Control of gene expression is mediated partly by recruitment of transcriptional factors to the promoter elements. Recruitment of these factors is further regulated by the chromatin structure surrounding the promoter region. If the chromatin structure is in an """"""""open"""""""" conformation, the promoter element is accessible to the transcriptional factors but if it is in a """"""""closed"""""""" conformation, the promoter element is inaccessible. Using a novel yeast strategy, we cloned a human serine-threonine kinase that appears to convert chromatin from a """"""""closed"""""""" conformation to an """"""""open"""""""" conformation. This kinase is expressed in all tissue types and is localized to the nucleus of human cells. The mechanism by which this kinase """"""""opens"""""""" up the chromatin is currently being investigated. In addition to chromatin opening activity, it appears to respond to DNA damage and may be important for DNA damage response. Once the chromatin is opened, transcriptional factors are recruited to activate or suppress transcription. Although a great deal is known about the transcriptional factors, study of their recruitment would be greatly enhanced by a methodology that allows detection of their recruitment in vivo, a methodology that does not exist yet. Toward this end, we developed a novel methodology PIN*POINT (protein position identification with nuclease tail). In this method, a fusion protein composed of a chosen protein linked to a non-sequence-specific nuclease is expressed in vivo and the binding of the protein to DNA is made detectable by the nuclease-induced cleavage near the binding site. Using PIN*POINT, we have been able to demonstrate that the beta-globin LCR (Locus Control Region) promotes recruitment of transcriptional activator EKLF to the beta-globin promoter in MEL (murine erythroleukemia) cells. We have also studied recruitment of erythroid specific transcription factor EKLF and chromatin remodelling factor BRG1 using this technique. EKLF, which binds to the CACCC box, is required for the expression of the beta-globin gene but not the gamma-globin gene. To understand EKLF's preference for the beta-globin promoter, we used PIN*POINT to study the recruitment of EKLF to the two types of globin promoters. We discovered that EKLF is recruited to the beta-globin CACCC box but not gamma-globin CACCC box. One of the reasons appears to be that the gamma-globin promoter binds to a protein that suppresses the recruitment of EKLF to the gamma-globin promoter. Chromatin remodelling factor BRG1 is also recruited to the beta-globin promoter and this requires the proximal promoter elements, the CACCC and TATA boxes. Interestingly, the ability to recruit BRG1 does not appear to be related to the strength of the promoter. The CMV promoter/enhancer, which has greater activity than the beta-globin/LCR, cannot recruit BRG1 consistent with the notion that the LCR unlike other enhancers has chromatin opening capacity.
