Abstract

Chromatin is folded at different levels of organisation from the fundamental nucleosomal fibre to large-scale chromatin structures. We have recently developed a method for analysing patterns of supercoiling and have shown that the human genome is organised into large supercoiling domains that are regulated by transcription and topoisomerase activity. To extend this work we have now investigated DNA supercoiling around transcription start sites in human cells at high resolution and reveal that CpG island and non-CpG island genes have different patterns of DNA structure. Our data indicates that one role of CpG islands is to stabilise DNA structure and act as a buffer to resist the topological alterations caused by excess DNA supercoiling which could otherwise impact on transcription factor binding. Previously we argued that DNA supercoiling and transcription could influence large scale chromatin structure however the molecular mechanisms linking these processes remain poorly understood. SAF -A (scaffold attachment factor A) was identified as a nuclear scaffold component and encodes both DNA binding and RNA binding domains. By immunofluorescence we observed that SAF -A is associated with many euchromatic loci but excluded from highly condensed heterochromatin regions, suggesting that it might be important for regulating global chromatin structures. To better understand the role of SAF -A, we analysed chromatin compaction using DNA -FISH and RNAi. Depletion of SAF -A caused significant compaction of both the X chromosome and chromosome 11 territories. Further higher resolution analysis showed that gene-rich regions enriched in “open” chromatin were compacted, but gene-poor regions were unaffected. Consistently, SAF -A binds to large domains around gene-rich regions but appears excluded from gene poor areas. Previously we showed that gene-rich regions were decompacted by transcription; we now show that SAF -A depletion also promotes the compaction of gene-rich regions indicating they may operate in the same pathway. We therefore propose that SAF -A is recruited to gene-rich regions by RNA and facilitates transcriptional regulation by maintaining an “open” chromatin structure, possibly by modulating large scale chromatin topology.