Figure 5. PRDM9 multimer formation is mediated by the ZF domain in an allele-biased manner.
(a) Overview of the different C-terminally tagged PRDM9 constructs used. Both an HA and a V5 version of each construct were generated for co-IP experiments. (b) Barplot showing the relative intensity of western blot co-IP bands normalized to input bands (from 50-μg of total lysate protein) for each combination of bait and prey constructs. Whenever both bait and prey contain the zinc-finger domain (green bars), the co-IP signal is much stronger than when either or both constructs lack a ZF domain (orange bars). See Figure 5—figure supplements 1 and 2 for complete westerns with mock controls. (c) Barplot showing the results of competitive co-IP experiments performed in cells transfected with both Human and Chimp as prey (with the same epitope tag) and either Human or Chimp as bait (with a complementary epitope tag). Bars indicate the relative co-IP band intensity for Human and Chimp prey constructs when pulled down with either Chimp or Human bait. When Human is used as bait, more Human prey is pulled down; when Chimp is used as bait, more Chimp prey is pulled down (and this holds for both directions of HA/V5 tagging).
Figure 5—figure supplement 1. Confirmation of PRDM9 multimer formation.
Left: Western blots illustrating controls and experimental results. Samples were split and run on two blots separately, one imaged using an anti-HA antibody (upper) and one using an anti-V5 antibody (lower). Exposure time was 4 min. Ladder lanes are overlaid on the left, with approximate sizes in kiloDaltons noted. Lanes are labeled according to which full-length Human construct (HA or V5) was used, as well as which antibody was used for immunoprecipitation. IgG heavy chains are visible around 50 kDa, while the Human allele is visible as a band around 100 kDA with two or three smaller bands beneath it, likely representing degradation products (Grey et al., 2011; Cole et al., 2014). ‘-' is a short-hand label for input lanes, for which 50 μg of input chromatin was loaded in each well. The first six lanes demonstrate the specificity of the antibodies and their lack of cross-reactivity. The last two lanes show the co-IP experimental results confirming multimerization. Right: Two independent replicates were performed to confirm the formation of multimers with the full-length human constructs, using IgG mock control lanes to rule out nonspecific co-precipitation. Images were cropped to include only the PRDM9 bands. Input lane bands appear to have run lower than expected due to the use of a higher concentration of loading buffer in the IP lanes, an issue which was avoided in subsequent experiments.
Figure 5—figure supplement 2. Multimerization is mediated primarily by ZF-ZF binding.
Western blots illustrating co-IP results for various combinations of full-length Human, noZF, and ZFonly constructs. (a) The third and fourth blots show only a very faint co-IP signal despite strong input expression of the noZF construct, indicating that the non-ZF portion of PRDM9 cannot form multimers efficiently with itself or full-length PRDM9. The first and second blots show strong co-IP signals for the ZFonly construct, indicating that the ZF domain binds itself and binds the full-length Human construct. The fifth plot shows that the ZFonly and noZF constructs do not bind each other and confirms that multimerization is not mediated by the C-terminal tags. (b) A replication of the experiment shown in the first blot above, but performing the IPs and western blots with both tag combinations. This confirms that the full-length Human construct can pull down the ZFonly construct, and the ZFonly construct is sufficient to pull down the full-length Human construct.
Figure 5—figure supplement 3. Benzonase treatment does not affect co-IP results.
(a) Replication of ZFonly-ZFonly co-IP western experiment with and without benzonase treatment. Lanes 1 and 7 are protein ladder. Lane 4 is empty. Lanes 2 and 5 are input lysate (50 μg protein measured by BCA). Lanes 3 and 6 are elutions from IP with anti-V5. Upper image shows the blot using a western antibody against the HA tag. A strong band corresponding to the ZFonly-HA construct appears in the IP lanes with and without benzonase treatment, confirming multimerization by this domain. Lower image shows the same blot after stripping and re-probing with anti-V5 antibody. (b) Replication of Chimp-Human competition co-IP western experiment with and without benzonase treatment. Lanes 1–3 are input lysate (50 μg protein measured by BCA) from untransfected cells, and transfected cells without or with benzonase treatment, respectively. Lanes 4–6 are IP elutions from untransfected cells, and transfected cells without or with benzonase treatment, respectively. Lane 7 is a protein ladder. Lower image shows the same blot after stripping and re-probing with anti-V5 antibody. (c) Relative quantification of band intensities for lanes 2–6 from b showing increased pull-down of the human prey relative to the chimp prey by the human bait. (d) co-IP prey band intensity ratios (similar to Figure 5c), though normalized to input band intensity ratios, with and without benzonase treatment. This confirms that the human allele preferentially multimerizes with the human allele over the chimp allele, and that this multimerization is not directly mediated by DNA. (e) 2% agarose DNA gel confirming complete digestion of DNA in transfected cell lysates (2 μg DNA per lane). Lane 1 contains a 1 kb DNA ladder. Lanes 2–3 correspond to cells transfected with ZFonly-ZFonly (used to make blot a) without or with benzonase treatment. Lanes 3–4 correspond to cells transfected with Human-V5 plus Chimp-HA plus Human-HA (used to make blot b) without or with benzonase treatment. Lane 6 contains a 100 bp DNA ladder.