Identifying membrane-bound transcriptional regulatory proteins from rare but evolutionarily conserved domain combinations
Transcriptional regulatory proteins, including transcription factors (TFs) and chromatin modifiers, perform their crucial cellular functions inside cell nuclei. However, unlike most TFs, membrane-bound transcription factors (MBTFs) are first anchored in membranes before nuclear translocation. Known MBTFs are vital for processes from myelin expression (MYRF) to cholesterol homeostasis (SREBP), yet their overall diversity remains uncharted. We hypothesized that additional membrane-bound transcriptional regulators (MBTRs) might exist and developed a bioinformatics screen that prioritizes membrane proteins likely to regulate transcription. Our approach leverages domain composition, assuming that surprising domain combinations suggest novel biological functions. We searched for rare but evolutionarily conserved combinations of transmembrane domains paired with domains likely involved in transcriptional regulation. Our method rediscovered known MBTFs and membrane-bound histone kinases, and identified novel MBTR candidates, including histone N-acetyltransferases and the prolactin regulatory element-binding protein PREB. Experimental validation indicates that PREB may undergo cleavage. Without its transmembrane domain, PREB exhibits nuclear localization and chromatin association in mouse embryonic stem cells. Furthermore, we identified a putative class of new MBTFs that share MYRF's DNA-binding domain. These findings underscore the utility of our method and provide a framework for investigating MBTRs that likely facilitate the integration of extracellular signals with transcriptional responses. Overall design: bioChIP-seq was performed on PREB- and PREB?TM-expressing ES cells treated with 0.5 µg/mL Dox for 24 hours as previously described (21). Cells were cross-linked with 1% formaldehyde (Fisher Bioreagents, BP531-500) for 7 minutes at room temperature, and the reaction was quenched by adding glycine (Fisher Scientific, BP381-1) to a final concentration of 0.125 M. The chromatin was sheared to an average fragment size of ~200 bp using a Bioruptor sonicator (Diagenode). Cross-linked chromatin was pre-cleared with Protein A agarose beads (Thermofisher, 10008D) and subsequently subjected to immunoprecipitation using Dynabeads MyOne Streptavidin T1 (Invitrogen, 65602). Immunoprecipitated complexes were washed twice with 2% SDS, once with buffer A (0.1% Deoxycholate, 1% Triton X-100, 1mM EDTA, 50mM HEPES pH 7.5, 500mM NaCl), once with buffer B (250mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1mM EDTA, 10mM Tris-Cl pH 8.1), and twice with TE buffer. The crosslinked protein-DNA complexes were reversed with an elution buffer (1% SDS, 10mM EDTA, 50mM Tris-Cl 8) to the washed beads and were incubated at 65°C overnight. RNase A (Thermo Scientific, EN0531) was then treated for 30 minutes, followed by treatment of proteinase K (NEB, P8107S) for 2 hours. DNA was extracted with phenol:chloroform:isoamyl alcohol (Invitrogen, 15593031), then precipitated and re-suspended in the TE buffer. Library preparation was performed using the NEBNext Ultra DNA Library Prep Kit (NEB, 7103L), and sequencing was conducted on an Illumina NovaSeq 6000 platform to generate 50-bp paired-end reads. Comparison of the samples with the BirA-only control allowed identification of target-specific binding sites, and IgG ChIP served as a negative control, providing a background signal.