Joanna R. DiSpirito

Lipoatrophy and severe metabolic disturbance in mice with fat-specific deletion of PPARγ.

Significance Obesity, due to increased adipose (fat) tissue, predisposes to metabolic diseases, including diabetes. Thus, it is important to understand adipose development and function. Peroxisome proliferator-activated receptor gamma (PPARγ) is widely believed to be the master regulator of adipocyte biology. Surprisingly, however, previous studies attempting to delete the PPARγ gene specifically in mouse adipose tissue did not demonstrate a dramatic phenotype. By using newer methods, the present study reports that fat-specific loss of PPARγ causes dramatic loss of adipose tissue, severe insulin resistance and diabetes, fatty liver, and abnormalities of bone, skin, and mammary glands, all of which contain adipose tissue. We show that adipocyte PPARγ is required for normal fat development and metabolic function in vivo. Adipose tissue is an important metabolic organ, the dysfunction of which is associated with the development of obesity, diabetes mellitus, and cardiovascular disease. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is considered the master regulator of adipocyte differentiation and function. Although its cell-autonomous role in adipogenesis has been clearly demonstrated in cell culture, previous fat-specific knockouts of the murine PPARγ gene did not demonstrate a dramatic phenotype in vivo. Here, using Adipoq–Cre mice to drive adipose-specific recombination, we report a unique fat-specific PPARγ knockout (PPARγ FKO) mouse model with almost no visible brown and white adipose tissue at age 3 mo. As a consequence, PPARγ FKO mice had hugely enlarged pancreatic islets, massive fatty livers, and dramatically elevated levels of blood glucose and serum insulin accompanied by extreme insulin resistance. PPARγ FKO mice also exhibited delayed hair coat formation associated with absence of dermal fat, disrupted mammary gland development with loss of mammary fat pads, and high bone mass with loss of bone marrow fat, indicating the critical roles of adipose PPARγ in these tissues. Together, our data reveal the necessity of fat PPARγ in adipose formation, whole-body metabolic homeostasis, and normal development of fat-containing tissues.

Genetic Variation Determines PPARγ Function and Anti-diabetic Drug Response In Vivo.

SNPs affecting disease risk often reside in non-coding genomic regions. Here, we show that SNPs are highly enriched at mouse strain-selective adipose tissue binding sites for PPARγ, a nuclear receptor for anti-diabetic drugs. Many such SNPs alter binding motifs for PPARγ or cooperating factors and functionally regulate nearby genes whose expression is strain selective and imbalanced in heterozygous F1 mice. Moreover, genetically determined binding of PPARγ accounts for mouse strain-specific transcriptional effects of TZD drugs, providing proof of concept for personalized medicine related to nuclear receptor genomic occupancy. In human fat, motif-altering SNPs cause differential PPARγ binding, provide a molecular mechanism for some expression quantitative trait loci, and are risk factors for dysmetabolic traits in genome-wide association studies. One PPARγ motif-altering SNP is associated with HDL levels and other metabolic syndrome parameters. Thus, natural genetic variation in PPARγ genomic occupancy determines individual disease risk and drug response.

The orphan nuclear receptors at their 25-year reunion

The nuclear receptor superfamily includes many receptors, identified based on their similarity to steroid hormone receptors but without a known ligand. The study of how these receptors are diversely regulated to interact with genomic regions to control a plethora of biological processes has provided critical insight into development, physiology, and the molecular pathology of disease. Here we provide a compendium of these so-called orphan receptors and focus on what has been learned about their modes of action, physiological functions, and therapeutic promise.

Epigenetic manipulation restores functions of defective CD8⁺ T cells from chronic viral infection.

Functional exhaustion of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T cell dysfunction are not well understood. Epigenetics plays an important role in the control of T cell development, differentiation, and function. To examine if epigenetics also plays a role in T cell exhaustion, we analyzed chromatin remodeling in CD8+ T cells from mice with chronic lymphocytic choriomeningitis virus infection. We observed downregulation of diacetylated histone H3 in both virus-specific and total CD8+ T cells, and functional defects not only in virus-specific CD8+ T cells but also within the total CD8+ T cell population. In vitro treatment of these exhausted CD8+ T cells with histone deacetylase inhibitors restored diacetylated histone H3 levels, and improved their immune functions. Upon adoptive transfer, these treated CD8+ T cells developed into functional memory T cells in vivo that enhanced protective immunity. These results define a role of epigenetics in T cell exhaustion and suggest epigenetic manipulation as a novel molecular therapy to restore immune functions.Functional exhaustion of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T cell dysfunction are not well understood. Epigenetics plays an important role in the control of T cell development, differentiation, and function. To examine if epigenetics also plays a role in T cell exhaustion, we analyzed chromatin remodeling in CD8(+) T cells from mice with chronic lymphocytic choriomeningitis virus infection. We observed downregulation of diacetylated histone H3 in both virus-specific and total CD8(+) T cells, and functional defects not only in virus-specific CD8(+) T cells but also within the total CD8(+) T cell population. In vitro treatment of these exhausted CD8(+) T cells with histone deacetylase inhibitors restored diacetylated histone H3 levels, and improved their immune functions. Upon adoptive transfer, these treated CD8(+) T cells developed into functional memory T cells in vivo that enhanced protective immunity. These results define a role of epigenetics in T cell exhaustion and suggest epigenetic manipulation as a novel molecular therapy to restore immune functions.

Histone Acetylation at the Single-Cell Level: A Marker of Memory CD8+ T Cell Differentiation and Functionality

Following stimulation, memory T (TM) cells rapidly express many effector functions, a hallmark feature that allows them to provide protective immunity. Recent studies suggest that genes involved in this rapid recall response may maintain an open chromatin structure in resting TM cells via epigenetic modifications. However, these studies have mostly focused on a few loci, and the techniques used required a large number of cells. We have developed a flow cytometric assay measuring histone modifications in individual murine T cells in combination with lineage-specific markers. In this study, we show that the per-cell level of a marker of open chromatin, diacetylated histone H3 (diAcH3), increases as naive CD8+ T cells develop into TM cells, demonstrating a novel correlation between the differentiation state of a CD8+ T cell and its abundance of a specific histone modification. Furthermore, our results show that TM cells defective in rapid recall ability have less diAcH3 than their fully functional counterparts, indicating that the diAcH3 level of individual TM cells is a useful marker for assessing their functionality.

Targeting PPARγ in the epigenome rescues genetic metabolic defects in mice

Obesity causes insulin resistance, and PPAR&ggr; ligands such as rosiglitazone are insulin sensitizing, yet the mechanisms remain unclear. In C57BL/6 (B6) mice, obesity induced by a high-fat diet (HFD) has major effects on visceral epididymal adipose tissue (eWAT). Here, we report that HFD-induced obesity in B6 mice also altered the activity of gene regulatory elements and genome-wide occupancy of PPAR&ggr;. Rosiglitazone treatment restored insulin sensitivity in obese B6 mice, yet, surprisingly, had little effect on gene expression in eWAT. However, in subcutaneous inguinal fat (iWAT), rosiglitazone markedly induced molecular signatures of brown fat, including the key thermogenic gene Ucp1. Obesity-resistant 129S1/SvImJ mice (129 mice) displayed iWAT browning, even in the absence of rosiglitazone. The 129 Ucp1 locus had increased PPAR&ggr; binding and gene expression that were preserved in the iWAT of B6x129 F1–intercrossed mice, with an imbalance favoring the 129-derived alleles, demonstrating a cis-acting genetic difference. Thus, B6 mice have genetically defective Ucp1 expression in iWAT. However, when Ucp1 was activated by rosiglitazone, or by iWAT browning in cold-exposed or young mice, expression of the B6 version of Ucp1 was no longer defective relative to the 129 version, indicating epigenomic rescue. These results provide a framework for understanding how environmental influences like drugs can affect the epigenome and potentially rescue genetically determined disease phenotypes.

Pruning of the Adipocyte Peroxisome Proliferator-Activated Receptor γ Cistrome by Hematopoietic Master Regulator PU.1

ABSTRACT “Master” transcription factors are the gatekeepers of lineage identity. As such, they have been a major focus of efforts to manipulate cell fate for therapeutic purposes. The ETS transcription factor PU.1 has a potent ability to confer macrophage phenotypes on cells already committed to a different lineage, but how it overcomes the presence of other master regulators is not known. The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of the adipose lineage, and its genomic binding pattern in adipocytes is well characterized. Here we show that, when expressed at macrophage levels in mature adipocytes, PU.1 bound a large fraction of its macrophage sites, where it induced chromatin opening and the expression of macrophage target genes. Strikingly, PU.1 markedly reduced the genomic binding of PPARγ without changing its abundance. PU.1 expression repressed genes with nearby adipocyte-specific PPARγ binding sites, while a common macrophage-adipocyte gene expression program was retained. Together, these data reveal unexpected lability within the adipocyte PPARγ cistrome and show that, even in terminally differentiated cells, PU.1 can remodel the cistrome of another master regulator.

Transcriptome Signatures Reveal Rapid Induction of Immune-Responsive Genes in Human Memory CD8 + T Cells

Memory T cells (TM) play a prominent role in protection and auto-immunity due to their ability to mount a more effective response than naïve T cells (TN). However, the molecular mechanisms underlying enhanced functionality of TM are not well defined, particularly in human TM. We examined the global gene expression profiles of human CD8+ TN and TM before and after stimulation. There were 1,284, 1,373 and 1,629 differentially expressed genes between TN and TM at 0 hr, 4 hr and 24 hr after stimulation, respectively, with more genes expressed to higher levels in TM. Genes rapidly up-regulated in TN cells were largely involved in nitrogen, nucleoside and amino acid metabolisms. In contrast, those in CD8+ TM were significantly enriched for immune-response-associated processes, including cytokine production, lymphocyte activation and chemotaxis. Multiple cytokines were rapidly up-regulated in TM cells, including effector cytokines known to be produced by CD8+ T cells and important for their functions, as well as regulatory cytokines, both pro- and anti-inflammatory, that are not typically produced by CD8+ T cells. These results provide new insights into molecular mechanisms that contribute to the enhanced functionality of human CD8+ TM and their prominent role in protection and auto-immunity.

PPARγ is a nexus controlling alternative activation of macrophages via glutamine metabolism

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is known to regulate lipid metabolism in many tissues, including macrophages. Here we report that peritoneal macrophage respiration is enhanced by rosiglitazone, an activating PPARγ ligand, in a PPARγ-dependent manner. Moreover, PPARγ is required for macrophage respiration even in the absence of exogenous ligand. Unexpectedly, the absence of PPARγ dramatically affects the oxidation of glutamine. Both glutamine and PPARγ have been implicated in alternative activation (AA) of macrophages, and PPARγ was required for interleukin 4 (IL4)-dependent gene expression and stimulation of macrophage respiration. Indeed, unstimulated macrophages lacking PPARγ contained elevated levels of the inflammation-associated metabolite itaconate and express a proinflammatory transcriptome that, remarkably, phenocopied that of macrophages depleted of glutamine. Thus, PPARγ functions as a checkpoint, guarding against inflammation, and is permissive for AA by facilitating glutamine metabolism. However, PPARγ expression is itself markedly increased by IL4. This suggests that PPARγ functions at the center of a feed-forward loop that is central to AA of macrophages.

Molecular diversification of regulatory T cells in nonlymphoid tissues

Different tissue microenvironments differentially refine Treg cell transcriptional regulatory modules already primed in lymphoid organs. Mapping Treg regulomes Technological advances are allowing immunologists to study rare populations of immune cells that take residence in various tissues including adipose tissue, skin, and the lung. Here, DiSpirito et al. have generated transcriptomes and chromatin accessibility maps of mouse regulatory T cells (Tregs) that reside in visceral adipose tissue, muscle, and the colon and compared them with the profiles generated from splenic Tregs. They have used these data sets to define transcriptional networks that are shared by all these populations and to identify networks that are unique to one or more tissue-resident Treg populations. Foxp3+CD4+ regulatory T cells (Tregs) accumulate in certain nonlymphoid tissues, where they control diverse aspects of organ homeostasis. Populations of tissue Tregs, as they have been termed, have transcriptomes distinct from those of their counterparts in lymphoid organs and other nonlymphoid tissues. We examined the diversification of Tregs in visceral adipose tissue, skeletal muscle, and the colon vis-à-vis lymphoid organs from the same individuals. The unique transcriptomes of the various tissue Treg populations resulted from layering of tissue-restricted open chromatin regions over regions already open in the spleen, the latter tagged by super-enhancers and particular histone marks. The binding motifs for a small number of transcription factor (TF) families were repeatedly enriched within the accessible chromatin stretches of Tregs in the three nonlymphoid tissues. However, a bioinformatically and experimentally validated transcriptional network, constructed by integrating chromatin accessibility and single-cell transcriptomic data, predicted reliance on different TF family members in the different tissues. The network analysis also revealed that tissue-restricted and broadly acting TFs were integrated into feed-forward loops to enforce tissue-specific gene expression in nonlymphoid-tissue Tregs. Overall, this study provides a framework for understanding the epigenetic dynamics of T cells operating in nonlymphoid tissues, which should inform strategies for specifically targeting them.