. Condensins I/IDC purified from nematodes can induce the formation of supercoils in vitro. In vivo, we observe differential bTMP enrichment at X-linked TSS, coinciding with the location of the condensin IDC. This suggests that supercoiling may link the X-specific transcriptional repression and condensin loading.
[
International Worm Meeting,
2019]
Transcription initiation is a dynamic, multi-step process, requiring the ordered recruitment of multiple factors to the core promoter. The highly dynamic nature of transcription means that at any given moment, a particular promoter might be in one of multiple states in different cells, resulting in heterogeneous signals from a population of cells. Most techniques that study transcription initiation, such as chromatin-IP or GRO-seq provide population average measurements of the transcriptional process. To really understand transcription regulation, it is necessary to examine changes in promoter state at the single molecule level. To achieve this, we applied to C. elegans a technique known as "dual enzyme Single Molecule Footprinting (dSMF)". The method involves in vitromethylation of chromatin in intact nuclei with bacterial CpG and GpC methyltransferases. Closed chromatin bound by proteins is protected from methylation, whereas accessible DNA is methylated. After bisulfite conversion, specific amplicons, or genome-wide DNA libraries are sequenced to identify footprints of accessible DNA. Correlation of dSMF data with publicly available genome wide datasets shows that dSMF footprints coincide with nucleosome free regions in promoters and other genomic regions such as HOT sites. To identify what the different footprints represent in terms of promoter states in the transcription cycle, we then inhibit individual steps of the transcription cycle either with chemicals or by blocking engineered cyclin-dependent kinases with ATP analogs. Upon mapping the promoter occupancy states in transcription inhibited conditions, we are able to correlate the different promoter footprints with specific protein complexes present during the different steps of transcription initiation. Our data strengthen our understanding of how chromatin and gene structure regulate gene expression at the level of transcription initiation.