A dynamic CTCF chromatin binding landscape promotes DNA hydroxymethylation and transcriptional induction of adipocyte differentiation

Julie Dubois-Chevalier 0 1 2 3 Fr ed erik Oger 0 1 2 3 H el e`ne Dehondt 0 1 2 3 Fran cois F. Firmin 0 1 2 3 C eline Gheeraert 0 1 2 3 Bart Staels 0 1 2 3 Philippe Lefebvre 0 1 2 3 J ero me Eeckhoute 0 1 2 3 0 Institut Pasteur de Lille , F-59019 Lille, France 1 Universit e Lille 2 , F-59000 Lille, France 2 Inserm UMR U1011, F-59000 Lille, France 3 European Genomic Institute for Diabetes (EGID) , FR 3508, F-59000 Lille, France - CCCTC-binding factor (CTCF) is a ubiquitously expressed multifunctional transcription factor characterized by chromatin binding patterns often described as largely invariant. In this context, how CTCF chromatin recruitment and functionalities are used to promote cell type-specific gene expression remains poorly defined. Here, we show that, in addition to constitutively bound CTCF binding sites (CTS), the CTCF cistrome comprises a large proportion of sites showing highly dynamic binding patterns during the course of adipogenesis. Interestingly, dynamic CTCF chromatin binding is positively linked with changes in expression of genes involved in biological functions defining the different stages of adipogenesis. Importantly, a subset of these dynamic CTS are gained at cell type-specific regulatory regions, in line with a requirement for CTCF in transcriptional induction of adipocyte differentiation. This relates to, at least in part, CTCF requirement for transcriptional activation of both the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) and its target genes. Functionally, we show that CTCF interacts with TET methylcytosine dioxygenase (TET) enzymes and promotes adipogenic transcriptional enhancer DNA hydroxymethylation. Our study reveals a dynamic CTCF chromatin binding landscape required for epigenomic remodeling of enhancers and transcriptional activation driving cell differentiation. INTRODUCTION Eukaryotic cell differentiation is a multi-step process that ultimately leads to the establishment of cell type-specific transcriptomes from a shared genetic template. This involves a mutual influence between transcription factor (TF)/cofactor genomic binding and chromatin remodeling events to specify the transcriptional regulatory outputs of promoters/enhancers (1,2). During adipogenesis, preadipocytes convert into mature adipocytes, a differentiation process extensively studied in vitro using 3T3-L1 fibroblasts as a model (3,4). This process involves activation of cell type-specific TFs including notably the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG), which is instrumental to the acquisition and maintenance of mature adipocyte functions such as lipid handling and storage (35). The transcriptional regulatory activities of PPARG require cooperating factors including its heterodimerization partner Retinoid X receptor (RXR), members of the CCAAT/enhancer binding protein (CEBP) family as well as transcriptional coactivators such as Mediator complex subunit 1 (MED1) and CREB binding protein (CBP) (69). PPARG and its collaborating factors bind to transcriptional regulatory regions, including both promoters and enhancers, whose functionalization is linked to chromatin remodeling during adipocyte differentiation (10,11). These remodeling events include nucleosome destabilization/eviction and changes in histone posttranslational modifications (1113). For instance, acetylation of histone H3 lysine 27 (H3K27ac) and methylation of H3K4 (H3K4me) is co-ordinately induced with PPARG recruitment (12). Additionally, methylated cytosines in DNA (5mC) are subjected to oxidation to give rise to hydroxymethylated cytosines (5hmC) through the action of TET methylcytosine dioxygenases (TET) (14,15). In contrast, the role of ubiquitous TF in establishing cell type-specific transcriptional programs often remains more elusive. CCCTC-binding factor (CTCF) is a ubiquitously expressed TF characterized by multiple functions (16). Indeed, CTCF is well known for its role at insulators, which restrict enhancer-mediated transcriptional inductions. It can also serve as a chromatin barrier delimitating active and repressive domains. Finally, CTCF can act as a transcriptional activator/repressor at gene promoters or enhancers (16). These context-dependent activities often rely on interactions with different collaborating proteins including TFs, transcriptional cofactors, RNA polymerase II and the cohesin complex (17,18). Interaction with the cohesin complex is thought to confer global chromatin organization properties to CTCF through chromatin looping. However, the exact role exerted by CTCF in the threedimensional folding of chromatin is still elusive. Indeed, loss-of-function studies yielded conflicting results regarding the role of CTCF in local chromatin interactions and higher order topological domain structures (1921). Hence, while CTCF is required for embryonic development and neuronal and hematopoietic cell differentiation (22), the mechanisms involved remain only partially understood. The CTCF chromatin binding landscape (defined as its cistrome) has been described as invariant across different tissues/cell types (2325) and well conserved across divergent species (26,27). This led to propose a conserved role for CTCF across tissues (28). However, recent studies challenged this view and revealed that tissue-specific CTCF binding occurs, correlates with tissue-specific DNA methylation patterns (29) and is characterized by lower occupancy and degenerated CTCF recognition motifs when compared to ubiquitous binding sites (30). Altogether, these data suggest that the CTCF cistrome is more versatile than initially thought even though the extent and functional importance of cell type-specific CTCF chromatin binding is poorly understood. Here, we used the 3T3-L1 adipogenesis model to thoroughly study CTCF cistrome plasticity and dynamics during the course of cell differentiation. We report that the CTCF cistrome is highly dynamic during adipogenesis, with lost and gained CTCF binding sites (CTS) that are linked to dynamic gene regulation associated with the different stages of the differentiation process. Moreover, we show that gained cell type-specific CTCF binding to transcriptional regulatory regions allows for PPARG-mediated transcriptional induction of adipocyte differentiation. Finally, we establish that these activities involve a functional link between CTCF and enhancer DNA hydroxymethylation through an interaction with TET. MATERIALS AND METHODS Cell culture and transfection 3T3-L1 pre-adipocyte cells were grown and differentiated according to the MDI standard protocol as described in (7). Briefly, 2 days after reaching confluence, adipocyte differentiation of 3T3-L1 cells was induced by treating cells for 2 days with 0.5 mM IBMX, 1 M dexamethasone and 10 g/ml insulin. Alternatively, 2 M rosiglitazone in the presence of 1 M dexamethasone was used in experiments performed to validate CTCF role in PPARG-mediated ind (...truncated)