Fig. 6: ANKRD1 forms a complex with AP-1.

a Motif analysis of ANKRD1 ChIP-seq, assessed and quantified using MEME and DREME software (https://meme-suite.org/meme/tools/dreme). Values are expressed as log10 E-value. Shown are the top three transcription factor families enriched in ANKRD1 peak profile. b Prediction of transcription factors binding using the GIGGLE score. GIGGLE represents the similarity between user-defined peak profile with deposited ChIP-seq profiles in the Cistrome DB toolkit (http://dbtoolkit.cistrome.org/). GIGGLE scores for top-ranking AP1 family members on ANKRD1-bound CAF genes are represented with box plots, showing the interquartile range, and the center line representing the median value; the minimum and maximum values delineate the range of data points. Each point represents a separate ChIP-seq dataset. c Predicted 3D structure of ANKRD1-AP1(JUN/FOS)-DNA complex. ANKRD1 3D structure was predicted using Alphafold (https://alphafold.ebi.ac.uk/), the partial crystal structure of JUN/FOS/DNA complex was available at PDB protein databank (https://www.rcsb.org/structure/1FOS⁵⁵. HADDOCK software (https://wenmr.science.uu.nl/haddock2.4/) was used to dock the two structures. Shown are the clusters with the lowest HADDOCK score. d Van der Waals energy and Electrostatics energy scores for the top eight protein clusters derived from HADDOCK docking of the ANKRD1-AP1(JUN/FOS)-DNA complex. Represented with box plots, showing the interquartile range, and the center line representing the median value; the minimum and maximum values delineate the range of data points. e Schematic view of ANKRD1-JUN predicted interacting residues. The predicted ANKRD1-AP1(JUN/FOS)-DNA complex was used in 3DBionote (https://3dbionotes.cnb.csic.es/ws) for predicting the interacting residues between ANKRD1 and JUN protein. ANKRD1 is predicted to interact with JUN through the DNA-binding domain (DBD) and Leucine zipper domain (bZip) of JUN. f Glutathione-conjugated beads were used to immunoprecipitate GST-tagged ANKRD1 (100 ng) recombinant protein mixed with the following recombinant proteins, DNA or AP1 inhibitor: Heat-denatured JUN (100 ng), native JUN (100 ng), HIS-tagged FOS (100 ng), DNA oligo enriched with AP1 consensus motif (50 ng), or T-5224 (20 µM). Western blot analysis for ANKRD1, JUN, and FOS. The experiment was repeated once. g In vitro protein interactions. Glutathione-conjugated beads were used to immunoprecipitate GST-tagged ANKRD1 (100 ng) recombinant protein mixed with the following recombinant proteins: HIS-tagged JUN (truncated form 1-241aa, 100 ng), full-length JUN (100 ng), or HIS-tagged FOS (100 ng). Western blot analysis for ANKRD1, JUN, and FOS. All Co-IP proteins were run in the same nitrocellulose membrane. Similarly, all the inputs (1%) were blotted on the same membrane (also for 6f). Experiment was repeated once.