Question

You are interested in the interactions of histone deacetylase (HDAC) with nucleosomes. Using a DNA template that is end labelled with 32P and has two nucleosomes already bound to it, you perform a gel-shift experiment with HDAC and the chromatin from above. You also assemble chromatin with nucleosomes that have been methylated at a particular amino acid residue of Histone H4. The results are below. Based on the data, (A) explain the autoradiogram below – what are all the bands? (B) Propose a model for the interaction between the HDAC and the chromatin.

4. You are interested in the interactions of histone deacetylase (HDAC) with nucleosomes. Using a DNA template that is end labelled with and has two nucleosomes already bound to it, you perform a gel-shift experiment with HDAC and the chromatin from above. You also assemble chromatin with nucleosomes that have been methylated at a particular amino acid residue of Histone H4. The results are below. Based on the data, (A) explain the autoradiogram below- what are all the bands? (B) Propose a model for the interaction between the HDAC and the chromatin. 8 points histone deacetylase (the wedges or ramps indicate increasing concentration of the HDAC) Lane 1 2 3 4 methylation methylation

Answer 1

A. The lower bands (3rd level bands) correspond to just nucleosomes unbound to any HDAC molecules, as this band is seen in lanes where HDAC has not been added. The second level bands correspond to nucleosomes bound to HDAC molecules. The third level bands correspond to nucleosomes bound to and even higher amount of HDAC enzymes. As more HDAC binds to the histones, the bands get shifted to a higher position in the gel.

B. Increasing HDAC concentration increases HDAC binding to histones (unmethylated), as is evident from the presence of the upper band in this lane. But HDAC binding increases when Histone H4 is methylated, especially when the HDAC concentration is increased (the upper band is evident in methylated histone lanes).

Thus methylation on histones enhances the recruitment of HDAC proteins.