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Polycomb and Trithorax Group Proteins: Master Regulators of Adipose Tissue Function?

Final Report Summary - ATACC (Polycomb and Trithorax Group Proteins: Master Regulators of Adipose Tissue Function?)

Current estimates place the prevalence of diabetes and obesity in the range of 300 million to beyond 1 billion by the year 2030. As critical risk factors for heart disease and stroke, obesity and diabetes currently represent one of the world’s chief economic and health care challenges.

While grossly divergent in their appearance, adipose excess (obesity) and adipose paucity (lipodystrophy) both present a number of parallel metabolic phenotypes, including insulin resistance, diabetes and cardiovascular disease, highlighting the critical homeostatic role played by a properly controlled, metabolically active adipose tissue mass. Adipose tissue is a dynamic metabolic sensory organ with numerous endocrine activities and functionally diverse sub-compartments (ie. sub-cutaneous, visceral and intra-muscular white adipose tissues (WAT) as well as the highly thermogenic brown adipose tissue depots). Mature adipocytes secrete a multitude of factors that play central roles in the regulation of energy balance, insulin sensitivity, immunological response, cardiovascular disease, and even cancer. While we currently have a well-developed genetic framework for understanding of obesity and the underlying adipose tissue function and development, the contribution of critical regulatory layers, in particular epigenetic regulation, remain poorly understood. Indeed, very recent evidence highlights the importance of epigenetic mediators in adipocyte differentiation and function. In short, a deeper understanding of the epigenetic basis of adipose tissue (dys)function is paramount to the development of new therapies for metabolic disease.

Polycomb and Trithorax Group (PcG/TrxG) proteins were first identified in D. melanogaster for their roles in silencing homeotic (Hox) genes and are now recognized as constituting a chromatin-based transcriptional regulatory system with key roles in multicellular development, stem cell biology and cancer.
Two main PcG complexes have been identified, Polycomb Repressive Complex 1 (PRC1) and PRC2. Each exerts independent but fundamental roles in PcG-silencing of gene expression. PRC2 methylates histone H3 at Lys27 (H3K27), via the methyltransferase activity of the SET domain containing Ezh2 protein. Of note Ezh2 is inactive alone and requires core complex assembly with Suz12 and Eed for robust enzyme activity. H3K27-modified histone then recruits PRC1, that ubiquitinates histone H2A and impedes transcription elongation. Finally, as a transcriptional balance to the system, there are the TrxG proteins. Discovered for their ability to suppress PcG mutation phenotypes, the TrxG proteins, including the Mll (mixed-lineage leukemia) H3K4 methyltransferases, are generally transcription permissive and comprise a more diverse collection of complexes and activities.

Data coming from genome-wide profiling of PRC1 and PRC2 target genes, genome-wide obesity screening in D. Melanogaster and targeted genetic studies in cultured adipocytes indicate a prominent role for the PcG/Trx system in adipose tissue biology and metabolic function.
To date, little is known about PcG/TrxG function in metabolic control in vivo. The aim of this proposal was to understand the role of the PcG/Trx regulatory system in the development, function and maintenance of the adipose organ in vivo. Our multi-pronged approach focussed on the functional characterization of conditional knockout mice bearing mutant alleles of either Ezh2 (the core PRC2 methyltransferase) or Mll (the core TrxG methyltransferase) and combined detailed in vivo metabolic characterization with profiling of gene expression, chromatin state, and protein-DNA interactions. The overall goal of this project was defined in two primary objectives:
1. To generate inducible, tissue-specific PcG/Trx knockout mice.
2. To characherize the roles of PcG/Trx proteins in adipose tissue function and metabolic control.

Generation of inducible, tissue-specific PcG/Trx knockout mice.
Due to an uncontrolled swapping of our Cretg mouse lines (Alb-Cre instead of Ap2-Cre) we had to focus our work on liver, rather than adipose tissue-specific knockouts. Serendipitously, this “mistake” led to intriguing and completely new findings. Studies on adipose tissue specific knockouts have been started.
All genetically modified animals are viable, fully developed and fertile.

Characterization of the roles of PcG/Trx proteins in adipose tissue function and metabolic control
The genetically modified animals have no developmental defect. On adipogenesis, ex vivo studies using the stromo-vascular fraction (SVF) from each fat depot (epiWAT, iWAT and BAT), as well as gain and loss-of-fuction experiments in 3T3-L1 preadipocytes cell lines showed, differently from what was previously reported, no evident developmental defect.

Our understanding of the role of chromatin regulatory systems in terminally differentiated tissues is still poor. To understand the role(s) of PcG and TrxG proteins in liver function and metabolic control, we performed a systematic metabolic analysis of our knockout mice including 1) energy homeostasis (indirect calorimetry, feeding behaviour, cold exposure and activity); 2) glucose metabolism (pyruvate, glucose and insulin tolerance tests, and euglycemic clamps if necessary); and 3) lipid homeostasis (lipid tolerance test, serum adipokines and lipid profiling) on normal chow diet and at two intervals of high-fat diet (HFD) treatment.
Interestingly, Alb-Ezh2-/- and Alb-Mll-/- did not show any relevant metabolic phenotype. Either these proteins are dispensible therefore or, perhaps more likely, alternate redundant enzymatic activities may be compensating for their loss. Indeed Mll has at least 5 homologs and Ezh2 has Ezh1 as a potential functional replacement. In support of the latter hypothesis, Alb-Eed-/- mice display a striking metabolic phenotype.
On either normal chow diet or HFD, Alb-Eed-/- mice show an improved metabolic profile (glucose tolerance, insulin sensitivity and energy expenditure) when compared to their wild-type littermates. Additionally, knockout mice are protected from diet-induced obesity (DIO) and metabolic syndrome on HFD.

Hedgehog (Hh) is a morphogen essential for body patterning and proper embryonic development. Previous data from our group have shown that Hh not only controls white vs brown adipocyte development, but also energy handling in mature, fully differentiated, adipocytes. Active Hh peptides are dually lipid modified in the producing cells, carried on very-low density lipoproteins (VLDL), and released to act on receiving cells.
Here, we show that PcG inhibition in the liver increases expression and secretion of Hh peptides, which selectively signal to subcutaneous white adipocytes and induce functional browning, as indicated by whole transcriptome (RNA-Seq) analysis and functional studies like acute cold challenge and exposure to thermoneutrality.
Corroborated by preliminary in vivo immune-neutralization experiments, these data indicate the PcG-Hh axis as a novel liver-to-adipose tissue crosstalk important for normal metabolic control.