TLR7訊號所驅動的表觀遺傳重塑源自共同淋巴前驅細胞的樹突細胞發育

Abstract

樹突細胞 (Dendritic cells, DCs)，包含漿狀樹突細胞 (plasmacytoid DCs, pDCs) 與傳統樹突細胞 (conventional DCs, cDCs)是免疫反應的關鍵調節者。這群細胞的生命週期短，而且不斷地由造血幹細胞與先驅細胞 (HSPCs) 持續補充。先前我們發現，Flt3 配體 (Flt3 ligand, FL) 在穩定狀態下會促進共同淋巴先驅細胞 (common lymphoid progenitors, CLPs) 優先分化成 pDC 。然而，當使用 TLR7 促效劑 R848刺激或感染流感病毒時，無論在體內或體外，反而會促進 cDC 的發育但抑制 pDC 的生成。對 CLPs 下游的 DC 前驅細胞進行分析後發現，R848 的刺激會阻斷所有淋巴先驅細胞 (ALPs) 向 pre-pDCs 的分化進程，並將這些先驅細胞的發育命運轉向 cDC。轉錄組分析顯示，由 TLR7 訊息傳遞所引發的前 100 個下調基因中，有 70% 是 pDC 的專一表達基因，包括關鍵轉錄因子Tcf4、Runx2 和 Irf8。由於表觀遺傳(Epigenetic)調控在發育過程中對基因表現的活化(Transcriptionally active)或靜默異染色質(Silencing heterochromatin)扮演關鍵的角色，我們假設它也參與了TLR7訊息傳遞所誘導的DC發育重塑。整體的表觀遺傳分析顯示，與單獨使用 FL 相比，R848 刺激 6 小時後，CLPs 中的 H3K27ac、H3K27me3、H3K9ac 和 H3K9me3 組蛋白標記皆顯著增強。然而，只有H3K9ac和H3K9me3的標記在R848處理 24小時後仍然持續增加，這暗示了與轉錄活化或靜默異染色質相關的H3K9ac及H3K9me3標記可能參與其中。有趣的是，調控H3K9me3 的數個表觀遺傳修飾因子的轉錄程度，包括甲基轉移酶(Methyltransferase)如 Setdb1、Setdb2 以及組蛋白讀取蛋白(Reader)如 Cbx1，在 R848 刺激 6 小時後均顯著上調。我們結合不同組蛋白標記的 CUT&Tag 數據，並使用ChromHMM 對「染色質狀態」進行了綜合分析。最佳化後的14 種狀態ChromHMM 模型顯示，TLR7 誘導的表觀遺傳的重編程在6小時便在 pDC 相關的專一基因位點建立了抑制性的染色質狀態。相反的，在cDC 相關專一基因上則誘導產生活化性的染色質狀態。而此時的CUTAC分析顯示，pDC 與 cDC 基因位點的染色質開放區域與 48 小時的轉錄組分析結果呈現正相關，這些結果顯示 TLR7 誘導的表觀遺傳重塑在早期就已確立了發育的路徑。透過 ROSE 演算法從 CUT&Tag 數據中鑑定出的超級增強子 (super enhancer) 顯示，H3K9ac及H3K27ac 超級增強子可能各自主導了 DC 發育重塑的不同迴路。最後，利用 SCENIC （單細胞調控網絡推斷與聚類） 從single-cell RNA sequencing推斷出的基因調控網路 (GRNs)，以及從 CUT&Tag 預測的 DNA 結合序列皆顯示存在數群細胞類型特異性的 GRNs 可能引導了此發育重塑。總體而言，我們的研究結果強調，TLR7 訊息傳遞誘導的 CLPs 發育重塑可能是由表觀遺傳重編所驅動，而此過程又可能受到不同超級增強子群集與細胞類型特異性 GRNs 之間的交互作用來調控。

Dendritic cells (DCs), comprising plasmacytoid DCs (pDCs) and conventional DCs (cDCs), are essential mediators of the immune response. These short-lived cells are continuously replenished from hematopoietic stem and progenitor cells (HSPCs). Previously, we found that Flt3 ligand (FL) preferentially promoted pDC development from common lymphoid progenitors (CLPs) at steady state. However, the administration of a TLR7 agonist R848 or infection with influenza viruses favored cDC development but suppressed pDC generation from CLPs in vitro and in vivo. Analysis of the DC precursors downstream of CLPs revealed that the progression of all lymphoid progenitors (ALPs) to pre-pDCs was blocked, and the developmental fate of these progenitors was also skewed toward cDCs upon R848 stimulation. Transcriptomic profiling revealed that 70% of the TLR7 signaling-induced top 100 downregulated genes were pDC-specific, including key transcription factors Tcf4, Runx2, and Irf8. Since epigenetic regulation plays a critical role in activating or silencing gene expression during development, we hypothesized that it may also be involved in TLR7 signaling-induced reprogramming of DC development from CLPs. Total epigenetic profiling revealed that FL plus R848 stimulation enhanced the establishment of histone marks H3K27ac, H3K27me3, H3K9ac, and H3K9me3 in CLPs after 6 h of treatment compared to FL treatment alone. However, only H3K9ac, H3K9me3, but not H3K27ac or H3K27me3, were maintained for up to 24 h post-treatment, suggesting that H3K9ac and H3K9me3, a mark known to induce transcriptionally active regions and silent heterochromatin, respectively, may play a role in this process. Interestingly, the transcriptional levels of several epigenetic modifiers for H3K9me3, including a methyltransferase Setdb1 and Setdb2, and histone readers Cbx1, were significantly upregulated by R848 stimulation at 6 h. A comprehensive analysis of the “chromatin state” was performed by combining the CUT&Tag of different histone marks by using the chromatin hidden Markov model (ChromHMM). The optimized 14-state ChromHMM model revealed that the TLR7-induced epigenetic reprogramming established the suppressive chromatin states on pDC-specific gene loci and induced the active chromatin states on cDC-specific gene loci at 6 h. Chromatin accessible data on pDC and cDC gene loci from cleavage under targeted accessible chromatin (CUTAC) at 6 h post-treatment showed a positive correlation with transcriptomic profiling at 48 h post-treatment, suggesting that TLR7-induced epigenetic reprogramming establishes the developmental pathway early on. Moreover, we identified the super enhancers of the CUT&Tag dataset by Rank Ordering of Super-Enhancers (ROSE). We found that H3K9ac/H3K27ac super enhancer may govern different circuits of reprogrammed DC development from CLPs. Gene regulatory networks (GRNs) inferred from single-cell RNA sequencing using the single-cell regulatory network inference and clustering (SCENIC) and DNA binding motif predicted from CUT&Tag demonstrated the presence of groups of cell-type-specific GRNs that may guide the reprogrammed DC development. Together, our findings emphasize that TLR7 signaling-induced reprogrammed DC development from CLPs is driven by epigenetic reprogramming, which is governed by the interaction between various clusters of super enhancers and cell-type-specific GRNs.