The 855 bp region of the +3 kb enhancer and +11 kb intronic region were amplified with PCR using HotFirePol polymerase (Solis Biodyne, Tartu, Estonia) and primers listed in Supplementary file 1, with the reverse primers having a 5' biotin modification (Microsynth, Balgach, Switzerland). PCR products were purified using DNA Clean and Concentrator−100 kit (Zymo Research, Irvine, CA) using a 1:5 ratio of PCR solution and DNA binding buffer. The concentration of the DNA was determined with Nanodrop 2000 spectrophotometer (Thermo Scientific).

The preparation of nuclear lysates was performed as follows. Cortices from 8-day-old Sprague Dawley rat pups of both sexes were dissected and snap-frozen in liquid nitrogen. Nuclear lysates were prepared with high salt extraction as in Wu, 2006 and Lahiri and Ge, 2000 with minor modifications. Briefly, cortices were weighed and transferred to pre-cooled Dounce tissue grinder (Wheaton). Also, 2 ml of ice-cold cytoplasmic lysis buffer (10 mM HEPES, pH 7.9 [adjusted with NaOH], 10 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, 300 mM sucrose, 1x cOmplete Protease Inhibitor Cocktail [Roche, Basel, Switzerland], and phosphatase inhibitors as follows: 5 mM NaF [Fisher Chemical, Pittsburgh, PA], 1 mM beta-glycerophosphate [Acros Organics, Pittsburgh, PA], 1 mM Na3VO4 [ChemCruz, Dallas,TX], and 1 mM Na4P2O7 [Fisher Chemical, Pittsburgh, PA]) was added, and tissue was homogenized 10 times with tight pestle. Next, the lysate was transferred to a 15 ml tube and cytoplasmic lysis buffer was added to a total volume of 1 ml per 0.1 g of tissue. The lysate was incubated on ice for 10 min with occasional inverting. Next, the lysate was transferred to a 100 µm nylon cell strainer (VWR) to remove tissue debris and the flow-through was centrifuged at 2600 × g at 4°C for 1 min to pellet nuclei. The supernatant (cytoplasmic fraction) was discarded and the nuclear pellet was resuspended in 1 ml per 1 g of tissue ice-cold nuclear lysis buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 2.5% glycerol, 1x cOmplete Protease Inhibitor Cocktail [Roche, Basel, Switzerland], and phosphatase inhibitors) and transferred to a new Eppendorf tube. To extract nuclear proteins, the pellet was rotated at 4°C for 30 min and finally centrifuged at 11,000 × g at 4°C for 10 min. The supernatant was collected as nuclear fraction, and protein concentration was measured with BCA Protein Assay Kit (Pierce).

In vitro DNA pulldown was performed as follows. Two biological replicates were performed using nuclear lysates of cortices from pups of different litters. Pierce Streptavidin Magnetic Beads (50 µl per pulldown reaction) were washed 2 times with 1× binding buffer (BB, 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), resuspended in 2× BB, and an equal volume of 50 pmol biotinylated DNA (in 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA) was added and incubated at room temperature for 30 min with rotation. To remove the unbound probe, the beads were washed three times with 1× BB. Finally, 400 µg of nuclear proteins (adjusted to a concentration of 1.6 mg/ml with nuclear lysis buffer) and an equal volume of buffer D (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 8% glycerol, 1x cOmplete Protease Inhibitor Cocktail [Roche, Basel, Switzerland], and phosphatase inhibitors) were added and incubated with rotation at 4°C overnight. The next day the beads were washed three times with 1× PBS, once with 100 mM NaCl and once with 200 mM NaCl. Bound DNA and proteins were eluted with 16 mM biotin (Sigma-Aldrich, Saint Louis, MO) in water (at pH 7.0) at 80°C for 5 min, and the eluate was transferred to a new tube and snap-frozen in liquid nitrogen.

Mass-spectrometric analysis of the eluates was performed with nano-LC-MS/MS using Q Exactive Plus (Thermo Scientific) at Proteomics core facility at the University of Tartu, Estonia, as described previously (Mutso et al., 2018) using label-free quantification instead of SILAC and Rattus norvegicus reference proteome for analysis. The full lists of proteins obtained from mass-spectrometric analysis are shown in Supplementary file 3. Custom R script (available at Tuvikene, 2021) was used to keep only transcription factors based on gene symbols of mammalian genes from gene ontology categories ‘RNA polymerase II cis-regulatory region sequence-specific DNA binding’ and ‘DNA-binding transcription factor activity’ from http://geneontology.org/ (obtained March 16, 2020). At least 1.45-fold enrichment to the +3 kb enhancer probe compared to the +11 kb intronic probe was used as a cutoff for specific binding. The obtained lists were manually curated to generate Venn diagram illustration of the experiment.

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