Global mapping of cell type-specific open chromatin by FAIRE-seq reveals the regulatory role of the NFI family in adipocyte differentiation.

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type-specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA-mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type-specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation.

CDK5 Regulatory Subunit-Associated Protein 1-like 1 Negatively Regulates Adipocyte Differentiation through Activation of Wnt Signaling Pathway.

CDK5 Regulatory Subunit-Associated Protein 1-like 1 (CDKAL1) was identified as a susceptibility gene for type 2 diabetes and body mass index in genome-wide association studies. Although it was reported that CDKAL1 is a methylthiotransferase essential for tRNALys(UUU) and faithful translation of proinsulin generated in pancreatic ß cells, the role of CDKAL1 in adipocytes has not been understood well. In this study, we found that CDKAL1 is expressed in adipose tissue and its expression is increased during differentiation. Stable overexpression of CDKAL1, however, inhibited adipocyte differentiation of 3T3-L1 cells, whereas knockdown of CDKAL1 promoted differentiation. CDKAL1 increased protein levels of ß-catenin and its active unphosphorylated form in the nucleus, thereby promoting Wnt target gene expression, suggesting that CDKAL1 activated the Wnt/ß-catenin pathway-a well-characterized inhibitory regulator of adipocyte differentiation. Mutant experiments show that conserved cysteine residues of Fe-S clusters of CDKAL1 are essential for its anti-adipogenic action. Our results identify CDKAL1 as novel negative regulator of adipocyte differentiation and provide insights into the link between CDKAL1 and metabolic diseases such as type 2 diabetes and obesity.

NFIA co-localizes with PPARγ and transcriptionally controls the brown fat gene program.

Brown fat dissipates energy as heat and protects against obesity. Here, we identified nuclear factor I-A (NFIA) as a transcriptional regulator of brown fat by a genome-wide open chromatin analysis of murine brown and white fat followed by motif analysis of brown-fat-specific open chromatin regions. NFIA and the master transcriptional regulator of adipogenesis, PPARγ, co-localize at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes and facilitates the binding of PPARγ, leading to increased chromatin accessibility and active transcription. Introduction of NFIA into myoblasts results in brown adipocyte differentiation. Conversely, the brown fat of NFIA-knockout mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of muscle genes. Finally, expression of NFIA and the brown-fat-specific genes is positively correlated in human brown fat. These results indicate that NFIA activates the cell-type-specific enhancers and facilitates the binding of PPARγ to control the brown fat gene program.

Clinical heterogeneity of adult Japanese diabetes depending on titers of glutamic acid decarboxylase autoantibodies.

UNLABELLED: Aims/Introduction: We examined whether levels of glutamic acid decarboxylase autoantibodies (GADAb) might show the clinical heterogeneity of adult Japanese diabetes. MATERIALS AND METHODS: In this cross-sectional study, the serum levels of GADAb were measured in a total of 1857 consecutive adult diabetic patients aged 20 years or older. The patients with positive GADAb, arbitrarily defined as ≥1.5 U/mL, were divided into quartiles according to the number of patients. The age- and sex-matched diabetic patients without GADAb were selected as a control group. RESULTS: A total of 103 (5.5%) of the diabetic patients had GADAb, and showed higher HbA1c and serum high-density lipoprotein (HDL) cholesterol levels, lower body mass index (BMI), urinary C-peptide immunoreactivity (CPR), serum triglycerides (TG) and uric acid (UA) levels, and lower prevalence of metabolic syndrome than the control group (P < 0.05). Quartiles 3 and 4 (i.e. GADAb ≥4.6 U/mL) showed a higher HbA1c level, lower BMI, urinary CPR, serum TG and UA levels, quartile 2 (2.5 ≤ GADAb < 4.6 U/mL) showed a lower BMI level than the control group (P < 0.05). Among the clinical parameters, we observed significant upward trends for both HbA1c and serum HDL cholesterol levels, and significant downward trends for BMI, serum TG and UA, urinary CPR levels, and prevalence of metabolic syndrome across GADAb quartiles (P < 0.05 for trend). CONCLUSIONS: These results show that the clinical phenotype of adult Japanese diabetes correlates with GADAb levels, and that patients with GADAb (≥2.5 U/mL) show different characteristics from those without GADAb, although further longitudinal studies are required. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00190.x, 2011).

Usefulness of serum cystatin C in Japanese patients with type 2 diabetes mellitus and nephropathy.

We examined usefulness of serum cystatin C to detect chronic kidney disease (CKD) stage >or=3, which was defined by Modification of Diet in Renal Disease formula. Serum cystatin C could detect CKD stage >or=3 with high efficacy in 289 Japanese patients with type 2 diabetes and nephropathy.

A SWI2/SNF2-type ATPase/helicase protein, mDomino, interacts with myeloid zinc finger protein 2A (MZF-2A) to regulate its transcriptional activity.

BACKGROUND: The myeloid zinc finger protein 2A (MZF-2A) is a Krüppel-type C2H2 zinc finger transcription factor expressed in myeloid cells and involved in the growth, differentiation and tumorigenesis of myeloid progenitors. Previously we identified a 180 amino acid domain in MZF-2A which is responsible for the transcriptional activation of MZF-2A. To understand the mechanism of the MZF-2A-dependent transcriptional activation, we screened for molecules that interact with the transactivation domain (TAD) of MZF-2A. RESULTS: By using the yeast Ras recruitment two-hybrid screening, we identified a novel SWI2/SNF2-related protein, termed mammalian Domino (mDomino), as an MZF-2A-binding partner. The mDomino protein, which shows a marked similarity to the Drosophila Domino protein, contains a SWI2/SNF2-type ATPase/helicase domain, a SANT domain, and a glutamine-rich (Q-rich) domain. The C-terminal Q-rich domain of mDomino physically associates with the TAD of MZF-2A in mammalian cells as well as in yeast. Expression of the mDomino Q-rich domain, together with MZF-2A in myeloid LGM-1 cells, enhanced the MZF-2A-mediated activation of a reporter gene. CONCLUSIONS: These results strongly suggest that an ATP-dependent chromatin-remodelling complex containing mDomino interacts with MZF-2A to regulate gene expression in myeloid cells.