5. Results
5.6 DNA-protein binding studies
5.6.2 EMSA
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Figure 34 a fluorescence picture of gels 1-4 (left to right) eYFP-mPRDM9cstΔZnF0 (expression #2) incubated at different temperatures for binding reaction b EMSA result. Gel 1 and 2: 20s exposure; Gel 3&4:30s exposure.
Looking at the fluorescence pictures in Figure 34a, clear differences are visible between the different buffer conditions. The samples dissolved in TKZN (gel 1 and 3) have more protein sticking in the slot than the samples in Patel300 (gel 2 and 4). Sarcosyl seems to improve the solubility of the protein further as less fluorescence signal is sticking in the slots for the sample with a final sarcosyl concentration of 0.15% (gel 3 and gel 4). In gel 1, the samples show a double shift and smear can be observed where the protein runs into the gel. The sample which is not heated up shows mostly a high shift and seems to bind best to the DNA as the band at height of free DNA is almost disappeared. The more it is heated up, the less protein binds, and the less double shift can be seen. In gel 2 (Patel without sarcosyl), no binding at all is visible even though less fluorescence is sticking in the slots compared to gel 1 (TKZN without sarcosyl). The free DNA bands have a similar intensity as the DNA only band meaning no protein has bound. In the samples in gel 3 (TKZN + sarcosyl), all DNA is bound and shifted to a high band where smaller shifts and some free DNA is visible for the same parameters except the added sarcosyl in gel 1. No difference is visible for the different temperatures during the binding in gel 3. Given the fact that the binding reactions
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were heated up, it indicates that sarcosyl might stabilize the DNA-protein binding. In gel 4 (Patel + sarcosyl), there are slight shifts visible for all samples. The higher the temperature, the weaker the bands get. Again, it looks like the Patel300 buffer is not promoting a binding as the same samples showed a much stronger binding with the TKZN buffer.
Looking at these results, it seems as if sarcosyl promotes the DNA-protein binding, possibly by enhancing the protein solubility and/or stabilizing the complex. TKZN seems to be a more appropriate binding buffer than Patel300. Looking at the buffer ingredients, the TKZN buffer has a lower total salt concentration (50mM KCl vs 300mM NaCl) but twice the amount of ZnCl2, which is thought to stabilize the ZnF of PRDM9. Additionally, Patel300 buffer contains glycerol and TCEP while TKZN only uses a very small amount of NP-40 to stabilize the protein in solution.
Next, a sarcosyl titration was performed with human PRDM9 samples to see if more sarcosyl enables a binding. Total sarcosyl concentrations of 0%, 0.05%, 0.15% and 0.2% were used in TKZN as a binding buffer. 100fmol Hlx1-75bp and 1µg polydIdC were used together with 1 µL protein. The binding reaction took place over night at 4°C.
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Figure 35 a fluorescence picture of sarcosyl titration with hFcIgG1-eYFP-hPRDM9A ΔZnF0 (expression #52 SN* in TKZN +0.3% sarcosyl in BL21-AI, 16°C) and eYFP- hPRDM9A ΔZnF0 (expression #2 SN* in TKZN +0.3% sarcosyl). mouse positive: YFP_mPRDM9cst-ZnF (expression #2 in TKZN +0.3% sarcosyl). 2s exposure. b EMSA result of sarcosyl titration. 5s exposure. c height profile of EMSA result. 5s exposure.
The different sarcosyl concentrations slightly impact the solubility of the proteins which can be observed in Figure 35a, the fluorescence picture. The more sarcosyl is added, the less fluorescence signal is visible in the gel slots. Looking at Figure 35b, there is no shift visible for the human samples but the unbound DNA fraction gets weaker the more sarcosyl is added in the reaction. In Figure 35c, the height profiles of the pixel intensities prove that an increased sarcosyl concentration leads to a decrease of free DNA. Given the fact that less protein seems to stick in the gel slots at an increased sarcosyl concentration, sarcosyl indeed seems to enhance the solubility of the protein and dissolution of huge conglomerates.
To optimize the protein concentration and incubation times used, a protein titration with 1, 2, 5 and 10 µL protein was performed at different incubation times of 1, 10 and 20 min. As reaction buffer, 1x TKZN without sarcosyl added was used together with 100fmol Hlx1-75bp DNA and 1µg polydIdC.
Figure 36 a EMSA result of protein titration and incubation time variation with hFcIgG1-eYFP-hPRDM9AΔZnF (expression
#9). 10s exposure time. b intensity 3D height profile of EMSA
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The intensity profile indicates that there is no difference between the incubation time variations.
However, the protein titration clearly shows that the more protein is added, the less unbound DNA is visible. There seems to be a binding, which is either not detectable or a huge complex is formed which doesn’t run into the gel.
Test of different membranes and ECL solutions
As binding was not detectable, EMSA-Membranes and ECL solutions of different companies were compared to investigate their sensitivity: membranes from Biorad and from GE Healthcare were tested as well as ECL solutions from Thermo Fisher and GE Healthcare. They were handled using the completely same protocol and applying the same samples. As protein, 1 µL hFcIgG1-YFP-h PRDM9AΔZnF 16°C expression, SN* (expression #3) was used for binding 100, 500, 1000 or 2000fmol of Hlx1-75bp DNA or Baudat-106bp DNA. The reaction took place in 1xTKZN buffer containing 1µg polydIdC but no sarcosyl.
Figure 37: membrane and ECL test: left to right: Biorad vs GE Healthcare membrane; up to down: Thermo Fisher vs GE Healthcare ECL
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Comparing the two different membranes, there are signals visible at the GE membrane where no signals are present at the Biorad Membrane. The height of shifts should be different for DNAs of a different size but is the same for the Hlx1 and Baudat fragment, which indicates an unspecific signal. Nevertheless, also the free DNA signal is considerably stronger for the same DNA amount loaded for the GE Healthcare membrane. When the two different ECL solutions are compared, one can see that the GE Healthcare ECL select produces a stronger signal than the Thermo Fisher ECL, which leads to a more sensitive result. The GE products will be used for the following EMSAs.
Adjusted protein and DNA amounts
According to the DeNovix concentration measurement of eYFP-labeled PRDM9 via UV-vis absorption, the same amount of protein, 3pmol, was tried to be used for binding reactions. No more than 10µl could be used in the binding reaction.
Table 20: volume for ~3pmol protein
sample conc. [µM] µl for ~3pmol
#9 hFcIgG1-eYFP-hPRDM9A ΔZnF 2.553 1.2
#2 eYFP-hPRDM9A ΔZnF 1.006 2.98
#3 16°C hFcIgG1-eYFP- hPRDM9A ΔZnF lysate prep. protocol 3
0.429 6.99
#3 16°C hFcIgG1-eYFP- hPRDM9A ΔZnF lysate prep. protocol 4
0.292 10
#3 RT hFcIgG1-eYFP- hPRDM9A ΔZnF lysate prep. protocol 3
0.274 10
#2 YFP- mPRDM9cst-ZnF 0.961 3.12
The DNA concentration was decreased significantly to only 4fmol (compared to 100fmol for the previous tests) of Hlx1-273bp or Baudat-106bp. 1µg polydIdC was added to each binding reaction which took place in 1xTKZN buffer without sarcosyl for 20min. For each protein, incubation with both DNAs and a protein only was applied.
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Figure 38 equal protein amount for all samples: hFcIgG1-eYFP-hPRDM9AΔZnF (expression #50 and #52 16°C old (lysate prep. 3)/ new (lysate prep 4) /RT), eYFP-hPRDM9AΔZnF (expression #51) and YFP- mPRDM9cst-Exon10 (expression
#51); negative control: eYFP (expression #56). 10s exposure
In lane 1-2, expression #9 hFcIgG1-eYFP-hPRDM9AΔZnF doesn’t show a shift and also no weaker unbound DNA bands compared to the DNA only in lane 4. No binding has occurred and a slight unspecific signal is visible in all lanes containing this protein lysate, also the protein only lane (lane 3). In lane 5-7, expression #2 eYFP-hPRDM9AΔZnF shows a clear shift for the Hlx1 binding as well as some smear for the Baudat binding. The free DNA signal gets significantly weaker for both DNA fragments compared to the DNA only bands. Only a very weak unspecific signal is visible in all three lanes containing this lysate. Expression #3 16°C hFcIgG1-eYFP-hPRDM9AΔZnF, which was lysed according to protocol 3, also shows a binding to both DNA fragments with less free DNA but it is even smearier for the Hlx1 fragment binding. The unspecific signal is also higher than the previous described. The same Expression #3 16°C hFcIgG1-eYFP-hPRDM9AΔZnF with lysate preparation according to protocol 4 shows a very strong unspecific signal for all three lanes (lanes 11-13). The free DNA for both Hlx1 and Baudat fragments is almost disappeared so a binding occurred. A high shift is visible in the Hlx1 lane. The unspecific binding could derive from the lysate preparation:
sonication, which is thought to sheer (bacterial) DNA fragments, was not used in this protocol.
Expression #3 RT hFcIgG1-eYFP-hPRDM9AΔZnF shows a smeary band for the Hlx1 lane (lane 14) which shift is lower than the other Hlx1 shifts. There is also less free DNA visible of the Baudat
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fragment but no shifted band is visible. An unspecific signal is visible for all three lanes (lanes 14-16). For the mouse positive control, there is no free Hlx1 DNA visible but a shift at very high molecular weight. The mouse protein only is empty. The negative control, eYFP, doesn’t show a binding to Hlx1 DNA.
In this EMSA, higher protein volumes were added (3-10 µL to achieve 3pmol, see Table 20). With a higher volume, the amount of unspecific protein increases as well and unspecific bindings can occur resulting in a smear.
Test of polydIdC and sarcosyl concentrations
To prevent the smearing probably deriving from unspecific binding, different polydIdC and sarcosyl concentrations were tested, as well as a protein titration. The different conditions used are listed in Table 21 below; the results can be seen in Figure 39.
Table 21: different conditions used for polydIdC and sarcosyl tests
Figure 39 a b
protein extract (SN*) 3pmol
#2:eYFP-hPRDM9AΔZnF
#3 16°C expr., lysate prep 3: hFcIgG1-eYFP-hPRDM9AΔZnF
#3 RT expr.: hFcIgG1-eYFP-hPRDM9AΔZnF
400nM – 120nM
#2:eYFP-hPRDM9AΔZnF
DNA Hlx1-273bp, 4fmol Fragment 1, 1fmol
(donor 1034, 9A)
polydIdC 1µg / 2µg / 4µg 0.3µg per 3µL protein / 0 sarcosyl no added sarcosyl const. 0.15% / const.
0.3% / no added sarcosyl
For the first time, a human HSII fragment (fragment 1) is used for binding in Figure 39b.
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Figure 39 different EMSAs to test polydIdC and sarcosyl conditions. a test of higher polydIdC concentrations and different protein extracts. 1s exposure. b test sarcosyl concentrations: constant vs titration. 25s exposure.
In Figure 39a, a weaker, more smeary binding can be observed for all protein extracts the more polydIdC is added. The clearest band is derived by adding just 1µg polydIdC. More polydIdC lowers the shift. The different protein extracts give a different strong binding signal under the same conditions. Expression #2 shows the strongest signal directly followed by expression #3 at room temperature, which gives a strong unspecific signal though. Expression #3 16°C lysate prep. 3 doesn’t give a clear band for any of the three polydIdC conditions but a smear and as well an unspecific signal. Figure 39 b shows different sarcosyl concentrations at different protein amounts each with and without 0.1µg/µl polydIdC. The DNA only shows 2 bands (lanes 10 and 16) of which one is overlayed with the unspecific protein signal in other lanes. In lanes 1-10, sarcosyl is hold constant at 0.15% which shows no difference in binding strength even though the protein amount is decreasing using a 1.5x dilution from 402 nM to 120 nM which could mean all DNA is bound even at
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the lowest protein concentration. A constant sarcosyl concentration of 0.3% also doesn’t show a difference in signal strength between 270 nM (lanes 11 and 12) and 120 nM (lanes 13 and 14).
Binding reactions without added sarcosyl (lanes 17 – 20) give a detectable signal as well. All binding reactions without added polydIdC give a more distinct band. The 0.3% sarcosyl concentration samples show the most distinct bands. A strong unspecific signal is visible in all protein-containing lanes, which could probably be avoided by using a shorter exposure time (here it was 25s). Therefore, more DNA would have to be added.
Protein binding titrations
The final experiment series was to detect and compare the binding strength of hPRDM9A ΔZnF to a real human Hotspot (HSII in Arbeithuber et al. 2015, see 2.3) which was divided into five fragments of ~300bp. Therefore, a protein titration of a 1.5x dilution starting with 402.4nM protein (expression
#2: YFP- hPRDM9A ΔZnF0, SN* in TKZN + 0.3% sarcosyl; estimated concentration: 1.01 µM) with a constant DNA amount of 4fmol was performed for each fragment to be able to compare them. A DNA only sample served as a comparison. As binding buffer, 1xTKZN was used with a constant final sarcosyl concentration of 0.3% and no added polydIdC. Three individual experiments with each of the five fragments were performed to be able to state a measurement inaccuracy. One of the three measurements is shown in Figure 40 below.
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Figure 40: EMSAs of Fragments 1-5 (F1 – F5). Exposure times: Fragment1 1.5s; Fragment2 3s; Fragment3 1.8s;
Fragment4 1.3s; Fragment5 0.7s.
DNA-protein complex formation is visible in the shifts in Fragments 1-5. With increasing protein concentration, more complex and less free DNA is visible. All fragments, also the ones without a Myers Motif show a binding to PRDM9.
As negative controls, unspecific DNAs were also tested in EMSA to exclude the possibility of an unspecific binding. A fragment of the mouse B6 Hotspot Pbx1 of 336bp in size as well as an unspecific human SNP DNA with a length of 75bp were incubated with the same conditions as the human Hotspot fragments (protein titration, 4fmol DNA, 1xTKZN buffer, final sarcosyl concentration 0.3%, no polydIdC).
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Figure 41: EMSA protein titration of eYFP-human PRDM9AΔZnF (expression #2) with unspecific DNA. Left: Pbx1 hotspot, 5s exposure. Right: unspecific SNP DNA, 1s exposure.
The short unspecific DNA fragment doesn’t show any binding to PRDM9 while the longer mouse Hotspot fragment Pbx1 shows a complex formation. The Pbx1 Hotspot contains a Myers Motif with 2 Mismatches in the end which is probably the reason for a binding. Otherwise, unspecific binding could have been detected which would question all bindings of human PRDM9 to DNA fragments.
The fraction bound of the EMSAs with the 5 Hotspot fragments was calculated from the quantitative readout of the pixel intensities of the shifted band compared to the free DNA (see Formula 1). The fraction bound was blotted against the hPRDM9AΔZnF concentration in a semi-logarithmic graph.
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Figure 42: fraction bound over protein concentration for fragments 1 – 5. experiments 1, 2 and 3 and mean (green) with standard deviation
The data points have quite a high standard deviation for some of the fragments.
To compare them, the means for all fragments were added into one plot (Figure 43).
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Figure 43: fraction bound over protein concentration for the means of all 5 Hotspot fragments. Standard deviations not shown for better clarity.
The data points for the 5 fragments don’t show much of a difference. A fit could be applied to the data resulting in the equilibrium dissociation constant (KD) but the experiments performed are not accurate enough for a reliable statement due to the high standard deviation (formula for fit according to Striedner et al. 2017). More data with higher accuracy would be needed and the same exposure time should be used for each experiment. In the simplest binding model in which PRDM9 acts as a monomer, an estimated 100nM protein are needed according to the plot to achieve 50%
fraction bound indicating a KD of roughly 100nM. Fragment 5 seems to have a slightly higher KD than the other fragments according to the plot (Figure 43)
5.5.3 Conclusion of human HSII fragment binding
As expected, the purified protein does not show any binding and is probably degraded (see section 5.4)
The binding buffer variations showed that in TKZN, a clear shifted band for mouse PRDM9 can be achieved in contrast to Patel300. This effect is enhanced when Sarcosyl is added; maybe by keeping the complex in solution as less fluorescence signal iss detected in the gel slots. Binding at room temperature shows a better result than at higher temperatures (see Figure 34). When performing binding reactions with human PRDM9, less free DNA is detected when more Sarcosyl is added to the binding reaction as a Sarcosyl titration from 0 – 2% final concentration shows, even though no shifted band consisting of a protein-DNA complex could be detected (see Figure 35). The incubation time does not seem to have an effect on binding; 1min incubation time shows a similar
10 100
0,0 0,2 0,4 0,6 0,8 1,0
F1 F2 F3 F4 F5
% Fraction bound
protein concentration [nM]
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signal to 20min binding A protein titration with rising human PRDM9 concentrations shows that more protein in the binding reaction results in less free DNA even though the binding complex is not visible (see Figure 36). It iss probably not in solution but sticking in the gel slots or can’t be detected with the membrane and ECL solution used (see Figure 35 and Figure 36). Therefore, ECL solution and EMSA membrane of different brands are compared: indeed, ECL solution and membrane manufactured by GE Healthcare are more sensitive (see Figure 37). Unfortunately, an unspecific signal deriving from protein without any bound DNA is detected as well. Finally, with adjusted protein and DNA amounts (3pmol protein and 4fmol DNA), a shifted band for various human PRDM9 samples becomes visible. The unspecific signal is still present for all samples and remains visible for all further experiments (see Figure 38). A test with different Sarcosyl concentrations and with or without Poly dIdC shows the clearest band without added Poly dIdC and with 0.3% final Sarcosyl concentration (see Figure 39). In the end, the 5 fragments of human HS DNA are used for affinity studies. The more protein is added, the less free DNA is visible and the better and higher a shifted band is visible.
Surprisingly, a binding is detected for all 5 fragments which rises with increased protein concentration, even though not all of them contain the Myer's motif (see Figure 40). The percentile fraction bound over protein concentration for all fragments was applied to a plot in which the data points do not differ significantly in their trends (see Figure 42). Applying a fit resulting in the KD is not performed due to the high standard deviations, which also derived from the difficulty of determining a clear shifted band. A fragment of similar length supposed to serve as unspecific negative control also shows a binding but contains a Myers motif with two mismatches which could explain the binding – nevertheless, an unspecific binding cannot be excluded as no "real" negative control of appropriate size (~300bp) is available (see Figure 41). The shorter negative control of 75bp length does not show a binding but Striedner et al. showed that PRDM9 binding increases with DNA length so a comparable negative control should have the same length as the HS fragments (Striedner et al. 2017).
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