Detection of H2Bub1 in lysates prepared using the modified boiling method. (A) Yeast whole cell lysates were prepared using the modified boiling procedure from the wild type or mutant (bre1Δ) strains harboring H2B (no tag control), Flag-H2B or Flag-H2B-K123R. Equal amounts of lysates (50 µg) were resolved in a 15% acrylamide-SDS polyacrylamide gel (15% SDS-PAGE), transferred to a PVDF membrane and probed with an antibody that detects the Flag epitope (α-Flag, 1:5000 dilution, Sigma, catalog no. F3165) or with an antibody that recognizes H3 dimethyl lysine 4 (α-H3K4me2, 1:5000 dilution, Millipore, catalog no. 07–030). (B) Yeast whole cell lysates were prepared from wild type (BY4742) or mutant (bre1Δ) strains and subjected to Western blotting as described for panel A, but the blots were probed with an antibody that detects yeast H2B (α-H2B, 1:2500 dilution, Active Motif, catalog no. 39237).

Antibody titration to determine the optimal amount required for high enrichment in ChIP assay. Soluble chromatin was prepared using a yeast strain expressing Swd2 with 3 copies of the HA epitope tag at the C-terminus (Swd2-3HA) or lacking the tag (no tag) subjected to double crosslinking (DMA and HCHO). The soluble chromatin (500 µg) was subjected to immunoprecipitation (IP) using 0.5 µl, 1 µl or 2 µl of an antibody that recognizes the HA epitope (α-HA, GenScript, catalog no. A00168). Quantitative PCR in real time (qPCR) was used to measure DNA associated with Swd2 in the immunoprecipitate and the total DNA in the soluble chromatin subjected to IP (input). Relative enrichment (IP/input) was initially calculated. For no tag control, the relative enrichment obtained represents the background signal (set as 1). Fold enrichment relative to the no tag control is shown for Swd2-3HA using different amounts of the antibody.

Determining the type of antibody suitable for effective enrichment of chromatin-bound factors in ChIP assays. Yeast strains expressing either Spp1 or Swd2 with 3 copies of the HA epitope tag at the C-terminus (Spp1-3HA or Swd2-3HA, respectively), expressing Set1 containing 9 copies of Myc epitope tag at the N-terminus (9Myc-Set1) or strains lacking the tags (no tag) were subjected to double crosslinking (DMA and HCHO) prior to soluble chromatin preparation. For panel A, soluble chromatin (500 µg) was subjected to IP using 2 µl α-HA, which is either a mouse monoclonal antibody (mAb) (kindly provided by Ethan Lee) or goat polyclonal antibody (pAb) (GenScript, catalog no. A00168). For panel B, soluble chromatin (1 mg) was subjected to IP using 2 µl of a mouse monoclonal α-Myc (clone 9E10, kindly provided by Ethan Lee) or a rabbit polyclonal antibody α-Myc (GenScript, catalog no. A00172). Enrichment of ChIP DNA relative to input DNA was calculated following qPCR as described in Fig. 2. Fold enrichment using different types of antibody are shown for Spp1-3HA and Swd2-3HA (panel A) or 9Myc-Set1 (panel B) relative to the no tag control (set as 1).

Increased washing increases the enrichment of chromatin-bound factors in ChIP assays. Soluble chromatin was prepared using a yeast strain expressing Bre2 with 9 copies of the Myc epitope tag at the C-terminus (Bre2-9Myc) or lacking the tag (no tag) subjected to double crosslinking (DMA and HCHO). 500 µg soluble chromatin was used in IP along with 2 µl mouse monoclonal α-Myc. Following IP, bead-bound immunoprecipitates were washed either with a small volume of buffers [comprised of sequential washes with 500 µl each of FA140 buffer (two times), FA500 buffer and LiCl/NP40 buffer] or a large buffer volume [comprised of sequential washes with 1 ml each of FA140 buffer (two times), FA500 buffer (two times) and LiCl/NP40 buffer]. Enrichment of ChIP DNA relative to input DNA was calculated following qPCR as described in Fig. 2. Fold enrichment using different wash volumes are shown for Bre2-9Myc relative to the no tag control (set as 1).

A combinatorial approach employing chromatin association and ChIP assays can reveal defects in protein-protein interactions on chromatin. Schematic representations of the chromatin association assay and ChIP assay are shown. A speculative scenario of destabilization in the interactions of a chromatin-bound protein under certain experimental condition (mutation or treatment) compared to the control (wild type or untreated) and discrepancy/difference in the detection of this destabilized interaction between the two assays are depicted. In (i) and (iv), a protein of interest (POI, oval or octagon) is shown stably associated with the wild type chromatin either by its interactions with a partner or bridging protein (gray circle) or via its direct interaction with the nucleosome (histone octamer, circle; DNA, solid line). These direct and indirect interactions of the POI may be destabilized or weakened in the mutant without any reduction in its overall levels on chromatin (oval or octagon with dashed line). (ii) The destabilized/weakened interactions might render the POI susceptible to dissociation from the chromatin during the multiple steps, with solutions of different ionic strengths, involved in the chromatin association assay (nuclei isolation and chromatin fractionation) and resulting in the reduced levels of POI on the chromatin in the mutant. (iii) Normalized soluble chromatin is subjected to Western blotting and an antibody (α-POI) is used to measure changes in the chromatin-bound levels of POI in the wild type or mutant. The amount of H3 is used to confirm that equal amounts of chromatin were used in the assay (detected using α-H3, loading control). As shown in the mock-up Western blot, chromatin-bound levels of POI in the mutant might be highly reduced (thin gray line) due to its dissociation as compared to the wild type. (v) Addition of a bifunctional crosslinking agent (DMA) and formaldehyde causes protein-protein and protein-DNA crosslinking (parallel black lines) and results in stable tethering of the POI to chromatin. Crosslinking stabilizes the “loose” or weakened interactions of POI in the mutant and prevents its dissociation during the several incubation and wash steps (including highly stringent conditions, such as the exposure to 500 mM NaCl and detergents) involved in the ChIP assay. (vi) Soluble chromatin is prepared from the wild type and mutant strains subjected to double crosslinking and IP is performed using α-POI. Changes in the chromatin-bound levels of POI (occupancy) in the wild type and mutant are determined using qPCR analysis of input and ChIP DNA. As depicted in the model graph, owing to crosslinking, occupancy of the POI on chromatin in the mutant might be similar to that in the wild type, unlike in chromatin association assay (iii).