semi-automated-isolation-stromal-vascular-fraction-from-murine-white

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

the university of tokyo

The transcription factor nuclear factor I-A (NFIA) is a regulator of brown adipocyte differentiation. Here we show that the C-terminal 17 amino acid residues of NFIA (which we call pro#3 domain) are required for the transcriptional activity of NFIA. Full-length NFIA-but not deletion mutant lacking pro#3 domain-rescued impaired expression of PPARγ, the master transcriptional regulator of adipogenesis and impaired adipocyte differentiation in NFIA-knockout cells. Mechanistically, the ability of NFIA to penetrate chromatin and bind to the crucial Pparg enhancer is mediated through pro#3 domain. However, the deletion mutant still binds to Myod1 enhancer to repress expression of MyoD, the master transcriptional regulator of myogenesis as well as proximally transcribed non-coding RNA called DRReRNA, via competition with KLF5 in terms of enhancer binding, leading to suppression of myogenic gene program. Therefore, the negative effect of NFIA on the myogenic gene program is, at least partly, independent of the positive effect on PPARγ expression and its downstream adipogenic gene program. These results uncover multiple ways of action of NFIA to ensure optimal regulation of brown and beige adipocyte differentiation.

NFIA differentially controls adipogenic and myogenic gene program through distinct pathways to ensure brown and beige adipocyte differentiation.

Thermogenic brown and beige adipocytes counteract obesity by enhancing energy dissipation via uncoupling protein-1 (Ucp1). However, the effect of genetic variation on these cells, a major source of disease susceptibility, has been less well studied. Here we examined beige adipocytes from obesity-prone C57BL/6J (B6) and obesity-resistant 129X1/SvJ (129) mouse strains and identified a -regulatory variant rs47238345 that is responsible for differential Ucp1 expression. The alternative T allele of rs47238345 at the  -12kb enhancer in 129 facilitates the allele-specific binding of nuclear factor I-A (NFIA) to mediate allele-specific enhancer-promoter interaction and  transcription. Furthermore, CRISPR-Cas9/Cpf1-mediated single nucleotide polymorphism (SNP) editing of rs47238345 resulted in increased Ucp1 expression. We also identified Lim homeobox protein 8 (Lhx8), whose expression is higher in 129 than in B6, as a acting regulator of Ucp1 in mice and humans. These results demonstrate the - and acting effects of genetic variation on Ucp1 expression that underlie phenotypic diversity.

Kaede Saito

Division for Health Service Promotion,
Department of Diabetes and Metabolic Diseases,
Graduate School of Medicine,
Department of Diabetes and Metabolic Diseases, Graduate School of Medicine

the university of tokyo

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

Kaede Saito

Division for Health Service Promotion,Department of Diabetes and Metabolic Diseases,Graduate School of Medicine,Department of Diabetes and Metabolic Diseases, Graduate School of Medicine

the university of tokyo

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

Division for Health Service Promotion,
Department of Diabetes and Metabolic Diseases,
Graduate School of Medicine,
Department of Diabetes and Metabolic Diseases, Graduate School of Medicine