XPF interacts with TOP2B for R-loop processing and DNA looping on actively transcribed genes

Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage

Contactomics : Exploring New Dimensions of 3D Genome Architecture

3D genome folding is increasingly recognized as an instrumental regulator of gene expression. This thesis contains two literature reviews in which the current knowledge regarding genome folding and function is described; chapter one focusses on the implications for gene regulation at each hierarchical level of genome folding and addresses the factors that are believed to fold the genome at each level. In chapter two, the unique 3D folding of the pluripotent genome is discussed. The three experimental chapters of this thesis describe the results of three studies, aimed to gain a better understanding of the relationship between genome form and function. Chapter three describes specific features of the 3D genome of pluripotent stem cells, and how these are lost and gained during differentiation and reprogramming. Chapter four focusses on the development of a novel method to study genome conformation: multi-contact chromosome conformation capture sequencing, which allows studying of multi-way chromatin conformation and chromatin hubs. The final chapter describes a collaborative study in which the chromatin dynamics of enhancer-promoter contacts are followed during in vitro cardiomyocyte differentiation. Together, the chapters of this thesis illustrate the importance of studying gene expression in the context of the 3D nucleus