BackgroundHypoxia-inducible factors (HIF)1 and 2 are transcription factors that regulate the homeostatic response to low oxygen conditions. Since data related to the importance of HIF1 and 2 in hematopoietic stem and progenitors is conflicting, we investigated the chromatin binding profiles of HIF1 and HIF2 and linked that to transcriptional networks and the cellular metabolic state.MethodsGenome-wide ChIPseq and ChIP-PCR experiments were performed to identify HIF1 and HIF2 binding sites in human acute myeloid leukemia (AML) cells and healthy CD34+ hematopoietic stem/progenitor cells. Transcriptome studies were performed to identify gene expression changes induced by hypoxia or by overexpression of oxygen-insensitive HIF1 and HIF2 mutants. Metabolism studies were performed by 1D-NMR, and glucose consumption and lactate production levels were determined by spectrophotometric enzyme assays. CRISPR-CAS9-mediated HIF1, HIF2, and ARNT−/− lines were generated to study the functional consequences upon loss of HIF signaling, in vitro and in vivo upon transplantation of knockout lines in xenograft mice.ResultsGenome-wide ChIP-seq and transcriptome studies revealed that overlapping HIF1- and HIF2-controlled loci were highly enriched for various processes including metabolism, particularly glucose metabolism, but also for chromatin organization, cellular response to stress and G protein-coupled receptor signaling. ChIP-qPCR validation studies confirmed that glycolysis-related genes but not genes related to the TCA cycle or glutaminolysis were controlled by both HIF1 and HIF2 in leukemic cell lines and primary AMLs, while in healthy human CD34+ cells these loci were predominantly controlled by HIF1 and not HIF2. However, and in contrast to our initial hypotheses, CRISPR/Cas9-mediated knockout of HIF signaling did not affect growth, internal metabolite concentrations, glucose consumption or lactate production under hypoxia, not even in vivo upon transplantation of knockout cells into xenograft mice.ConclusionThese data indicate that, while HIFs exert control over glycolysis but not OxPHOS gene expression in human leukemic cells, this is not critically important for their metabolic state. In contrast, inhibition of BCR-ABL did impact on glucose consumption and lactate production regardless of the presence of HIFs. These data indicate that oncogene-mediated control over glycolysis can occur independently of hypoxic signaling modules.

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HIF1/2 对糖酵解基因表达的控制在功能上与人白血病干/祖细胞中的糖酵解无关

背景缺氧诱导因子 (HIF)1 和 2 是调节对低氧条件的稳态反应的转录因子。由于与 HIF1 和 2 在造血干细胞和祖细胞中的重要性相关的数据相互矛盾，我们研究了 HIF1 和 HIF2 的染色质结合谱，并将其与转录网络和细胞代谢状态联系起来。方法全基因组 ChIPseq 和 ChIP-PCR 实验是用于鉴定人急性髓性白血病 (AML) 细胞和健康 CD34+ 造血干/祖细胞中的 HIF1 和 HIF2 结合位点。进行转录组研究以鉴定由缺氧或氧不敏感 HIF1 和 HIF2 突变体的过表达引起的基因表达变化。通过 1D-NMR 进行代谢研究，葡萄糖消耗和乳酸产生水平通过分光光度酶测定法确定。产生了 CRISPR-CAS9 介导的 HIF1、HIF2 和 ARNT-/- 细胞系，以研究在异种移植小鼠体内移植敲除细胞系后在体外和体内丧失 HIF 信号传导的功能后果。 结果全基因组 ChIP-seq 和转录组研究表明，重叠的 HIF1 和 HIF2 控制位点高度富集了各种过程，包括代谢，特别是葡萄糖代谢，而且还用于染色质组织、细胞对压力的反应和 G 蛋白偶联受体信号传导。ChIP-qPCR 验证研究证实，在白血病细胞系和原发性 AML 中，糖酵解相关基因而非与 TCA 循环或谷氨酰胺分解相关的基因受 HIF1 和 HIF2 控制，而在健康的人类 CD34+ 细胞中，这些基因座主要受 HIF1 而非 HIF2 控制。然而，与我们最初的假设相反，CRISPR/Cas9 介导的 HIF 信号敲除不影响缺氧条件下的生长、内部代谢物浓度、葡萄糖消耗或乳酸产生，甚至在将敲除细胞移植到异种移植小鼠体内时也不影响体内。数据表明，虽然 HIF 控制人类白血病细胞中的糖酵解而不是 OxPHOS 基因表达，但这对其代谢状态并不重要。相比之下，无论是否存在 HIF，BCR-ABL 的抑制都会影响葡萄糖消耗和乳酸产生。这些数据表明，癌基因介导的糖酵解控制可以独立于缺氧信号模块发生。