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It is unclear whether the core promoter is involved in developmental regulation. To address this question, we mutated the TATA box of the human [gamma]-globin gene, produced transgenic mice, and examined the effect of the mutation during the course of mouse development. In our test system, the [gamma]-globin gene is expressed at similar levels in the embryonic and adult erythroid cells. The TATA box mutation dramatically reduced expression of the [gamma]-globin gene in the adult but not in embryonic erythroid cells. In addition, the disruption of the [gamma] TATA box significantly reduced the recruitment of TATA box-binding protein (TBP) in the adult cells, but not in embryonic cells, suggesting that the recruitment of TBP to the [gamma] gene promoter is developmentally specific. Similarly, the recruitment of transcription factor II B and RNA polymerase II to the [gamma] promoter was affected in the adult but not in embryonic cells. The distinct effects of the TATA mutation in the embryonic and adult developmental stages suggest that the basal transcription apparatus can be recruited to a core promoter in a developmental stage-dependent manner. The TATA mutation resulted in a shift of transcription initiation site 6 bp or longer upstream to the cap site both in the embryonic and adult erythrocytes. We conclude that the TATA box determines the initiation site but not the efficiency of transcription of the [gamma]-globin gene.

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and costeffective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications. [ABSTRACT FROM AUTHOR]

Examines the effect of TATA box mutation during the course of mouse development. Observation of embryonic stem cells; Identification of the polyacrylamide gel; Formation of the murine erythroleukemia cells.

Neuromedin U (NMU) is a highly conserved neuropeptide with a variety of physiological functions mediated by two receptors, peripheral NMUR1 and central nervous system NMUR2. Here we report the generation and phenotypic characterization of mice deficient in the central nervous system receptor NMUR2. We show that behavioral effects, such as suppression of food intake, enhanced pain response, and excessive grooming induced by intracerebroventricular NMU administration were abolished in the NMUR2 knockout (KO) mice, establishing a causal role for NMUR2 in mediating NMU's central effects on these behaviors. In contrast to the NMU peptide-deficient mice, NMUR2 KO mice appeared normal with regard to stress, anxiety, body weight regulation, and food consumption. However, the NMUR2 KO mice showed reduced pain sensitivity in both the hot plate and formalin tests. Furthermore, facilitated excitatory synaptic transmission in spinal dorsal horn neurons, a mechanism by which NMU stimulates pain, did not occur in NMUR2 KO mice. These results provide significant insights into a functional dissection of the differential contribution of peripherally or centrally acting NMU system. They suggest that NMUR2 plays a more significant role in central pain processing than other brain functions including stress/anxiety and regulation of feeding. [ABSTRACT FROM AUTHOR]

We have previously reported that DNase I hypersensitive site 5 (5'HS5) of the human beta-globin locus control region functions as a chromatin insulator in stable transfection assays. In this report we show that a 3.2 kb DNA fragment containing the entire 5'HS5 region can protect a position-sensitive (A)gamma-globin gene against position effects in transgenic mice. Bracketing is required for function of 5'HS5 as an insulator. The 5'HS5 insulator operates in adult as well as in embryonic murine erythroid cells. The insulator has no significant stimulatory effects of its own. These results indicate that 5'HS5 can function as a chromatin insulator in vivo.

The beta-globin locus control region (LCR) is a powerful regulatory element required for high-level globin gene expression. We have generated transgenic mouse lines carrying a beta-globin locus yeast artificial chromosome lacking the LCR to determine if the LCR is required for globin gene activation. beta-Globin gene expression was analyzed by RNase protection, but no detectable levels of epsilon-, gamma- and beta-globin gene transcripts were produced at any stage of development. These findings suggest that the presence of the LCR is a minimum requirement for globin gene expression. Next, we tested whether the LCR is necessary to activate globin gene expression in a gamma-globin promoter mutant that causes hereditary persistence of fetal hemoglobin (HPFH). beta-YAC transgenic mice carrying the -117 HPFH mutation and a HS3 core deletion that specifically abolishes gamma-globin gene expression during definitive erythropoiesis were produced to test whether the -117 (A)gamma promoter is activated in the absence of interaction with the LCR. In four transgenic mouse lines, gamma-globin gene expression was absent in adult erythrocytes, suggesting that an interaction between the gamma-globin gene promoter and the LCR is required for gamma gene activation even when the promoter contains an HPFH mutation.