Enhancer bound proteome of intronic Ig Emu CORE enhancer with reverse DNA sequence polarity control bait

The specificity of humoral immune responses depends on the functional rearrangement and expression of only one allele of immunoglobulin (Ig) genes. Here, we analyzed the comprehensive proteome of the murine Ig Emu CORE enhancer, which governs the rearrangement and expression of the Ig mu heavy chain allele. The Ig Emu consists of a CORE enhancer (harboring a multitude of transcription factor binding sites) and two 5’ and 3’ flanking MAR (matrix attachment region) elements. By mass spectrometry of proteins bound at wild type versus mutant Emu enhancers, we identified Emu-binding proteins and associated multi-protein complexes. We found that the MSL/MOF complex, a regulator of gene dosage compensation in flies, binds Emu via transcription factor YY1 and facilitates Emu-driven chromatin looping and promoter interaction. Msl2 gene knockout in primary pre-B cells or Mof heterozygosity in mice reduced mu gene expression. In this data set we compare proteins binding to the wild-type Emu versus a DNA bait control for which the Emu CORE sequence was switched to its reverse polarity sequence (the flanking MARs sequence are wild-type). The latter conserves the DNA GC content but virtually destroys all sequence-specific transcription factor binding sites. Of note, DNA repetitive sequences that can also be bound by DNA interacting proteins are kept functional by this control bait. SILAC quantitative proteomics was employed in a label swap approach incubating wild-type and control DNA with labeled and non-labled protein extracts, respectively.

体液免疫应答的特异性，仅取决于免疫球蛋白（immunoglobulin, Ig）基因单个等位基因的功能性重排与表达。本研究针对调控Ig μ重链等位基因重排与表达的小鼠Ig Emu核心增强子（Ig Emu CORE enhancer），开展了全面蛋白质组学分析。Ig Emu元件由核心增强子（包含大量转录因子结合位点）以及5’、3’侧翼的MAR（基质附着区，matrix attachment region, MAR）元件组成。本研究通过对野生型与突变型Emu增强子结合的蛋白质进行质谱分析，鉴定出了Emu结合蛋白及其相关多蛋白复合物。研究发现，果蝇中调控基因剂量补偿的MSL/MOF复合物，可通过转录因子YY1结合Emu元件，并介导Emu驱动的染色质环化与启动子相互作用。在原代前B细胞中敲除Msl2基因，或在小鼠中使Mof基因呈杂合状态，均可下调μ基因的表达水平。本数据集对比了结合野生型Emu的蛋白质与结合DNA诱饵对照的蛋白质：该对照的Emu核心序列被替换为反向极性序列，但其侧翼MAR序列仍为野生型。该对照保留了DNA的GC碱基组成，但几乎完全破坏了所有序列特异性转录因子结合位点。值得注意的是，该对照仍保留了可结合DNA互作蛋白的DNA重复序列的功能。本研究采用SILAC定量蛋白质组学技术，通过标记互换策略：分别将野生型与对照DNA与标记及未标记的蛋白质提取物共孵育。