The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 ) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C. J. Hetherington, H.-M. Chen, and D. G. Tenen, Mol. Cell. Biol. 14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to -59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor.

With the use of photoresponsive T7 promoters tethering two 2′‐methylazobenzenes or 2′,6′‐dimethylazobenzenes, highly efficient photoregulation of DNA transcription was obtained. After UV‐A light irradiation (320–400 nm), the rate of transcription with T7 RNA polymerase and a photoresponsive promoter involving two 2′,6′‐dimethylazobenzenes was 10‐fold faster than that after visible light irradiation (400–600 nm). By attaching a nonmodified azobenzene and 2′,6′‐dimethylazobenzene at the two positions, respectively, and by utilizing the different cis→trans thermal stability between cis‐nonmodified azobenzene and cis‐2′,6′‐dimethylazobenzene, four species of T7 promoter (cis–cis, trans–cis, cis–trans, and trans–trans) were obtained. The four species showed transcriptional activity in the order of cis–cis > cis–trans > trans–cis > trans–trans. Kinetic analysis revealed that the Km for the cis–cis promoter (both of the introduced azobenzene derivatives were in the cis form) and T7 RNA polymerase was 68 times lower than that for the trans–trans form, indicating that high photoregulatory efficiency was mainly due to a remarkable difference in affinity for RNA polymerase. The present approach is promising for the creation of biological tools for artificially controlling gene expression, and as a photocontrolled system for supplying RNA fuel for RNA‐powered molecular nanomachines.

Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90% in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis.

A photoresponsive GFP gene was constructed by attaching a T7 promoter that involves two azobenzene moieties as the photoswitch. The azobenzene moieties tethered on D-threoninol were inserted precisely into the sequence of T7 promoter at two positions in the nontemplate strand. By using azobenzene-tethered DNA as one primer, azobenzene was attached to GFP gene after PCR amplification. However, a single-stranded overhang involving azobenzene was formed because primer extension stopped at the position of azobenzene moiety. Interestingly we found that oligonucleotide complementary to the overhang could be ligated by T4 DNA ligase at the stopped position, and the intact photoresponsive T7 promoter was attached onto GFP gene. Furthermore, the in vitro expression of the constructed photoresponsive GFP gene was successfully switched on and off with light irradiation.

The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. Biol. 14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to -59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor.