Optimization of cell lysis for ChIP assay

To optimize plasma membrane lysis and improve overall yield of crosslinked DNA-p53 material, an optional sonification step can be included after douncing (Prokesch et al., 2016). Since excessive sonication can lead to an interference of protein binding to DNA, the effectiveness of an additional sonification step was tested Therefore, four

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protocols were performed without and with five sonication cycles at two different times during processing, respectively, to investigate whether an effect on the overall yield could be observed. Sonication to lyse the plasma membrane and sonication to fragment DNA were analyzed by agarose gel electrophoresis in the following combination (Figure 6): Un-sonicated for lysis and fragmentation; un-sonicated for lysis and sonicated for fragmentation; sonicated for lysis and un-sonicated for fragmentation; sonicated for lysis and fragmentation. Five cycles of sonication were used for lysis and/or DNA - fragmentation. No significant increase in yield could be observed in the amount of isolated DNA (L-/F+: 58ng/µl and L+/F+: 70ng/µl) and the fragment size was not reduced as seen in Figure 6.

Figure 6. Lysis efficiency was analyzed according to fragment length by running the DNA on a 1% agarose gel. Five cycles of sonication were used after douncing crosslinked iBACs (L+) to enhance plasma membrane lysis and/or after nuclei lysis to fragment DNA material (F+). By omitting sonication cycles (“L-“ or “F-“) different protocols were used to determine lysis efficiency.

23 3.2. Adjusting the ChIP protocol

Next, ChIP-qPCR was used to amplify chromatin derived from immunoprecipitation with p53 antibody. P21, a well described target gene of p53 (Benson et al., 2013), has been used as control locus. The locus for miR92a was our locus of interest, as we previously identified a potential interaction of p53 with miR92a. Primer pairs for negative controls have been designed at loci that are several kbs distant of p53 binding sides.

Using Eppendorf tubes with the highest number of sonification cycles (shown in Figure 5A) for DNA fragmentation showed no recognizable difference in enrichment (Figure 7). This led to the assumption that insufficient DNA fragmentation results in precipitation of too long DNA fragments, delivering qPCR signals from regions far distant from the locus of interest, including negative control loci. This is shown in Figure 7, where miR92a locus and p21 target regions (positive control) showed no increased enrichment over negative control.

Since p53 signalling has been shown to be increased upon starvation, in an effort to increase binding efficiency, iBACs have been starved for 24h in HBSS and HEPES before crosslinked material was harvested. Additionally, bioruptor tubes have been used for sonicating the DNA-protein complexes, as they have been validated to improve fragmentation of the DNA.

Figure 7. ChIP-qPCR precipitated with 3.54µg antibody and 20µl magnetic tubes. Eppendorf tubes were used for DNA fragmentation. After harvesting mature iBACs, cultivated in maintenance media, 40 cycles of sonication were used for DNA fragmentation.

miR92a IgG p21 IgG 0.0

0.5 1.0 1.5

ChIP-qPCR

%Input over neg. ctrl

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Figure 8. qPCR of starved iBACs. Cells were starved for 24hrs in HBSS with 10mM Hepes (stv) while control sample remained in maintenance medium before harvest. 2.5µg of antibody combined with 20µl magnetic beads were used for precipitation after DNA fragmentation by 10 cycles of sonication.

While a slight enrichment over the negative control region was measured, comparing control with starved iBACs, ChIP-qPCR signal indicated no remarkable difference in enrichment of the p21 and miR92a loci (Figure 8).

As an alternative method, we used idasanutlin, a frequently used pharmacological agent that leads to stabilization of p53 at the protein level, resulting in activation of p53 target genes such as p21. In line, idasanutlin-treated samples showed increased pulldown of p21 and miR92a loci, when precipitated with 1.25µg antibodies (not shown). However, negative controls also increased noticeably, resulting in an overall reduced "%Input over negative control" enrichment compared to control sample (Figure 9).

Figure 9. qPCR of nutlin treated iBACs precipitated with 1.25µg

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To test whether increasing the amount of antibody could increase the overall specific enrichment, we doubled the amount of antibody for pull down. This showed an overall increase in enrichment for the miRNA92 as well as for the p21 region. However, negative control signal proportionally increased as well. This ultimately shows reduced enrichment of idasanutlin treated sample over control sample, using 1.5 µg antibody (Figure 9) and no discernible change in enrichment of target regions miRNA92a and p21 using 2.5 µg antibody (Figure 10), depicted in “%Input over negative control”.

Ctrl Nutlin

0.0 0.5 1.0 1.5

ChIP- qPCR

%Input over neg. ctrl _miR92a

p21 IgG

Figure 10. ChIP-qPCR of chromatin derived from control or nutlin treated iBACs precipitated with 2.5µg antibody. Cells were treated for 24 hours with 5µM idasanutlin (Nutlin) in maintenance medium before harvesting Control sample (Ctrl) was solely cultivated in maintenance medium before harvest. 2.5µg of antibody (D2H9O) combined with 20µl magnetic beads were used for precipitation after 5 cycles of sonication.

Taken together, using pharmacological agents to stabilize p53 or adjusting the antibody concentration did not result in improved binding efficiency. Moreover, overall enrichment was the highest using untreated samples (control) with less antibody (1.25µg) (Figure 10).

Since insufficient amounts of magnetic beads might result in high unspecific binding, different amounts of beads were tested for ChIP. Thus, 100µl (Figure 11A) or 200 µl (Figure 11B) (as suggested by the manufacturer) beads per sample were used for precipitation.

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One hundred µl bead precipitation showed no change in enrichment of the control over idasanutlin treated sample (Figure 11A) in any of the examined loci. Using 200µl beads, miR92a and p21 loci showed high “%Input over negative control” enrichment in control compared to idasanutlin treated sample (Figure 11B). However, the dramatically increased enrichment of target loci pulled down by IgG antibodies suggests that increased unspecific binding is the reason for the increased overall enrichment of the control sample. In contrast, idasanutlin-treated samples showed lower enrichment of "%Input over negative control" pulled down with the p53 antibody, but enrichment of the same loci was drastically lower in comparison when the IgG antibody was used for pulldown. Taken together, while 100µl beads treatment showed no change in “%Input over negative control”, when using 200µl beads idasanutlin-treated samples seem to be lower compared to the control. However, the high enrichment of the control sample could be due to unspecific binding as indicated by high enrichment of loci precipitated with IgG. Since the negative control loci also showed the lowest %Input enrichment when using 200µl beads, further experiments were performed using this parameter (Figure 11B).

Figure 11. Bead analysis of cell precipitated with 100 µl (A) and 200µl (B) beads. After 24 hours incubation of iBACs with indasanutlin (Nutlin), harvested cells were precipitated with magnetic beads combined with 2.5µg D2H9O antibodies. Control sample (Ctrl) was harvested after cultivation in maintenance medium.

Ctrl Nutlin

27 medium and precipitated with 2.5µg antibody (D2H9O)

serving as control (Ctrl) and IgG control (IgG). By changing the wash buffer of the finals wash step from 150mM to 500mM NaCl stringency was increased (high salt). By reducing the wash steps with wash buffer by half (3 times) the stringency was lowered (reduced wash steps).

Another approach to optimize antibody specificity was done by using different numbers of washing steps or increased stringency of the washing buffer. By increasing stringency of the wash process (e. g. increasing wash steps, higher salt concentration in wash buffer) weak, unspecific bindings with antibodies might be reduced, while stronger bindings remain. No recognizable increase in miR92a enrichment could be observed when increasing the stringency of the wash buffer to salt concentrations of 500mM (Figure 12). Decreasing stringency however, increased %Input enrichment at the p21 locus while signals from the negative control and miR92a loci were unchanged.

However, inspection of the negative control signal revealed a drastically reduced p21 locus at reduced stringency, while miR92 remained the same ("reduced wash step" in Figure 12). Furthermore, all the other negative controls used (not shown in this Figure) showed increased enrichment comparable to p21 expression. All in all, lowering stringency seems to increase unspecific binding, while increasing it suggests a slight improvement of unspecific binding events.

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Combining the results of several independent experiments of iBACs cultivated in maintenance media, miR92a locus and positive control loci in the gene promoter of p21 showed significantly increased %Input, when pulling down p53 (Figure 13).

However, other negative controls used in these experiments (not shown) scatter drastically across runs and in some cases even surpass the enrichment values of target and positive control loci. Due to these conflicting results, further adjustment of the ChIP protocol may be required.

3.3. Slc2a5 downregulation by miR92a 3.3.1. Luciferase Assay in iBACs

To examine possible binding between Slc2a5 and miR92a using luciferase assay the 3’UTR of Slc2a5, harbouring predicted miR92a seed sequences, was cloned in a luciferase reporter vector (PsiCHECK2_Slc2a5_UTR_3). To establish an efficient co-transfection of reporter vector and miR92a in iBACs, three conditions with varying amount of the generated vector-construct was electroporated with miR92a mimic or non-targeting sequence control (ntc). Comparison of ntc and miRNA mimics of each condition (1-, 3- and 5µg PsiCheck2 vector product) showed that cells transfected with the highest amount of PsiCHECK2- vector had an approximately 20% reduction of luciferase signal, when transfected with miR92a (Figure 14A). This data indicated that miR92a interaction with Slc2a5 3’UTR could lead to reduced luciferase signal when 5

Figure 13. ChIP- qPCR of Chromatin derived from iBACs cultivated in maintenance medium. After DNA was fragmented upon 10 cycles of sonication, samples were precipitated with 2.5µg antibody (D2H90) and 200µl magnetic beads. Six wash steps were performed with “wash buffer”. Results are depicted in “%Input over negative control”. (n=3). The results shown are the mean ± SD.

IP-p53

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µg of reporter vector is used.

Figure 14. Luciferase Assay of electroporated iBACs. (A) shows the pre-experiment, where the different amounts of PsiCHECK2_Slc2a5_UTR_3 product were transfected, are noted below the graph. All samples were co-transfected with 1µM of miR92a or non-targeting sequence as a negative control. (B) shows 5µg of the PsiCHECK2 vector product, like in (A), repeated with 3 samples each. (C) depicts the same condition as (B), with 1.5µMof miR92a/ntc co-transfected with it.

Therefore, iBACs were electroporated with 5µg of PsiCHECK- vector. Nevertheless, subsequent luciferase assay showed no effect (Figure 14B). Additionally, increasing the amount of co-transfected miRNA mimic 92a from 1µM to 1.5µM (Figure 14C) did not result in reduction of the luciferase signal upon miRNA92a mimic co-transfection.

3.3.2. Optimization of miRNA92a mimic overexpression

To validate sufficient overexpression of miR92a in iBACs transfected with miR92a-mimics in Figure 14B, gene expression of miR92a after electroporation was analyzed.

qPCR revealed increased miR92a expression upon overexpression of miR92a mimic compared to electroporation with non-targeting sequence control.

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miR92a mimic ntc 0

50 100 150 200

miR92a overexpression

Relative experssion level

Figure 15. qPCR of iBACs overexpressed with miR92a. Around 500 000 iBACs were electroporated with 5µg

PsiCHECK2_Scl2a5_UTR_3 vector and 1µM mimic miR92a or non-targeting sequence (ntc). The normalized ratio between miR92a mimic and ntc,expressed is 176,5 to 1, indicating successful transfection of miRNA.

Overall Figure 15 suggests successful overexpression of miR92a.

To test transfection efficiency of electroporated iBACs an EGFP vector instead of the generated PsiCHECK2 construct was transfected. Observation of electroporated cells under bright-field fluorescence microscopy revealed a transfection efficiency of less than 10% (Figure 16).

Taking the results together, low transfection efficiency could obscure binding of miR92a to Slc2a5, leading to inconsistent results.

Figure 16. Transfection efficiency of electroporated iBACs. Around 500 000 mature iBACs were electroporated in a 100µl solution containing 3µg of EGFP_LC3 vector. Under the bright- field fluorescence microscope, the bright spots shown represent the successful transfection in cells.

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To improve transfection efficiency, cationic lipid transfection was done in HEK cells.

Three different conditions were examined according to “lipofectamine3000” protocol, on three different cell densities.

Figure 17. Examination of Transfection efficiency with Lipofectamine3000. 100 000 HEK 293 cells were seeded in the first row (A-C), 60 000 cells in the second (D-F) and 40 000 cells in the third (G-I), before transfection. EGFP_LC3 Plasmid vector was transfected with lipofectamine reagent at following ratios: The first column (A-G) was transfected with 1:1.25 [0.2µg:0.25µl], second column H) 1:2 [0.2µg:0:4] and third column (C-I) with double the amount of the second column (B-H) [0.4µg:0.8µl].

Figure 17 depicts varying cell number and Lipofectamine 3000 ratios. We observed that with fewer cell numbers, transfection showed a trend of improved efficacy. Further improvement can be seen when increasing the DNA:reagent ratio to 1:2. However, by using twice the DNA and Lipofectamine3000 reagent amount as in Figure 17B, E and H, no apparent increase in transfection efficacy was observed. Another important observation under these conditions was the noticeable onset of cell death, as a marked decrease in cells adhering to the surface was observed under the microscope.

Ultimately, confirming the higher the amount Lipofectamine3000 reagent in relation to cell number the higher the toxicity (as mentioned in manufacturers protocol).

Nevertheless, lipofectamine used at the lowest ratio on the highest cell count (10⁵ cells as shown in Figure 17A) showed the worst transfection efficiency with 30-40%. While every transfection of conditions using 40 000 and 60 000 cells, reached at least 70%

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(but in general 90%) efficacy. Subsequently, the condition with the highest transfection efficiency and no recognizable cell death was: 1:1.25 DNA: reagent ratio in 40 000 HEK cells (as shown in Figure 17G, reaching 80-90%).

3.2.5. Luciferase Assay in HEK 293

To validate direct binding of miR92a to Slc2a5 3’UTR, luciferase assay was performed.

We found that the luciferase signal was markedly reduced when miR92a was overexpressed in HEK cells indicating that miR92a binds to Slc2a5 mRNA, leading to the degradation of Slc2a5 mRNA (see Figure below).

ntc miR92a 0.0

0.5 1.0 1.5

0.2µg PsiCHECK2 +1µM miR92a/ntc

RLU (normalized to Renilla) 0.0588

Figure 18. miR92a downregulating Slc2a5. Luciferase assay was performed in HEK 293 cells according to lipofectamine3000 manual. 40 000 cells were transfected with 1µM miR92a or non-targeting control sequence and 0.2µg

PsiCHECK2_Slc2a5_UTR_3 in a ratio 1:1.25 with lipofectamine reagent. n=5. Two-tailed unpaired Student’s t test was performed. The results shown are the mean ± SEM.

3.4. Fructose uptake and catabolism in brown adipocytes

As Slc2a5 is a known fructose transporter (Barone et al., 2009), we next compared iBACs incubated for 24 hours in maintenance medium containing 1.5 or 5 g/L fructose with a control sample without added fructose. Nuclear magnetic resonance (NMR) measurements detected increasing amounts of fructose intracellularly, positively correlated with the amount of supplemented fructose (Figure 19). Similar effects could be observed, when stimulating b-adrenergic signaling with Isoproterenol. No difference in fructose amount with and without isoproterenol (see Figure 19) was observed.

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Figure 19. Intracellular fructose measurement of mature iBACs via Nuclear Magnetic Resonance (NMR) measurement.

iBACs were incubated for 24 hours in Maintenance Media containing 1.5g/L (1.5Fru) and 5g/L fructose(5Fru), whereas no fructose was added to the maintenance medium in the control. With the same of each conditions other samples were treated with 1µM isoproterenol (depicted “…_iso”) for 1 hour before harvesting.

Fructolysis feeds into the glycolysis pathway after aldolase converts fructos-1-phosphate into glyceraldehyde and DHAP (Heinz et al., 1968). Although both usually metabolize to glyceraldehyde-3-phosphate, DHAP has an alternative pathway generating glycerol in order to ultimately produce triglycerides (Heinz et al., 1968).

What is unknown is whether and under which conditions brown adipocytes utilize fructose for thermogenesis. Hence, the next experiment aims to examine fructose metabolization in iBACs.

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3.5. Brown adipocytes might use fructose as substrate for glycolysis

To elucidate how fructose is utilized in BAT, extracellular acidification rate (ECAR) measurement was performed in mature iBACs. Comparing ECAR of iBACs upon acute injection of glucose or fructose, an increase in ECAR is observed with both glucose and fructose albeit to a lesser extent (Figure 20).

Increased acidification of the medium indicates increased glycolysis of cells after substate injection. By going through glycolysis protons are released, resulting in the measured acidification of the media. Since oligomycin injection inhibits ATP synthase, the resulting increase in acidification precludes oxidative phosphorylation while shifting energy production to glycolysis, revealing cellular maximum glycolytic capacity (see Figure 20A). Injection of glucose analog, 2-deoxy-glucose (2-DG), inhibits glycolysis through competitive binding to glucose hexokinase as antagonist. Therefore, the resulting decrease in ECAR after 2-DG injection confirms that the produced ECAR level was due to glycolysis (seen in Figure 20A) (as mentioned in the manufacturers manual).

Although, fructose does not seem to be as efficiently catabolized as glucose, it still suggests that fructose may feed into glycolysis to generate ATP and might ultimately lead to NST (see Figure 20).

Figure 20. Glycolytic stress test of mature iBACs cultivated in maintenance medium and measured via Seahorse XF. (A) Extracellular acidification rate curve after glucose or fructose injection “GLU/FRU”, “Oligomycin” and 2- deoxy-glucose “2-DG”. Increase in ECAR indicating glycolysis is depicted in the bar charts (B) for glucose & (C) for fructose injection. n=3. Two tailed paired t-test was performed on these results. The results shown are the mean ± SEM.

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4. Discussion & Outlook

This thesis aimed to elucidate the role of miR92a and p53 as regulators in BAT activity.

Based on previous findings, fasted mice maintained at mild cold stress showed p53 signaling as top upregulated pathway and miRNA-92a-1-5p as most upregulated miRNA in BAT. This, together with a predicted p53 binding site in the mouse miRNA-92a-1-5p locus, led to the hypothesis that p53 directly activates miR92a transcription.

Further bioinformatics predictions revealed a potential miR92a binding site on the 3’UTR of the mRNA of fructose transporter Slc2a5. Because downregulation of the fructose transporter would result in a decreased amount of intracellular fructose, fructose was hypothesized to be a potential energy source in BAT.

Since the organism needs to conserve energy during nutrient deprivation, fructose reduction could be an important factor in reducing/ switching off NST in BAT, which would be consistent with our hypothesis. Therefore, a ChIP-qPCR assay was performed in order to elucidate a possible interaction between p53 and the miR92a locus. Subsequently, the putative interaction of miR92a with Slc2a5 was investigated using a luciferase assay.

The herein conducted ChIP experiments are insufficient to reliably confirm that p53 directly binds to miR92a locus, since only one of several negative controls used (shown throughout this thesis) for ChIP shows low enrichment for the most part compared to positive control targets, pointing to problems of unspecific binding of the antibodies in this method. Other negative controls used, strongly scatter throughout pulldown experiments and often exceed miRNA- target and positive control loci indicative of unspecific binding. However, using the setups established through this thesis, significant enrichment of the miR92a target locus as well as the positive control locus over one negative control locus could be shown.

However, luciferase assay results suggest an interaction between miR92a and Slc2a5, indicating downregulation of Slc2a5 via miR92a in vivo. Since NMR measurements indicate fructose uptake while ECAR measurements suggests fructose metabolism in brown adipocytes, both results also support the hypothesis that fructose is an energy source in mature brown adipocytes.

4.1. Regulation of miRNA92a by p53: Troubleshooting the ChIP protocol

Since data of our group previously showed increased p53 signaling upon fasting, one of the first adjustments done for pulldown was cultivating iBACs 24 hours with

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starvation medium. It was expected that increased ChIP enrichment of p53 targets would be observed when compared to cells maintained in full medium. However, most experiments done with starvation medium in this thesis showed lower enrichment compared to control.

One explanation might be a different efficiency of the crosslinking process when done in starvation medium compared to maintenance medium, resulting in weak binding of proteins on DNA.

Subsequently, we chose treatment with idasanutlin, a known p53 stabilizer, as alternative to increase p53 on protein level. However, results turned out difficult to reproduce as well. No increased enrichment or reduction of unspecific binding could be observed. A possible explanation could be an oscillatory effect in the p53 response, as shown for many other protein responses (Nelson et al., 2004). Therefore, p53 response might already subside when cells are harvested.

Furthermore, results show different levels of “%Input” enrichment throughout experiments with some experiments showing unexpectedly high levels of enrichment (not shown). Lack of reproducibility and high levels of enrichment might hint to problems with the ChIP procedure.

Since p53’s recognition site of D2H9O antibody is C-terminal, the occurring problems with unspecific binding could also be the result of insufficient antibody-epitope

Since p53’s recognition site of D2H9O antibody is C-terminal, the occurring problems with unspecific binding could also be the result of insufficient antibody-epitope

Im Dokument Dissecting a novel p53-mirna axis in brown adipocytes (Seite 22-0)