Assessing Self-interaction of Mammalian Nuclear Proteins by Co-immunoprecipitation.

Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins such as transcription factors are spatially distributed into local high-concentration clusters in mammalian cells, suggesting that many nuclear proteins self-interact. These observations have further underscored the need for orthogonal biochemical approaches for testing if self-association occurs, and if so, what the mechanisms are. Here, we describe a CoIP protocol specifically optimized to test self-association of endogenously tagged nuclear proteins (self-CoIP), and to evaluate the role of nucleic acids in such self-interaction. This protocol has proven reliable and robust in our hands, and it can be used to test both homotypic and heterotypic (CoIP) protein-protein interactions.

. Testing protein self-interaction by CoIP. Different strategies to genetically engineer cells and test the self-association of the POI by co-immunoprecipitation. A. A single plasmid encoding the tagged POI is transfected into cells in addition to the wild type protein expressed from the endogenous promoter (1). If the POI self-interacts, the wild type POI will associate both with itself (not shown) and with the exogenously expressed tagged version (2). A CoIP experiment that uses an antibody directed against the tag will also pull-down the wild type POI (3). If the tag is large enough, the tagged POI can be distinguished from the wild type one by size in a Western blot experiment using an antibody against the POI (4). B. Same as in (A) but this time two separate plasmids are transfected, each encoding the POI tagged with different epitopes (TAG1 and TAG2) (1). The immunoprecipitated material is subject to SDS-PAGE followed by Western blotting and self- 4 www.bio-protocol.org/e3526 interaction is assessed with antibodies against each tag (4). C. Our strategy to tag the endogenous POI with 2 different epitopes. A CRISPR/Cas9 targeting construct (for the expression of both Cas9 and the gRNA of choice) is transfected along with two donor plasmids, each containing the tag of choice (TAG1 or TAG2) flanked by genomic sequences homologous to the endogenous POI (LHR, left homology region, and RHR, right homology region) (1). Clones are selected and characterized that contain each endogenous allele tagged with one of the two epitopes (1'). If the POI self-interacts (2), CoIP experiments with antibodies against the TAG1 will also pulldown the TAG2-POI (3). The immunoprecipitated material is subject to SDS-PAGE followed by Western blotting and selfinteraction is assessed using antibodies against each tag (4). Nucleases specific for either RNA  Figure 1C). The dual tagging can be attempted by providing 2 separate homology repair constructs together in parallel with CRISPR/Cas9, but we generally have higher success rate tagging each allele sequentially in a separate experiment, by using heterozygous clones from the first tagging and re-targeting the "wild type" allele (in fact, this is often a pseudo-wt allele, where Cas9 introduced indels at the cut site; this can be exploited to design another set of guide RNAs that will specifically target the untagged allele). Using the dual-tagged alleles we can then probe whether the TF self-interacts by co-immunoprecipitation (self-CoIP), and further investigate whether such self-association is mediated by protein-protein interactions and/or nucleic acids performing the experiment with or without specific nucleases. We provide here detailed instructions to perform the self-CoIP, while you can refer to our previous publications for the endogenous dual tagging strategy Hansen et al., 2019). This method cannot distinguish between a direct and an indirect self-interaction, and because it requires cell lysis and long incubation times, it cannot accurately estimate the amount of self-association happening in live cells, but rather give lower bounds. Nevertheless, endogenous tagging avoids artifacts originating from overexpression (Hansen et al., 2017;Shao et al., 2018), and, provided that all the deployed antibodies are specific (see Note 1), the self-CoIP method detailed here only requires standard lab equipment and reagents, and it has been working robustly and reproducibly in our hands with several TFs. While this protocol reliably detects self-association of nuclear proteins, we routinely use it to also test heterotypic protein-protein interaction in a standard CoIP setting, both in the nucleus and in the cytoplasm (see Note 2). 5 www.bio-protocol.org/e3526  2. Resuspend each pellet in 700 μl of the freshly prepared cell lysis buffer using a P1000 pipet.
3. Transfer the lysate to low-retention 1.5-ml tubes and rock at 4 °C for 8 min to allow lysis (e.g., using a rotator in the cold room). 4. Spin in a refrigerated microfuge at 4 °C and 3,000 x g for 3 min. 5. Optional: keep the supernatant as the "cytoplasmic fraction".
Note: This protocol is specifically optimized for nuclear proteins, but you can also perform the self-CoIP on the cytoplasmic fraction.
6. Remove the supernatant with a P1000 first, and then with a P200, without disturbing the nuclear pellet. 11. Measure the samples' volume while transferring them back to a 1.5-ml low retention tube.
12. Bring the volume to 2 ml with 0.1 M CoIP buffer plus protease inhibitors. 13. Equally divide the sample to two tubes, 1 ml to be left untreated and 1 ml to treat with Benzonase (1 μl per ml of lysate).
Note: Benzonase digests both DNA and RNA, and thus will tell you whether the self-interaction is mostly mediated by protein-protein interactions or it requires nucleic acids. If the selfassociation is dependent on nucleic acids, you can test whether RNA or DNA are involved replacing benzonase with either RNaseI or DNaseI (you will need to adjust the digestion buffer for DNaseI).
14. Rock at 4 °C for 3 to 4 h to achieve complete Benzonase digestion. 15. Spin briefly in a refrigerated microfuge at 4 °C to pull liquid down.  Supplemental Table S1 to build a standard curve with the BSA readings and to interpolate the concentration of your samples.

E. Setup your self-CoIP experiment and pre-clear lysates and beads
We are here illustrating a typical self-CoIP experiment to probe the self-association of the cohesin subunit Rad21, and its dependency on nucleic acids. Specifically, we are using nuclear lysates from mouse embryonic stem cells with an endogenously dually tagged Rad21 protein (the B4 clone originally described in Cattoglio et al. (2019). This cell line contains a Rad21-SNAPf-3xFLAG allele and a Rad21-Halo-V5 allele (Figure 2A). We will pulldown the protein expressed from the first allele with a FLAG antibody ( Figure 2B) and check whether the V5-tagged protein produced from the second allele is also immunoprecipitated using a V5 antibody during the Western blot ( Figure 2C), both in untreated lysates and in lysates treated with benzonase to digest nucleic acids. We will also check the total IP efficiency of the experiment blotting against the immunoprecipitated FLAG protein ( Figure 2D).   In this case, you will need 2.2 mg of nuclear lysates per condition (untreated and benzonasetreated).
Note: The normal IgG sample (immunoglobulins purified from pre-immune serum) controls for non-specific pulldown and it must be of the same species of your αFLAG antibody (e.g., mouse anti-FLAG antibody and normal mouse IgG).
2. Dilute the amount of lysates needed for your experiment to 1 mg/ml with 0.2 M CoIP buffer added of protease inhibitors (PMSF, aprotinin, benzamidine and Roche cOmplete inhibitors). If your volume exceeds 1.5 ml, move the diluted lysates to round-bottom tubes (Figure 3a).
In our illustrative experiment:

Condition
Concentration (  3. Preclear lysates with beads to reduce non-specific binding (note that these beads will be discarded at the end of the preclearing) (Figure 3b) ii. For each condition, pipet 20 μl of Dynabeads per mg of lysate (e.g., 44 μl for 2.2 mg) to a low-retention 1.5 ml tube.
iv. Insert the tube in the magnet separation rack and wait for 5 min for the solution to clear.
v. Aspirate the CoIP buffer with a pipet, remove the tube from the magnet and repeat the wash a second time (Steps E3a-iii to E3a-v).
b. Remove the tube from the magnet and use the diluted lysate to resuspend the beads and transfer them to the round-bottom tubes with the rest of your nuclear proteins.
c. Rock the lysates with the beads for at least 2 h at 4 °C. 4. Preclear beads with BSA (note that you will use these beads to perform the actual pulldown experiment) (Figures 3e-3f) a. Wash protein A or G Dynabeads; you will prepare a single pool of Dynabeads to be used with all your samples.

Note: You will not pipet Dynabeads in your input samples, but we include inputs when
preclearing beads to accommodate pipetting errors later on. c. Rock the beads with BSA at 4 °C overnight.
Note: For convenience, we preclear beads with BSA overnight while incubating the lysates with antibodies, but a couple of hours are also sufficient.
1. Briefly spin down the precleared lysates at 4 °C to remove liquid drops from the tubes' lids. 5. Prepare as many 1.5-ml low-retention tubes as needed for the IP (e.g., for our 6 samples). 6. Distribute the precleared lysates to the new tubes. In our illustrative experiments this will correspond to: 16 www.bio-protocol.org/e3526

Note: We obtain best results when incubating the antibody overnight, but you can test your
antibody and try to shorten the time.
1. Retrieve the samples and the BSA-precleared beads and briefly spin them down at 4 °C to remove liquid drops from the tubes' lids.
2. Place the tube with precleared beads in the magnetic separator rack. 9. After the last wash, also remove any residual buffer with a P20. (Figures 3k-3l).

I. Elute immunoprecipitated material and prepare inputs and samples for SDS-PAGE
1. Remove tubes from the magnet and put them back on ice.
Note: The elution volume depends on how many samples you have. We are here illustrating an experiment that uses a 10-well acrylamide gel, which can easily accommodate 17 μl. We also use 12-or 15-well gels, and adjust the elution volumes accordingly or load less material.
3. Vortex to resuspend.   e. Mix well pipetting up and down and finally add APS and TEMED.
f. Prompty mix and pipet into the gel cassettes, leaving enough space for the stacking gel.
g. Immediately layer 70% ethanol on top of the gel, using a squirt bottle. This will make your gel front neat and straight.
h. Wait for polymerization to complete.
Note: When solidified, the gel front will appear as a neat line easily distinguishable from the ethanol layered above.
i. Aspirate the ethanol and wash the gel extensively with ddH2O using a squirt bottle.
k. In a new 15 ml conical tube, start preparing the stacking gel, adding reagents in the order specified in Table 1 (Bis-Tris, acrylamide and water). n. Slowly place the selected combs (in our case 10-well combs), making sure no bubbles form below the wells. 19 www.bio-protocol.org/e3526  ii. Take a tray that will fit the gel holder cassettes.
iii. Add a gel holder cassette, black side facing down, and pour enough transfer buffer to cover it. iv. Add one sponge, and make sure it gets fully covered in transfer buffer.
v. Pre-wet in transfer buffer 2 pieces of blotting paper, cut only slightly larger than the gel to transfer.  c. Pipet the ECL reagent to fully cover the membrane. d. Incubate for 2 min gently swirling the container, still making sure that the membrane is fully covered.
e. Discard the ECL reagent and image your Western blot.
Note: You can either use X-ray films (our preferred choice) or chemiluminescent imaging instruments (e.g., Chemidoc).

Notes
1. It is of primary importance that you test the antibodies used to immunoprecipitate and/or detect each tag to exclude cross-reactivity with the other tag and also with the POI (using untagged, wild type cells). For example, while probing self-interaction between a V5-tagged and a FLAGtagged Rad21 protein, we found that one of the FLAG antibodies did detect a band of the same size of Rad21 in untagged cells, suggesting a possible cross-reactivity with the wild type Rad21