Osteocytes represent the most abundant cellular component of mammalian bones with important functions in bone mass maintenance and remodeling. To elucidate the differential gene expression between osteoblasts and osteocytes we completed a comprehensive analysis of their gene profiles. Selective identification of these two mature populations was achieved by utilization of visual markers of bone lineage cells. We have utilized dual GFP reporter mice in which osteocytes are expressing GFP (topaz) directed by the DMP1 promoter, while osteoblasts are identified by expression of GFP (cyan) driven by 2.3kb of the Col1a1 promoter. Histological analysis of 7-day-old neonatal calvaria confirmed the expression pattern of DMP1GFP in osteocytes and Col2.3 in osteoblasts and osteocytes. To isolate distinct populations of cells we utilized fluorescent activated cell sorting (FACS). Cells suspensions were subjected to RNA extraction, in vitro transcription and labeling of cDNA and gene expression was analyzed using the Illumina WG-6v1 BeadChip.Following normalization of raw data from four biological replicates, 3444 genes were called present in all three sorted cell populations: GFP negative, Col2.3cyan + (osteoblasts), and DMP1topaz + (preosteocytes and osteocytes). We present the genes that showed in excess of a 2-fold change for gene expression between DMP1topaz + and Col2.3cyan + cells. The selected genes were classified and grouped according to their associated gene ontology terms. Genes clustered to osteogenesis and skeletal development such as Bmp4, Bmp8a, Dmp1, Enpp1, Phex and Ank were highly expressed in DMP1topaz + cells. Most of the genes encoding extracellular matrix components and secreted proteins had lower expression in DMP1topaz + cells, while most of the genes encoding plasma membrane proteins were increased. Interestingly a large number of genes associated with muscle development and function and with neuronal phenotype were increased in DMP1topaz + cells, indicating some new aspects of osteocyte biology. Although a large number of genes differentially expressed in DMP1topaz + and Col2.3cyan + cells in our study have already been assigned to bone Contact Information: Ivo Kalajzic, Department of Reconstructive Sciences, MC 3705, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06032. Tel.: 860-679-6051; Fax: 860-679-2910; ikalaj@neuron.uchc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBone. Author manuscript; available in PMC 2010 October 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NI...

The forkhead transcription factor FoxO1 is a critical regulator of hepatic glucose and lipid metabolism, and dysregulation of FoxO1 function has been implicated in diabetes and insulin resistance. We globally identified FoxO1 occupancy in mouse hepatic chromatin on a genome-wide level by chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq). To establish the specific functional significance of FoxO1 against other FoxO proteins, ChIP-seq was performed with chromatin from liver-specific FoxO1 knockout and wild-type mice. Here we identified 401 genome-wide FoxO1-binding locations. Motif search reveals a sequence element, 5′ GTAAACA 3′, consistent with a previously known FoxO1-binding site. Gene set enrichment analysis shows that the data from FoxO1 ChIP-seq are highly correlated with the global expression profiling of genes regulated by FoxO1, demonstrating the functional relevance of our FoxO1 ChIP-seq study. Interestingly, gene ontology analysis reveals the functional significance of FoxO1 in retinoid metabolic processes. We show here that FoxO1 directly binds to the genomic sites for the genes in retinoid metabolism. Notably, deletion of FoxO1 caused a significantly reduced induction of Pck1 and Pdk4 in response to retinoids. As Pck1 and Pdk4 are downstream targets of retinoid signaling, these results suggest that FoxO1 plays a potential role in linking retinoid metabolism to hepatic gluconeogenesis.

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