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.

转录调控蛋白，包括转录因子（transcription factors, TFs）与染色质修饰因子，在细胞核内执行关键的细胞功能。与多数转录因子不同，膜结合转录因子（membrane-bound transcription factors, MBTFs）需先锚定在膜上，再发生核易位。已知的MBTFs对髓鞘表达（髓鞘调节因子MYRF）、胆固醇稳态（固醇调节元件结合蛋白SREBP）等诸多生理过程至关重要，但其整体多样性仍未被系统阐明。
我们推测可能存在更多的膜结合转录调控因子（membrane-bound transcriptional regulators, MBTRs），并开发了一款生物信息学筛选流程，优先筛选具备转录调控潜能的膜蛋白。本方法依托结构域组成特征，即异常的结构域组合往往预示着全新的生物学功能，以此搜寻罕见但进化保守的跨膜结构域与转录调控相关结构域的组合。
该方法不仅重新鉴定出已知的MBTFs与膜结合组蛋白激酶，还发现了全新的MBTR候选蛋白，包括组蛋白N-乙酰转移酶以及催乳素调节元件结合蛋白PREB。实验验证结果表明，PREB可能经历剪切加工过程。当缺失跨膜结构域时，PREB会在小鼠胚胎干细胞中呈现核定位与染色质结合特性。此外，我们还鉴定出一类潜在的新型MBTFs，它们共享MYRF的DNA结合结构域。上述发现凸显了本筛选方法的实用性，同时为研究介导细胞外信号与转录应答整合的MBTRs提供了标准化研究框架。
总体实验设计：参照此前报道的方法（21），对经0.5 µg/mL强力霉素（Dox）处理24小时的表达PREB及PREBΔTM的胚胎干细胞（ES细胞）开展生物染色质免疫沉淀测序（bioChIP-seq）实验。具体流程如下：用1%甲醛（Fisher Bioreagents, BP531-500）在室温下交联细胞7分钟，随后加入终浓度为0.125 M的甘氨酸（Fisher Scientific, BP381-1）终止交联反应。使用Bioruptor超声破碎仪（Diagenode）将染色质剪切至平均片段大小约200 bp。将交联染色质用Protein A琼脂糖磁珠（Thermofisher, 10008D）进行预澄清，再通过Dynabeads MyOne链霉亲和素T1磁珠（Invitrogen, 65602）完成免疫沉淀。免疫沉淀得到的复合物依次以2% SDS洗涤2次、缓冲液A（0.1%脱氧胆酸钠、1% Triton X-100、1mM EDTA、50mM HEPES pH 7.5、500mM NaCl）洗涤1次、缓冲液B（250mM氯化锂、0.5% NP-40、0.5%脱氧胆酸钠、1mM EDTA、10mM Tris-Cl pH 8.1）洗涤1次，最后用TE缓冲液洗涤2次。将洗脱缓冲液（1% SDS、10mM EDTA、50mM Tris-Cl pH 8）加入洗涤后的磁珠中，逆转交联的蛋白-DNA复合物，并于65℃孵育过夜。随后用RNase A（Thermo Scientific, EN0531）处理30分钟，再以蛋白酶K（NEB, P8107S）处理2小时。采用苯酚:氯仿:异戊醇（Invitrogen, 15593031）提取DNA，经沉淀后重悬于TE缓冲液中。使用NEBNext Ultra DNA文库制备试剂盒（NEB, 7103L）构建测序文库，并在Illumina NovaSeq 6000平台上完成测序，生成50 bp的双端读长序列。通过与仅表达BirA的对照样本进行比对，可鉴定出靶标特异性结合位点；IgG ChIP作为阴性对照，用于提供背景信号。