Rhythmic changes in histone acetylation at circadian clock genes suggest that temporal modulation of gene expression is regulated by chromatin modifications1-3. Furthermore, recent studies demonstrate a critical relationship between circadian and metabolic physiology4-7. The Nuclear Receptor Co-Repressor 1 (NCoR) functions as an activating subunit for the chromatin modifying enzyme histone deacetylase 3 (HDAC3)8. Lack of NCoR is incompatible with life, and hence it is unknown whether NCoR, and particularly its regulation of HDAC3, is critical for adult mammalian physiology9. Here we show that specific, genetic disruption of the NCoR-HDAC3 interaction in mice causes aberrant regulation of clock genes and results in abnormal circadian behavior. These mice are also leaner and more insulin sensitive due to increased energy expenditure. Unexpectedly, loss of a functional NCoR-HDAC3 complex in vivo does not lead to sustained elevations of known catabolic genes, but rather significantly alters the oscillatory patterns of several metabolic genes, demonstrating that circadian regulation of metabolism is critical for normal energy balance. These findings indicate that activation of HDAC3 by NCoR is a nodal point in the epigenetic regulation of circadian and metabolic physiology.Mammals display circadian rhythms in behavioral and physiologic processes, such as sleep, feeding, blood pressure, and metabolism10-12, guided by external light-dark signals that are integrated through intrinsic central and peripheral molecular clocks13, 14. Several critical clock and clock output genes display daily cycling of histone acetylation, suggesting that epigenetic regulation of chromatin plays a central role in circadian regulation1-3. Nuclear Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

To identify new regulatory elements within the mouse Ig locus, we have mapped DNase I hypersensitive sites (HSs) in the chromatin of B cell lines arrested at different stages of differentiation. We have focused on two regions encompassing 50 kilobases suspected to contain new regulatory elements based on our previous high level expression results with yeast artificial chromosome-based mouse Ig transgenes. This approach has revealed a cluster of HSs within the 18-kilobase intervening sequence, which we cloned and sequenced in its entirety, between the V gene closest to the J region. These HSs exhibit pro/pre-B cell-specific transcriptional silencing of a V gene promoter in transient transfection assays. We also identified a plasmacytoma cell-specific HS in the far downstream region of the locus, which in analogous transient transfection assays proved to be a powerful transcriptional enhancer. Deletional analyses reveal that for each element multiple DNA segments cooperate to achieve either silencing or enhancement. The enhancer sequence is conserved in the human Ig gene locus, including NF-B and E-box sites that are important for the activity. In summary, our results pinpoint the locations of presumptive regulatory elements for future knockout studies to define their functional roles in the native locus.The mouse immunoglobulin (Ig) gene locus has provided a paradigm to investigate many challenging and biologically relevant problems, including site-specific recombination (1-5), tissue-specific transcriptional regulation (1, 6, 7), somatic hypermutation (8 -11), DNA methylation (12-14), the relationship between chromatin structure and function (15-23), and the evolution of DNA sequence organization (24).The mouse locus is the largest multi-gene family locus thus far identified with respect to genomic length, spanning more than 3.5 megabases (25-30). The locus contains 96 potentially functional V genes that have been grouped into 18 families based on sequence homologies (29, 31, 32), 4 functional and 1 non-functional J regions, and a single C exon. The V families are semi-clustered but partially interspersed with other V families (25,29). The most 5Ј V gene is a member of the V24 family, some 3.5-megabases away from the J-C region (29). The most 3Ј V gene is V21G (29), 18 kb 1 away from J1 gene segment (this work).Previous studies have identified several cis-acting regulatory elements in the mouse Ig locus. All of these elements except for V gene promoter elements reside in a 16-kb segment near or within the J-C region toward the 3Ј end of the locus. These include two germ line promoter elements (33, 34), KI-KII sequences (35), two non-B cell-specific silencers (36, 37), a nuclear matrix association region (MAR) (38), an intronic enhancer (Ei) (39), and a 3Ј enhancer (E3Ј) (40). In some instances targeted deletions of these elements have been performed in cell lines or mice, permitting their functional significance to be addressed in the native locus. Deletion of a germ line promoter or KI-KII sequences or both res...

The mouse Igκ L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved. Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon Vκ-Jκ joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci. The largest minilocus (290 kb) possessed all members of the upstream Vκ2 gene family including their entire 5′ and 3′ flanking sequences, along with one member of a downstream Vκ21 gene family. In addition, again using yeast artificial chromosome-based technology, we created Igκ miniloci that contained differing lengths of sequences 5′ of the most distal Vκ2 gene family member. In transgenic mice, Igκ miniloci exhibited position-independent and copy number-dependent germline transcription. Igκ miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal Vκ gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects. The minilocus that contained the longest 5′ region exhibited significantly greater recombination of the upstream Vκ2 genes but not the downstream Vκ21 gene, providing evidence for a local recombination stimulating element. These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Igκ gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.

At a time of debate over physicians' fees and income, we describe the evolution of Blue Shield plans and programs to pay physicians' fees. We review how Medicare's "reasonable-charge" formulas fostered Blue Shield "usual, customary, and reasonable" (UCR) contracts. In a three-year period in the Washington, DC, area, Blue Shield UCR protocols permitted "customary" allowances for selected surgical procedures to rise 29 to 75 per cent; charges by two physicians increased allowances for coronary-artery bypass from $2000 to $3500. We find little justification for secrecy in fee-payment protocols. Physicians dominate the District of Columbia Blue Shield Board and its committees, and they control fee-payment formulas. Nationally, 61 per cent of Blue Shield boards have majorities of health-care providers; approximately two thirds of fee-related committees have physician majorities. We urge increased public debate, public representation, and accountability in monitoring and reforming the programs that we describe.