Martina I. Lefterova

New developments in adipogenesis

The obesity epidemic has focused attention on adipose tissue and the development of fat cells (i.e. adipocytes), which is known as adipogenesis. Peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding proteins have emerged as master regulators of adipogenesis, and recent genome-wide studies have indicated widespread overlap in their transcriptional targets. In addition, new evidence has implicated many other factors as positive and negative regulators of adipocyte development. This review highlights recent advances in the field of adipogenesis, including newly identified determinants of brown adipocytes, the function of which is to burn rather than store energy. Improved understanding of brown and white adipocyte origins and the integrative biology of adipogenesis might lead to more effective strategies for the treatment of obesity and metabolic disease.

DOT1L/KMT4 Recruitment and H3K79 Methylation Are Ubiquitously Coupled with Gene Transcription in Mammalian Cells

ABSTRACT The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recruitment is ubiquitously coupled with active transcription in diverse mammalian cell types. DOT1L preferentially occupies the proximal transcribed region of active genes, correlating with enrichment of H3K79 di- and trimethylation. Furthermore, Dot1l mutant fibroblasts lacked H3K79 di- and trimethylation at all sites examined, indicating that DOT1L is the sole enzyme responsible for these marks. Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. Conversion of H3K79 monomethylation into di- and trimethylation correlated with the transition from low- to high-level gene transcription. We also observed enrichment of H3K79 monomethylation at intergenic regions occupied by DNA-binding transcriptional activators. Our findings highlight several similarities between the patterning of H3K4 methylation and that of H3K79 methylation in mammalian chromatin, suggesting a widespread mechanism for parallel or sequential recruitment of DOT1L and MLL to genes in their normal “on” state.

Propagation of adipogenic signals through an epigenomic transition state.

The transcriptional mechanisms by which temporary exposure to developmental signals instigates adipocyte differentiation are unknown. During early adipogenesis, we find transient enrichment of the glucocorticoid receptor (GR), CCAAT/enhancer-binding protein beta (CEBPbeta), p300, mediator subunit 1, and histone H3 acetylation near genes involved in cell proliferation, development, and differentiation, including the gene encoding the master regulator of adipocyte differentiation, peroxisome proliferator-activated receptor gamma2 (PPARgamma2). Occupancy and enhancer function are triggered by adipogenic signals, and diminish upon their removal. GR, which is important for adipogenesis but need not be active in the mature adipocyte, functions transiently with other enhancer proteins to propagate a new program of gene expression that includes induction of PPARgamma2, thereby providing a memory of the earlier adipogenic signal. Thus, the conversion of preadipocyte to adipocyte involves the formation of an epigenomic transition state that is not observed in cells at the beginning or end of the differentiation process.

Cell-Specific Determinants of Peroxisome Proliferator-Activated Receptor γ Function in Adipocytes and Macrophages

ABSTRACT The nuclear receptor peroxisome proliferator activator receptor γ (PPARγ) is the target of antidiabetic thiazolidinedione drugs, which improve insulin resistance but have side effects that limit widespread use. PPARγ is required for adipocyte differentiation, but it is also expressed in other cell types, notably macrophages, where it influences atherosclerosis, insulin resistance, and inflammation. A central question is whether PPARγ binding in macrophages occurs at genomic locations the same as or different from those in adipocytes. Here, utilizing chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we demonstrate that PPARγ cistromes in mouse adipocytes and macrophages are predominantly cell type specific. In thioglycolate-elicited macrophages, PPARγ colocalizes with the hematopoietic transcription factor PU.1 in areas of open chromatin and histone acetylation, near a distinct set of immune genes in addition to a number of metabolic genes shared with adipocytes. In adipocytes, the macrophage-unique binding regions are marked with repressive histone modifications, typically associated with local chromatin compaction and gene silencing. PPARγ, when introduced into preadipocytes, bound only to regions depleted of repressive histone modifications, where it increased DNA accessibility, enhanced histone acetylation, and induced gene expression. Thus, the cell specificity of PPARγ function is regulated by cell-specific transcription factors, chromatin accessibility, and histone marks. Our data support the existence of an epigenomic hierarchy in which PPARγ binding to cell-specific sites not marked by repressive marks opens chromatin and leads to local activation marks, including histone acetylation.

PPARγ and the global map of adipogenesis and beyond

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor (NR) superfamily of ligand-dependent transcription factors (TFs) and function as a master regulator of adipocyte differentiation and metabolism. We review recent breakthroughs in the understanding of PPARγ gene regulation and function in the chromatin context. It is now clear that multiple TFs team up to induce PPARγ during adipogenesis, and that other TFs cooperate with PPARγ to ensure adipocyte-specific genomic binding and function. We discuss how this differs in other PPARγ-expressing cells such as macrophages and how these genome-wide mechanisms are preserved across species despite modest conservation of specific binding sites. These emerging considerations inform our understanding of PPARγ function as well as of adipocyte development and physiology.

Autoimmune Kidney Disease and Impaired Engulfment of Apoptotic Cells in Mice with Macrophage Peroxisome Proliferator-Activated Receptor γ or Retinoid X Receptor α Deficiency

Autoimmune glomerulonephritis is a common manifestation of systemic lupus erythematosus (SLE). In this study, we show that mice lacking macrophage expression of the heterodimeric nuclear receptors PPARγ or RXRα develop glomerulonephritis and autoantibodies to nuclear Ags, resembling the nephritis seen in SLE. These mice show deficiencies in phagocytosis and clearance of apoptotic cells, and they are unable to acquire an anti-inflammatory phenotype upon feeding of apoptotic cells, which is critical for the maintenance of self-tolerance. These results demonstrate that stimulation of PPARγ and RXRα in macrophages facilitates apoptotic cell engulfment, and they provide a potential strategy to avoid autoimmunity against dying cells and to attenuate SLE.

Absence of bacterially induced RELMβ reduces injury in the dextran sodium sulfate model of colitis

Although inflammatory bowel disease (IBD) is the result of a dysregulated immune response to commensal gut bacteria in genetically predisposed individuals, the mechanism(s) by which bacteria lead to the development of IBD are unknown. Interestingly, deletion of intestinal goblet cells protects against intestinal injury, suggesting that this epithelial cell lineage may produce molecules that exacerbate IBD. We previously reported that resistin-like molecule beta (RELMbeta; also known as FIZZ2) is an intestinal goblet cell-specific protein that is induced upon bacterial colonization whereupon it is expressed in the ileum and colon, regions of the gut most often involved in IBD. Herein, we show that disruption of this gene reduces the severity of colitis in the dextran sodium sulfate (DSS) model of murine colonic injury. Although RELMbeta does not alter colonic epithelial proliferation or barrier function, we show that recombinant protein activates macrophages to produce TNF-alpha both in vitro and in vivo. RELMbeta expression is also strongly induced in the terminal ileum of the SAMP1/Fc model of IBD. These results suggest a model whereby the loss of epithelial barrier function by DSS results in the activation of the innate mucosal response by RELMbeta located in the lumen, supporting the hypothesis that this protein is a link among goblet cells, commensal bacteria, and the pathogenesis of IBD.

Adipocyte-specific Expression of Murine Resistin Is Mediated by Synergism between Peroxisome Proliferator-activated Receptor γ and CCAAT/Enhancer-binding Proteins

Resistin antagonizes insulin action in mouse, making it a potential therapeutic target for treating metabolic diseases such as diabetes. To better understand how mouse resistin gene (Retn) expression is restricted to fat tissue, we identified an adipocyte-specific enhancer located ∼8.8-kb upstream of the transcription start site. This region contains a binding site for the master adipogenic regulator peroxisome proliferator-activated receptor γ (PPARγ), and binds endogenous PPARγ together with its partner retinoid-X receptor α (RXRα). It also contains three binding sites for CCAAT/enhancer-binding protein (C/EBP), and is bound by endogenous C/EBPα and C/EBPβ in adipocytes. Exogenous expression of PPARγ/RXRα and C/EBPα in non-adipocyte cells synergistically drives robust expression from the enhancer. Although PPARγ ligands repress Retn transcription in adipocytes, rosiglitazone paradoxically stimulates the enhancer activity, suggesting that the enhancer is not directly involved in negative regulation. Unlike expression of Retn in mouse, human resistin (RETN) is expressed primarily in macrophages. Interestingly, the region homologous to the mouse Retn enhancer in the human gene contains all three C/EBP elements, but is not conserved for the sequence bound by PPARγ. Furthermore, it displays little or no binding by PPARγ in vitro. Taken together, the data suggest that a composite enhancer binding both PPARγ and C/EBP factors confers adipocyte-specific expression to Retn in mouse, and its absence from the human gene may explain the lack of adipocyte expression in humans.

Retinol saturase promotes adipogenesis and is downregulated in obesity

Adipocyte differentiation is controlled by many transcription factors, but few known downstream targets of these factors are necessary for adipogenesis. Here we report that retinol saturase (RetSat), which is an enzyme implicated in the generation of dihydroretinoid metabolites, is induced during adipogenesis and is directly regulated by the transcription factor peroxisome proliferator activated receptor γ (PPARγ). Ablation of RetSat dramatically inhibited adipogenesis but, surprisingly, this block was not overcome by the putative product of RetSat enzymatic activity. On the other hand, ectopic RetSat with an intact, but not a mutated, FAD/NAD dinucleotide-binding motif increased endogenous PPARγ transcriptional activity and promoted adipogenesis. Indeed, RetSat was not required for adipogenesis when cells were provided with exogenous PPARγ ligands. In adipose tissue, RetSat is expressed in adipocytes but is unexpectedly downregulated in obesity, most likely owing to infiltration of macrophages that we demonstrate to repress RetSat expression. Thiazolidinedione treatment reversed low RetSat expression in adipose tissue of obese mice. Thus, RetSat plays an important role in the biology of adipocytes, where it favors normal differentiation, yet is reduced in the obese state. RetSat is thus a novel target for therapeutic intervention in metabolic disease.

Re-expression of GATA2 Cooperates with Peroxisome Proliferator-activated Receptor-γ Depletion to Revert the Adipocyte Phenotype

Nuclear peroxisome proliferator-activated receptor-γ (PPARγ) is required for adipocyte differentiation, but its role in mature adipocytes is less clear. Here, we report that knockdown of PPARγ expression in 3T3-L1 adipocytes returned the expression of most adipocyte genes to preadipocyte levels. Consistently, down-regulated but not up-regulated genes showed strong enrichment of PPARγ binding. Surprisingly, not all adipocyte genes were reversed, and the adipocyte morphology was maintained for an extended period after PPARγ depletion. To explain this, we focused on transcriptional regulators whose adipogenic regulation was not reversed upon PPARγ depletion. We identified GATA2, a transcription factor whose down-regulation early in adipogenesis is required for preadipocyte differentiation and whose levels remain low after PPARγ knockdown. Forced expression of GATA2 in mature adipocytes complemented PPARγ depletion and impaired adipocyte functionality with a more preadipocyte-like gene expression profile. Ectopic expression of GATA2 in adipose tissue in vivo had a similar effect on adipogenic gene expression. These results suggest that PPARγ-independent down-regulation of GATA2 prevents reversion of mature adipocytes after PPARγ depletion.