Epigenetic regulation is a key determinant of adipocyte fate, driving the differentiation toward white or thermogenic beige phenotypes in response to environmental cues. To dissect the mechanisms orchestrating this plasticity in human adipocytes, we conducted an integrative analysis of transcriptomic, epigenomic and enhancer connectome dynamics throughout white and beige adipogenesis. Using a machine learning approach, we show that the white transcriptional program is tightly linked to promoter-level modulation of H3K27ac and chromatin accessibility, whereas the beige-specific induction of mitochondrial genes is driven by promoter remodeling of H3K4me3, underscoring distinct epigenetic mechanisms for white or beige specification. Adipocyte beiging is accompanied by a targeted reorganization of the 3D genome, characterized by increased recruitment of short-range enhancers controlling thermogenesis genes, enriched for C/EBP transcription factor binding sites. Our findings highlight the multimodal regulation of the beige adipocyte fate, driven by the interplay of chromatin state transitions, enhancer rewiring, and transcription factor dynamics.

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 have 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 although the fundamental properties of topologically associating domains (TADs) are overall maintained, 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 is accompanied by the formation of de novo enhancer contacts and activation of MYC, illustrating how structural genomic variants can alter the 3D genome during oncogenesis. In summary, our findings provide evidence for the loss of genome organization at multiple scales during breast cancer progression, revealing novel relationships between genome 3D structure and oncogenic processes.

Early developmental cues exert lasting influence on the adipose tissue function and the metabolic health throughout adulthood. In mice, adipose tissue remodelling during the weaning transition programs long-term tissue plasticity, setting its capacity for expansion and thermogenesis in response to environmental stimuli. However, the mechanisms driving this remodelling, and the role of weaning-associated dietary changes in this process, are unknown. Here, we characterise the emergence of sexually dimorphic patterns of adipose tissue distribution and function during the post-weaning period. As subcutaneous adipocytes acquire a white phenotype, the tissue microenvironment undergoes a coordinated remodelling, including reduced innervation and an increase in immune cell content. Using a model of prolonged breastfeeding, we show that it promotes adipose tissue expansion through sex-dependent mechanisms, stimulating progenitor proliferation in males and adipocyte hypertrophy in females. In males, this is accompanied by accelerated tissue whitening, which we link to enhanced clearance of extracellular noradrenaline by mature adipocytes. Our findings reveal sex- and diet-dependent mechanisms governing adipose tissue maturation and highlight how early life nutritional cues shape the microenvironment and function of mature adipocytes. - The peri-weaning period associates with sexually dimorphic remodelling of the adipose tissue - Whitening of the adipose tissue associates with immune infiltration and decrease innervation in both sexes - Prolonged breastfeeding prevents drop in proliferation in males - Whitening is accelerated in males during prolonged breastfeeding

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

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. Characterization of nuclear and nucleolar dynamics during adipogenic differentiation of human adipose stem cells points to nucleolar remodeling as an active, mechanoregulated mechanism triggered by cytoskeleton rearrangement.

BackgroundThe 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.MethodsWe 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.ResultsWe 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.DiscussionThese results suggest that differential isoform expression through alternative TSS usage is an important regulatory mechanism for human adipocyte thermogenic specification.

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.

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.

Abstract 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.

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-metastatic 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 nuclear lamina provides a repressive chromatin environment at the nuclear periphery. However, whereas most genes in lamina-associated domains (LADs) are inactive, over ten percent reside in local euchromatic contexts and are expressed. How these genes are regulated and whether they are able to interact with regulatory elements remain unclear. Here, we integrate publicly available enhancer-capture Hi-C data with our own chromatin state and transcriptomic datasets to show that inferred enhancers of active genes in LADs are able to form connections with other enhancers within LADs and outside LADs. Fluorescence in situ hybridization analyses show proximity changes between differentially expressed genes in LADs and distant enhancers upon the induction of adipogenic differentiation. We also provide evidence of involvement of lamin A/C, but not lamin B1, in repressing genes at the border of an in-LAD active region within a topological domain. Our data favor a model where the spatial topology of chromatin at the nuclear lamina is compatible with gene expression in this dynamic nuclear compartment.

Lamins are the main constituents of the nuclear lamina, a protein meshwork underlining the inner face of the nuclear envelope (NE) and facing chromatin and nucleoplasm. A-type lamins (lamin A and C), encoded by the LMNA gene, have roles in the chromatin organization, through domains called Lamin-Associated Domains (LAD). Proper maintenance of chromatin organization is essential for normal cell function, and depends on nuclear envelope stability and tissue-specific nuclear envelope proteins. LMNA gene mutations are responsible for a wide spectrum of disorders called laminopathies, most of them affecting striated muscles. LMNA mutations have been associated with defects in LAD organization and gene expression at the nuclear periphery, contributing in the pathophysiological mechanisms of laminopathies. Laminopathies are characterized by a strong clinical variability and genetic or epigenetic factors, such as modifier genes or specific chromatin organization defects could explain such variability. We collected biological materials, through national and international collaborations and we received a majority of fibroblasts from LMNA-mutated patients. Genome organization being highly different between cell types, we myo-converted immortalized skin fibroblasts into myogenic cells, via overexpression of murine MyoD. We performed a study to validate this model as relevant to investigate LAD organization, by combining RNA-seq and ChIP-seq targeting Lamin A/C and histone marks. We showed that myotubes derived from these myo-converted fibroblasts underwent a clear phenotypic switch to myogenic cell type, at a transcriptomic and at a chromatin level, hence validating this cell system to study LAD organization in muscle cells. Lamins are the main constituents of the nuclear lamina, a protein meshwork underlining the inner face of the nuclear envelope (NE) and facing chromatin and nucleoplasm. A-type lamins (lamin A and C), encoded by the LMNA gene, have roles in the chromatin organization, through domains called Lamin-Associated Domains (LAD). Proper maintenance of chromatin organization is essential for normal cell function, and depends on nuclear envelope stability and tissue-specific nuclear envelope proteins. LMNA gene mutations are responsible for a wide spectrum of disorders called laminopathies, most of them affecting striated muscles. LMNA mutations have been associated with defects in LAD organization and gene expression at the nuclear periphery, contributing in the pathophysiological mechanisms of laminopathies. Laminopathies are characterized by a strong clinical variability and genetic or epigenetic factors, such as modifier genes or specific chromatin organization defects could explain such variability. We collected biological materials, through national and international collaborations and we received a majority of fibroblasts from LMNA-mutated patients. Genome organization being highly different between cell types, we myo-converted immortalized skin fibroblasts into myogenic cells, via overexpression of murine MyoD. We performed a study to validate this model as relevant to investigate LAD organization, by combining RNA-seq and ChIP-seq targeting Lamin A/C and histone marks. We showed that myotubes derived from these myo-converted fibroblasts underwent a clear phenotypic switch to myogenic cell type, at a transcriptomic and at a chromatin level, hence validating this cell system to study LAD organization in muscle cells.

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.

Abstract 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.

Different chromatin forms, or states, represent a fundamental means of controlling gene regulation. Chromatin states have been studied through either the distribution of histone modifications (e.g. 1–5 ) or more rarely via the occupancy of chromatin proteins 6–8 . However, these two approaches disagree on the nature and composition of active chromatin states 2,9 and modelling chromatin via both histone marks and chromatin proteins has been lacking. Here, combining protein and histone mark profiles, we show that chromatin in Drosophila melanogaster is organised into eight principle chromatin states that have consistent forms and constituents across cell types. These states form through the association of the Swi/Snf chromatin remodelling complex, Polycomb Group (PcG)/H3K27me3, HP1a/H3K9me3 or H3K36me3 complexes with either active complexes (RNA Pol/COM-PASS/H3K4me3/NuRF) or repressive marks (histone H1 and nuclear lamin occupancy). Enhancers, core promoters, transcription factor motifs, and gene bodies show distinct chromatin state preferences that separate by developmental and housekeeping/metabolic gene ontology. Within the 3D genome, chromatin states add an additional level of compartmentalisation through self-association of topologically associated domains (TADs) of the same state. Our results suggest that the epigenetic landscape is organised by the binding of chromatin remodellers and repressive complexes, and that through chromatin states the genome is fundamentally segregated into developmental and housekeeping/metabolic roles.

The three-dimensional (3D) organization of the genome is shaped by interactions with multiple structures within the nucleus, affecting gene expression outcomes. Technological breakthroughs in recent years have generated vast data reflecting various aspects of nuclear genome architecture in space and time. Integrating these datasets into comprehensive 3D genome models can reveal new insights into genome structure and regulation in normal and disease states. In this chapter, we provide a step-by-step guide on how to generate publication-ready integrated 3D genome models from (raw) Hi-C data and from lamin-genome (LAD) contacts.

BACKGROUND:LncRNAs are tissue-specific and emerge as important regulators of various biological processes and as disease biomarkers. HOTAIR is a well-established pro-oncogenic lncRNA which has been attributed a variety of functions in cancer and native contexts. However, a lack of an exhaustive, cell type-specific annotation questions whether HOTAIR functions are supported by the expression of multiple isoforms.RESULTS:Using a capture long-read sequencing approach, we characterize HOTAIR isoforms expressed in human primary adipose stem cells. We find HOTAIR isoforms population displays varied splicing patterns, frequently leading to the exclusion or truncation of canonical LSD1 and PRC2 binding domains. We identify a highly cell type-specific HOTAIR isoform pool regulated by distinct promoter usage, and uncover a shift in the HOTAIR TSS usage that modulates the balance of HOTAIR isoforms at differentiation onset.CONCLUSION:Our results highlight the complexity and cell type-specificity of HOTAIR isoforms and open perspectives on functional implications of these variants and their balance to key cellular processes.

The long non-coding RNA HOTAIR is the most differentially expressed gene between upper- and lower-body adipose tissue, yet its functional significance in adipogenesis is unclear. We report that HOTAIR expression is transiently induced during early adipogenic differentiation of gluteofemoral adipose progenitors and repressed in mature adipocytes. Upon adipogenic commitment, HOTAIR regulates protein synthesis pathways and cytoskeleton remodeling with a later impact on mature adipocyte lipid storage capacity. Our results support novel and important functions of HOTAIR in the physiological context of adipogenesis.

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.