The major distal regulatory sequence for the ␤-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.Genes in the ␤-globin gene complex (HBBC, containing HBE1, HBG2, HBG1, HBD, and HBB), together with those in the ␣-globin gene complex (HBZ2, HBA2, and HBA1) encode the developmentally regulated, erythroid-specific family of hemoglobins in vertebrates. Transcription of the mammalian HBBC is regulated both by proximal elements, such as promoters, and by a distal regulatory element known as the locus control region (LCR). The LCR is marked by several hypersensitive sites (HSs) in erythroid chromatin (18,45) and is required for high-level expression of genes within the HBBC in erythroid cells (reviewed in references 6 and 19). Transfection and transgenic mouse studies show that the LCR confers this high-level expression at many, but perhaps not all, ectopic sites of integration (1, 21, 31). Gain-of-function experiments examining multiple integrated copies of LCR constructs revealed expression that is copy number dependent and independent of the site of integration (e.g., see references 43 and 44), suggesting that the LCR contains a dominant chromatin-opening activity. However, deletion of HS1 to HS6 from the LCR of mouse Hbbc (3, 12), as well as deletion of HS2 to HS5 from the LCR of human HBBC on chromosome 11 (37), leaves the globin genes in an open chromatin domain, albeit expressed at very low to undetectable levels. Thus, the LCR is clearly required for enhancement but it is not necessary fo...

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The locus control region (LCR), marked by five DNasehypersensitive sites (HS) cores, is required for high level, expression of mammalian p-globin genes. Interspecies sequence alignments reveal many conserved blocks both within and between HS cores. The conserved blocks bind specific nuclear proteins in vitro. We tested the ability of the LCR fragments to enhance expression of a p-globin promoter-EGFP reporter after integration into the same marked site in MEL cells. The LCR fragments containing HS cores plus flanking sequences, or multiple HSs were compared in both orientations at three different chromosomal locations. No LCR construct, including a 9 kb fragment with HS2, HS3 and HS4 could overcome all the position effects. Single HSs enhance only 2-4 fold, but in combination can enhance up to 20-fold. However, this marked synergism requires sequences between and within the cores. These data support a model in which HS cores plus the flanking regions comprise a structural HS "unit" in which the flanking regions bound by the specific proteins, provide an optimal environment for activating and the domain-opening activities of the HS cores.for The enzyme inositol monophosphatase ( W A ) is a key enzyme in the phosphoinositide signaling system. W A is uncompetitively inhibited by therapeutically relevant concenhatiom of lithium (Li), a widely used mood-stabilizing medication, yet, the molecular mechanisn of its f l e a s is unknown. Bemdge first proposed that the physiological effect of Li's inhibition of IMF'A is depletion of brain free inositol and the consequent attenuation of neurohansmitkx driven phosphoinositide second messenger signal generation. Recently we have found that the activity of the IMPA in immortalized lymphoblastoid cell lines of bipolar patients is signiridy lower than those !?om a normal comparison group. When the bipolar patients were grouped acoording to clinical response to Li therapy, Li responders exhibited significantly lower IMPA activity compared to poor Li responden.Two human IMPA genes have recently been cloned. The M A 1 gene was found onchromosome 8q21.13-21.3 and W A 2 was located on chromosome lSpl1.2. Several studies have indicated the presence of a susceptibility locus for bipolar disorder on chromosome 18~11.2. Therefore, IMPA genes are candidates for genetic studies in bipolar disorder.We presently report the suuchue of mouse W A -2 cDNA including the 5' and 3' untranslated regions and the gnomic structure of both mouse IMPA genes. This is the first step toward the produaion of knockout mice lacking the Ih4PA genes. The role of histone acetylation in phosphate regulated1838 Regulation of the expression of 3-hydroxy-3-methylglutaryl