Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context.
We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how to measure the degree of chromatin assembly in the absence and presence of histone H1 using topological analysis and how to perform micrococcal nuclease digestion to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further, we describe the use of sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates).
Konesky
Kasey L
KL
Department of Biochemistry and Molecular Biology, 1870 Campus Delivery, Colorado State University, Fort Collins, CO 80523-1870, USA.
Laybourn
Paul J
PJ
D000818:Animals; D016367:Baculoviridae; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D018983:DNA Footprinting / Q000379:methods / Q000592:standards; D029721:Drosophila Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods* / Q000592:standards; D018832:Molecular Chaperones / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011786:Quality Control; D014158:Transcription, Genetic / Q000235:genetics*; D014461:Ultracentrifugation / Q000379:methods / Q000592:standards; D010948:Viral Plaque Assay / Q000592:standards; D015003:Yeasts / Q000235:genetics
D029721:Drosophila Proteins; D006657:Histones; D018832:Molecular Chaperones; D051548:Histone Acetyltransferases