Philippe Collas’s research while affiliated with University of Oslo and other places

Epigenetic regulation is a key determinant of adipocyte fate and function, conferring phenotypic plasticity to adipose tissue in response to metabolic and thermal challenges. To understand the spatiotemporal regulation of chromatin during the establishment of a beige thermogenic adipocyte phenotype, we analyzed the transcriptomic, epigenetic, and enhancer connectome dynamics during white and beige adipogenesis. Using a machine learning approach, we find that the white-specific transcriptional program is associated with promoter modulations of H3K2ac levels and chromatin accessibility. In contrast, beige-specific mitochondrial gene expression correlates with promoter changes in H3K4me3 levels. Adipocyte beiging is also mediated by a remodeling of the 3D genome involving the recruitment of short range enhancers targeting fatty acid oxidation and thermogenic genes. These increased promoter-enhancer contacts correlate with increased chromatin opening at sites enriched for C/EBP transcription factor motifs. We notably identify the C/EBP transcription factor NFIL3 as differentially bound between white and beige adipocytes at enhancers regulating PDK4, a key metabolic switch promoting fatty acid oxidation. Our results highlight a multimodal, pathway-specific regulation of the transcriptional program underlying the beige adipocyte phenotype.

Background: Obesity and related metabolic disorders have reached epidemic levels, calling for diverse therapeutic strategies. Altering nutrient intake, timing and quantity by intermittent fasting seems to elicit beneficial health effects by modulating endocrine and cell signaling networks. This study explores the impact of cyclic nutrient availability in the form of every-other-day fasting (EODF) on human adipose stem cells (ASCs). Methods: We subjected ASCs to repeated fasting/refeeding (F/R) cycles, mimicking low glucose/high fatty acid (LGHF) conditions, and assessed phenotypic and transcriptomic changes, lipid storage capacity, insulin sensitivity, and differentiation potential. Results: Four consecutive F/R cycles induced significant changes in adipogenic gene expression, with upregulation of FABP4 and PLIN1 during fasting, and increased lipid storage in the ASCs. Upon differentiation, ASCs exposed to LGHF conditions retained a transient increase in lipid droplet size and altered fatty acid metabolism gene expression until day 9. However, these changes dissipated by day 15 of differentiation, suggesting a limited duration of fasting-induced transcriptional and adipogenic memory. Despite initial effects, ASCs showed resilience, returning to a physiological trajectory during differentiation, with respect to gene expression and lipid metabolism. Conclusions: These findings suggest that the long-term effects of EODF on the ASC niche may be transient, emphasizing the ability of the adipose tissue to adapt and restore homeostasis.

Background The beneficial effect of thermogenic adipocytes in maintaining body weight and protecting against metabolic disorders has raised interest in understanding the regulatory mechanisms defining white and beige adipocyte identity. Although alternative splicing has been shown to propagate adipose browning signals in mice, this has yet to be thoroughly investigated in human adipocytes. Methods We performed parallel white and beige adipogenic differentiation using primary adipose stem cells from 6 unrelated healthy subjects and assessed differential gene and isoform expression in mature adipocytes by RNA sequencing. Results We find 777 exon junctions with robust differential usage between white and beige adipocytes in all 6 subjects, mapping to 562 genes. Importantly, only 10% of these differentially spliced genes are also differentially expressed, indicating that alternative splicing constitutes an additional layer of gene expression regulation during beige adipocyte differentiation. Functional classification of alternative isoforms points to a gain of function for key thermogenic transcription factors such as PPARG and CITED1, and enzymes such as PEMT, or LPIN1. We find that a large majority of the splice variants arise from differential TSS usage, with beige-specific TSSs being enriched for PPARγ and MED1 binding compared to white-specific TSSs. Finally, we validate beige specific isoform expression at the protein level for two thermogenic regulators, PPARγ and PEMT. Discussion These results suggest that differential isoform expression through alternative TSS usage is an important regulatory mechanism for human adipocyte thermogenic specification.

Differentiation of adipose progenitor cells into mature adipocytes entails a dramatic reorganization of the cellular architecture to accommodate lipid storage into cytoplasmic lipid droplets. Lipid droplets occupy most of the adipocyte volume, compressing the nucleus beneath the plasma membrane. How this cellular remodeling affects sub-nuclear structure, including size and number of nucleoli, remains unclear. We describe the morphological remodeling of the nucleus and the nucleolus during in vitro adipogenic differentiation of primary human adipose stem cells. We find that cell cycle arrest elicits a remodeling of nucleolar structure which correlates with a decrease in protein synthesis. Strikingly, triggering cytoskeletal rearrangements mimics the nucleolar remodeling observed during adipogenesis. Our results point to nucleolar remodeling as an active, mechano-regulated mechanism during adipogenic differentiation and demonstrate a key role of the actin cytoskeleton in defining nuclear and nucleolar architecture in differentiating human adipose stem cells.

Background Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. Results We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2—which are common events in adult gliomas and myeloid leukemias—also disrupt the formation of PML bodies. Conclusions We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.

Breast cancer entails intricate alterations in genome organization and expression. However, how three-dimensional (3D) chromatin structure changes in the progression from a normal to a breast cancer malignant state remains unknown. To address this, we conducted an analysis combining Hi-C data with lamina-associated domains (LADs), epigenomic marks, and gene expression in an in vitro model of breast cancer progression. Our results reveal that while the fundamental properties of topologically associating domains (TADs) remain largely stable, significant changes occur in the organization of compartments and subcompartments. These changes are closely correlated with alterations in the expression of oncogenic genes. We also observe a restructuring of TAD-TAD interactions, coinciding with a loss of spatial compartmentalization and radial positioning of the 3D genome. Notably, we identify a previously unrecognized interchromosomal insertion event, wherein a locus on chromosome 8 housing the MYC oncogene is inserted into a highly active subcompartment on chromosome 10. This insertion leads to the formation of de novo enhancer contacts and activation of the oncogene, illustrating how structural variants can interact with the 3D genome to drive oncogenic states. In summary, our findings provide evidence for the degradation of genome organization at multiple scales during breast cancer progression revealing novel relationships between genome 3D structure and oncogenic processes.

The promyelocytic leukemia (PML) protein organizes nuclear aggregates known as PML nuclear bodies (PML-NBs) , where many transcription factors localize to be regulated. In addition, associations of PML and PML-NBs with chromatin are described in various cell types, further implicating PML in transcriptional regulation. However, a complete understanding of the functional consequences of PML association to DNA in cellular contexts where it promotes relevant phenotypes is still lacking. We examined PML chromatin association in triple-negative breast cancer (TNBC) cell lines, where it exerts important oncogenic functions. We find that PML associates discontinuously with large heterochromatic PML-associated domains (PADs) that contain discrete gene rich euchromatic sub-domains locally depleted of PML. PML promotes heterochromatic organization in PADs and expression of pro-met ast atic genes embedded in these sub-domains. Importantly, this occurs outside PML-NBs, suggesting that nucleoplasmic PML exerts a relevant gene regulatory function. We also find that PML plays indirect regulatory roles in TNBC cells by promoting the expression of pro-metastatic genes outside PADs. Our findings suggest that PML is an important transcriptional regulator of pro-oncogenic metagenes in TNBC cells, via transcriptional regulation and epigenetic organization of heterochromatin domains that embed regions of local transcriptional activity.

The ability to recapitulate muscle differentiation in vitro enables the exploration of mechanisms underlying myogenesis and muscle diseases. However, obtaining myoblasts from patients with neuromuscular diseases or from healthy subjects poses ethical and procedural challenges that limit such investigations. An alternative consists in converting skin fibroblasts into myogenic cells by forcing the expression of the myogenic regulator MYOD. Here, we directly compared cellular phenotype, transcriptome, and nuclear lamina-associated domains (LADs) in myo-converted human fibroblasts and myotubes differentiated from myoblasts. We used isogenic cells from a 16-year-old donor, ruling out, for the first time to our knowledge, genetic factors as a source of variations between the two myogenic models. We show that myo-conversion of fibroblasts upregulates genes controlling myogenic pathways leading to multinucleated cells expressing muscle cell markers. However, myotubes are more advanced in myogenesis than myo-converted fibroblasts at the phenotypic and transcriptomic levels. While most LADs are shared between the two cell types, each also displays unique domains of lamin A/C interactions. Furthermore, myotube-specific LADs are more gene-rich and less heterochromatic than shared LADs or LADs unique to myo-converted fibroblasts, and they uniquely sequester developmental genes. Thus, myo-converted fibroblasts and myotubes retain cell type-specific features of radial and functional genome organization. Our results favor a view of myo-converted fibroblasts as a practical model to investigate the phenotypic and genomic properties of muscle cell differentiation in normal and pathological contexts, but also highlight current limitations in using fibroblasts as a source of myogenic cells.

Differentiation of adipose progenitor cells into mature adipocytes entails a dramatic reorganization of the cellular architecture to accommodate lipid storage into cytoplasmic lipid droplets. Lipid droplets occupy most of the adipocyte volume, compressing the nucleus beneath the plasma membrane. How this cellular remodeling affects sub-nuclear structure, including size and number of nucleoli, remains unclear. We describe the morphological remodeling of the nucleus and the nucleolus during in vitro adipogenic differentiation of primary human adipose stem cells. We find that cell cycle arrest elicits a remodeling of nucleolar structure which correlates with a decrease in protein synthesis. Strikingly, triggering cytoskeletal rearrangements mimics the nucleolar remodeling observed during adipogenesis. Our results point to nucleolar remodeling as an active, mechano-regulated mechanism during adipogenic differentiation and demonstrate a key role of the actin cytoskeleton in defining nuclear and nucleolar architecture in differentiating human adipose stem cells.