To understand the principles of control and selectivity in gene expression, the biochemical mechanisms by which promoter- and enhancer-binding factors regulate transcription by RNA polymerase II were analyzed. A general observed repressor of transcription was purified and identified as histone H1. Since many aspects of H1 binding to naked DNA resemble its interaction with chromatin, purified H1 bound to naked DNA was used as a model for the repressed state of the DNA template. Three sequence-specific transcription factors, Sp1, GAL4-VP16, and GAGA factor, were shown to counteract H1-mediated repression (antirepression). In addition, Sp1 and GAL4-VP16, but not the GAGA factor, activated transcription in the absence of H1. Therefore, true activation and antirepression appear to be distinct activities of sequence-specific factors. Furthermore, transcription antirepression by GAL4-VP16 was sustained for several rounds of transcription. These findings, together with previous studies on H1, suggest that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.

By using a DNase I footprinting assay, we have purified a factor by DNA affinity chromatography that binds to the minimal enhancer region of the Drosophila knirps gene and subsequently identified the protein as the core histone H2B. This inadvertent purification of a core histone as a putative sequence-specific DNA binding protein was due to a previously unknown property of H2B to interact with DNA in a periodic manner. Moreover, we found that each of the individual core histones, but not histone H1 or high mobility group protein 1, bound to the knirps enhancer to give a repetitive DNase I footprint pattern with a periodicity of about 10 base pairs, which is approximately one turn of the DNA helix. In addition, preparations containing the core histones H2A-H2B or H3-H4 yielded identical periodic DNase I footprint patterns on several different promoter and enhancer regions. These findings suggest that there are periodic, homotypic interactions between DNA-bound core histones that result from an alteration of the overall DNA structure such as the curvature rather than a specific sequence. We have also shown that histones H2A-H2B can repress initiation of transcription by RNA polymerase II. The phenomena described here may reflect histone-DNA interactions in non-nucleosomal stretches of chromatin and could be involved in some aspects of either rotational or translational positioning of nucleosomes. Furthermore, these findings indicate that a repeated 10 bp DNase I ladder, which has previously been considered to be a property of an intact nucleosome, can also be generated with subnucleosomal components. It will thus be necessary to reevaluate the criteria applied to the analysis of nucleosomes both in vivo and in vitro.

Affinity chromatography is a very effective and straightforward means of purifying a protein based on its sequence-specific DNA-binding properties. The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. The first basic protocol describes preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support. An provides a method to couple DNA to commercially available CNBr-activated Sepharose, and a support protocol describes how to purify crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin. The second basic protocol outlines the affinity chromatography procedure. A second support protocol describes determination of the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.

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The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. Preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support, is described, and an alternate protocol provides a method to couple DNA to commercially available CNBr-activated Sepharose. A method for purification of crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin is also provided. A detailed protocol for the actual affinity chromatography procedure is described and a support protocol allows the investigator to determine the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.