Quality control of reconstituted nucleosomes and nucleoso- nucleoso-mal arraysnucleoso-mal arrays

To obtain reproducible ubiquitylated nucleosome and chromatin templates, these were recon-stituted to full saturation of the DNA template with histone octamers. Mononucleosomes saturation was examined by native agarose gel electrophoresis. The presence of unbound template DNA or the unwanted assembly of subnucleosome complexes was resolved by over-titration of histone octamers into the reconstitution reaction. Fully saturated

mononucle-Figure 3.7: Preparation of DNA templates for mononucleosome and chromatin re-constitution. (A) Ethidium bromide (EtBr)-stained agarose gel of a pUC18-52x187 plasmid digested withAvaI andNotI restriction ezymes and gradient PEG 6000 purification of the result-ing 171 bp DNA insert away from the intact plasmid backbone. (B) EtBr-stained agarose gel of dephosporylation (Antarctic phosphatase) and biotinylation (T4 DNA ligase) reactions on the 171 bp DNA. (C) Dot blot using Streptavidin-HRP conjugate to detect biotinylated DNA templates.

(D) EtBr-stained agarose gel of a pUC18-12x200 plasmid digested with EcorI, HaeII, DdeI and BfuCI restriction ezymes and gradient PEG 6000 purification of the resulting 12x200 insert from the digested plasmid backbone. (E) EtBr-stained agarose gel of the biotinylation reaction of the 12x200 template. (F) Dot blot using Streptavidin-HRP conjugate to detect biotinylated DNA tem-plates. NC = negative control. After biotinitylation, the mononucleosomal DNA template was 187 bp long. M = DNA ladder marker [bp]

osomes appear on an agarose gel as a single band that is shifted from 187 bp to 400-500 bp (Figure 3.8C). Since octamer composition may change the surface properties of the nu-cleosomes (charge, folding), different mononunu-cleosomes travel slightly differently through a

Figure 3.8: Reconstitution of mononucleosomes and nucleosomal arrays. (A) EtBr-stained gel of the pure DNA templates used in the reconstitution reactions. (B) EtBr-EtBr-stained gel of biotinylated mononucleosomes, including the His.H2B, HA.H2B, His.H2BK120ub and HA.H2BK34ub reconstitutions used in affinity purification experiments for mass spectrometry analysis. (C) EtBr-stained gel of biotinylated nucleosomal arrays, including unmodified, H3K18ub, H3K23ub and H3K18/23ub2 reconstitutions used in affinity purification experiments for mass spec-trometry analysis. (D) EtBr-stained gel of biotinylated nucleosomal arrays, including unmodified, H2Amt, H3K18ub, H3K23ub, H3K23ub H2Amt and H3K18/23ub2 reconstitutions used in affin-ity purification experiments with recombinant proteins. (E) EtBr-stained gel of biotinylated nu-cleosomal arrays, including unmodified, His.H2B, HA.H2B, His.H2BK120ub and HA.H2BK34ub reconstitutions used in affinity purification experiments for mass spectrometry analysis.

Reconstitution of nucleosomal arrays was investigated with respect to two different parame-ters. The first parameter, nucleosome positioning, refers to the ability of the DNA sequence to prevent deviation (sliding or unwrapping) of a nucleosome from a set point (dyad axis) along the DNA sequence. If nucleosomes were misplaced, the desired uniform distribution conferred by the core positioning sequences would be skewed, which would result in the

for-Figure 3.9: Nucleosomal positioning control on reconstituted nucleosomal arrays. (A) Schematic annotation of cleavage sites for AvaI, BanII andNotI restriction enzymes with respect to two consecutive nucleosomal positioning sequences. (B) EtBr-stained gel of enzymatic digests of nucleosomal arrays including His.H2B, HA.H2B, His.H2BK120ub and HA.H2BK34ub-containing templates.

mation of unequal nucleosome spacing across the array. The second parameter, nucleosome occupancy, refers to the local density of nucleosomes within a given chromatin template. The nucleosome arrays used in this thesis contained twelve positioning sequences and were thus able to position strongly twelve nucleosomes. An undersaturated nucleosome array would contain less than twelve assembled nucleosomes.

Nucleosome positioning was addressed by restriction enzyme digestion, using enzymes that cleave unprotected DNA (Figure 3.9A). Restriction enzyme AvaI cleaves the 12x200 DNA template 34 bp upstream of the start of the core positioning sequence (108 bp away from the dyad axis). Restriction enzyme NotI cleaves the DNA template 7 bp upstream of the core positioning sequence (81 bp from the dyad axis). Restriction enzyme BanI cleaves the DNA template 13 bp downstream of the core positioning sequence (61 bp from the dyad axis).

Upon treatment with AvaI, the unmodified and ubiquitylated H2B chromatin arrays were fully digested to products which run to a front corresponding of 400 - 500 bp (Figure3.9B).

Figure 3.10: Nucleosomal occupancy control on reconstituted nucleosomal arrays.

(A) Schematic representation of the micrococcal nuclease (MNase) sensitive region between two consecutive nucleosomal positioning sequences. (B) EtBr-stained gel of time-course MNase digests of nucleosomal arrays containing His.H2B and His.H2BK120ub. (C) EtBr-stained gel of time-course MNase digests of nucleosomal arrays containing HA.H2B and HA.H2BK34ub. (D) EtBr-stained gel of time-course MNase digests of nucleosomal arrays containing unmodified and H3H23ub.

the nucleosome array. Upon treatment with NotI, the unmodified and ubiquitylated H2B chromatin arrays were fully digested to products which run to a front corresponding of 400 - 500 bp. This suggested that nucleosomes were not placed outside of the core positioning sequences. Together, the digestion reactions indicate that the nucleosomes were properly positioned after reconstitution.

Upon treatment with BanI, the His.H2B, HA.H2B and His.H2BK120ub chromatin fibers show protection against enzymatic digestion. In the case of HA.H2BK34ub, this protection is lost and several digestion products (mono-, di-, trinucleosomes) are observerd on the na-tive agarose gel. This is not an effect of nucleosomal positioning, but rather a consequence of spontaneous nucleosomal breathing [187]. H2BK34ub lowers the energy needed to brake the histone-DNA contacts at the two extremeties of the nucleosomal positioning sequence.

Nucleosome occupancy was addressed experimentally by digestion with micrococcal nuclease (MNase). In a MNase digestion experiment, unmodified chromatin is processed in parallel with ubiquitylated chromatin. Unprotected DNA (linker DNA or unbound nucleosomal po-sitioning sequence) is sensitive to MNase digestion (Figure 3.10A). All chromatin templates assayed produced twelve distinct cleavage bands in the first time-point of the digestion reac-tion (Figure 3.8B, Figure 3.10C, Figure 3.10D). Second, upon treatment with either AvaI, NotI orBanI, there was no evidence of unbound positioning sequences, which would appear as 200 bp DNA fragments on the native agarose gel (Figure3.10B, Figure3.10C). Together with the observation that MNase digests produced twelve cleavage products, this indicated that all nucleosome arrays that were produced for the chromatin affinity purification exper-iments were fully saturated.

The micrococcal nuclease digestion rate was slightly faster in the case of ubiquitylated nu-cleosomal arrays (Figure 3.10B, Figure 3.10C, Figure 3.10D). This was not an effect of nucleosome occupancy since the 147 bp or 200 bp digestion end products accumulate to the same extent in both unmodified and ubiquitylated arrays. This observation may have resulted from a higher order folding of ubiquitylated chromatin fibers that differed from that of the unmodified controls. Ubiquitylation of histones likely opened chromatin fibers locally, making the linker DNA more sensitive to MNase treatment.

3.2 Mapping of nuclear proteins recognising