The Study of Adipose Tissue Differentiation and Development

Abstract/nThe Study of Adipose Tissue Differentiation and Development/nby/nOlga Gulyaeva/nDoctor of Philosophy in Endocrinology/nUniversity of California, Berkeley/nProfessor Hei Sook Sul, Chair/nTwo functionally different adipose tissues exist in mammals: white (WAT) and brown/n(BAT). WAT is the primary energy storage site and a critical organ for regulation of/nenergy metabolism through secretion of various adipokines. In contrast, BAT is a key/nthermogenic organ, capable of burning chemical substrates for heat generation through/nunique protein UCP1. Interestingly, UCP1 is only expressed in brown or beige/nadipocytes found in WAT, however all the transcriptional activators of UCP1 promoter/nidentified by far are not tissue-specific and, therefore, cannot solely explain BAT-specific/nexpression of UCP1. BAT is believed to form early in embryogenesis from muscle-like/nprogenitors, while WAT depending on the anatomical locations, develops in late/nembryogenesis or postnatally from non-muscle–like progenitors. Although embryogenic/nBAT precursors have been well characterized, the molecular identity of WAT precursors/nremains controversial. The aim of this dissertation work was to identify novel tissuespecific/ntranscriptional regulator of UCP1 in BAT as well as to identify and characterize/nWAT precursors at various stages of adipogenic commitment and differentiation./nChapter 1 reviews developmental origin of adipose tissue. Most of subcutaneous WAT/ndepots develop perinatally, while visceral WAT develops postnatally from adipose/nprecursors expressing markers such as Myf5, Pref-1, Wt1, and Prx1 depending on the/nanatomical location. BAT develops in early embryogenesis form Myf5+, Pax7+ musclelike/nprecursors. Brown fat-like cells found in WAT upon cold exposure or b3-adrenergic/nstimulation may arise from transdifferentiation of white adipocytes or by de-novo/ndifferentiation of yet to be characterized precursors. Finally, transcriptional and/nepigenetic control of brown and beige adipocyte differentiation is discussed./nChapter 2 demonstrates my efforts to characterize the hierarchy of adipose progenitors/nin WAT in vivo. By using Pref-1 promoter-mediated conditional ablation of Sox9, one of/nPref-1 targets in inhibiting adipogenesis, coupled with fluorescent labeling, I identified/nand characterized a population of early adipose progenitors that express Pref-1, Sox9/nand CD24. This early WAT precursor population is highly proliferative and does not yet/nexpress adipogenic markers and rather resembles stem cell-like population. Sox9/nappeared to be critical for maintaining these early precursors in highly proliferative and/nundifferentiated state. Loss of Sox9 in Pref-1+ cells lead to a depletion of this precursor/npopulation and an increase in more committed PDGFRa+ preadipocytes that do not/nproliferate and express early adipogenic markers. This switch in precursor populations/nfrom Pref-1+ to PDGFRa+ in the absence of Sox9 leads to enhanced adipogenesis and/nincreased adiposity impairing insulin sensitivity in vivo./nChapter 3 describes a role of novel BAT-enriched transcription factor, Zfp516 in/nregulating UCP1 transcription and BAT differentiation and development. I found that/nZfp516 is induced upon cold exposure and directly binds and activates UCP1 and/nPGC1a promoters. I also showed that knockdown of Zfp516 in cultured cells impairs/nBAT-gene program and induces muscle-like gene signature. Thus, total body Zfp516/nKO mice exhibit severe defect in BAT development, while transgenic mice/noverexpressing Zfp516 in all adipose tissues show increased browning of WAT. This/nleads to improved cold tolerance and reduces body weight accumulation upon high fat/ndiet improving metabolic parameters./nChapter 4 focuses on my efforts to identify Zfp516 interacting partners for activation/nBAT-gene program resulting in discovery of lysine-specific demethylase 1, Lsd1, as a/ndirect binding partner of Zfp516. Lsd1 through interaction with Zfp516 is recruited to/nUCP1 and PGC1a promoters to demethylate H3K9 and therefore promoter/ntranscription. Lsd1 is induced during BAT differentiation and its ablation using Myf5-Cre/nresults in impaired embryonic BAT development. UCP1-Cre mediated ablation of Lsd1/nimpairs BAT gene program and results in formation of BAT that resembles WAT,/ntherefore reducing thermogenic capacity and causing obesity in mice. Lastly, Zfp516-/nLsd1 interaction is required for Lsd1 function for BAT gene program in vitro./nSupplement summarizes my effort to identify a novel gene critical for BAT function and/ncharacterizes one such gene, C11orf54. I found that C11orf54 is highly expressed in/nmurine BAT, kidney and liver and is highly induced during BAT cell line differentiation./nC11orf54 is localized to the nucleus and cytoplasm and appears to be important for BAT/ndifferentiation as CRISPR-mediated KO lines demonstrate dramatically impaired/nadipogenic differentiation. Overexpression of C11 in Cos7 cells results in increased/nformation of TAG through yet-to be identified mechanism./nFinally, chapter 5 summarizes my work describing importance of novel genes identified/nfor BAT and WAT development, future directions and remaining questions./nThus, these studies provide a better understanding of how BAT and WAT development/nand differentiation are regulated in vivo, as well as explain the hierarchy of adipose/nprogenitors with various degree of commitment and differentiation in WAT.

Advisor: Sul, Hei Sook