■通过激活诱导脱氨酶(AID)的免疫球蛋白可变(V)区的体细胞超突变(SHM)对于健壮至关重要,对病原体和疫苗抗原的长期体液免疫。AID优先在WRCH基序内突变胞嘧啶(其中W=A或T,R=A或G,H=A,C或T)。然而,一直观察到WRCH基序的可变性变化很大,即使在单个V区域内同一基序的多次出现之间,突变频率也存在很大差异。这导致了WRCH基序的直接序列上下文有助于可变性的概念。最近的研究强调了局部DNA序列特征在促进AGCT诱变中的潜在作用。一种常见突变的WRCH基序。有趣的是,AGCT基序更接近V区的5个末端,在框架1(FW1)子区域1内,突变频率较低,建议SHM抑制序列上下文。
■这里,我们用DeepSHM系统地研究了人类SHM数据集中AGCT位置偏差的基础,用于预测SHM模式的机器学习模型。这与综合梯度相结合,一种可解释性方法,询问DeepSHM预测的基础。
■DeepSHM以高精度预测了在AGCT基序处观察到的突变频率的位置差异。对于保守的人来说,FW1中AGCT基序的低突变,整合梯度预测5'C和3'G侧翼残基的负贡献很大,表明该位置的CAGCTG背景对SHM具有抑制作用。CAGCTG是E-box转录因子的识别基序,包括E2A,这与SHM有牵连。的确,我们发现了一个强大的,E盒基序保真度与突变频率成反比关系。此外,发现E2A与两种人B细胞系中的V区区域相关联。最后,对人类SHM数据集的分析显示,在3'G侧翼残基中自然发生的突变,这有效地废除了电子盒子的主题,与AGCT突变率显著升高相关。
■我们的结果表明,突变频率与E2A等E-box因子在特定AGCT基序环境下的结合之间存在拮抗关系,因此,突出一个新的,调节人V区局部SHM模式的抑制机制。
UNASSIGNED: Somatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, long-term humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5\' ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context.
UNASSIGNED: Here, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions.
UNASSIGNED: DeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5\'C and 3\'G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for E-box transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3\'G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation.
UNASSIGNED: Our results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions.