Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation-猫眼课题宝

DOI：

10.1182/blood.v126.23.841.841

发表时间：

2015-12

期刊：

Blood

影响因子：

20.3

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

Claire Mazumdar;Ying Shen;Seethu Xavy;Feifei Zhao;A. Reinisch;Rui Li;M. R. Corces-Zimmerman;Jason D. Buenrostro;Steven M. Chan;Daniel Thomas;J. Koenig;W. Hong;Howard Y. Chang;R. Majeti

粘附素复合体(RAD21、SMC1A、SMC3和STAG2)成分的反复突变已在人类AML和其他髓系恶性肿瘤中被发现，并已被证明发生在HSC中的白血病前突变。粘附素的功能是将染色质链固定在由四个核心成分组成的环状结构中，虽然它最好的作用是在有丝分裂期间维持姐妹染色单体的极性，但粘附素也参与双链DNA损伤修复和转录调节。由于对它们在白血病发生中的作用知之甚少，我们试图研究粘附素突变对人类造血的影响，特别是对造血干细胞和祖细胞(HSPC)的影响。将突变型粘附素导入AML细胞系和原代人HSPC后，分化受阻，CD34+祖细胞比例增加。在体外和体内都观察到了类似的表型，核心成分RAD21被敲除，表明突变的粘附素可以通过单倍体不足或显性负机制发挥作用。突变型粘附素增强了HSPC的体外系列复制能力，并对HSC和白血病干细胞的基因表达程序表现出丰富的作用，表明有增强干细胞功能的作用。此外，我们观察到在甲基纤维素中培养的粘附素突变克隆向髓系倾斜，这在体内的人类移植细胞中表现为强烈的髓系倾斜。因此，突变的粘附素执行干细胞程序，并损害人类造血祖细胞的分化。由于在我们调查的许多病例中，在白血病前期的HSC中发现了粘附素复合体突变，我们假设它们可能以一种细胞上下文相关的方式传递其表型。为了验证这一假设，从脐带血中分离出6个人HSPC亚群(HSC、MPP、LMPP、CMP、GMP和MEP)，并将这些细胞用粘附素WT、粘附素突变体、RAD21shRNA或对照慢病毒进行转导。然后在髓系分化或促进红系分化的条件下培养转导细胞。值得注意的是，只有凝集素突变转导的HSC和MPP才能观察到强烈的髓系分化障碍，而不是GMP。类似地，HSC和MPP也观察到强烈的红系分化障碍，但MEP没有观察到。这些结果表明，突变型粘附素的作用是上下文依赖的，并且仅限于最不成熟的HSPC。接下来，我们试图阐明粘附素突变对人类HSPC产生影响的机制。由于粘附素复合体具有建立和维持DNA可及性的功能，而粘附素的敲除可以导致转录因子(Tf)聚集区染色质可及性的降低(Yan等人，2013)，我们假设粘附素突变体通过调节染色质可及性来传递其表型效应。为了研究这一假设，我们使用了一种新开发的名为ATAC-Seq的方法(Buenrostro等人，2013)来评估粘连蛋白WT和突变的HSPC的全基因组可及性。正如预期的那样，我们发现粘附素突变体在转录调控元件上表现出染色质可及性的全局降低。然而，我们检测到已知在HSPC中高表达的转录因子以及包括ERG、GATA2和RUNX1在内的关键调控因子的染色质可及性增加。进一步的足迹分析表明，与WT细胞相比，突变细胞中这些因子的结合有很大的丰富。基于这些结果，我们建立了一个模型，在该模型中，突变的粘附素对人HSPC的功能影响是通过显示染色质可及性增加的转录因子如ERG、GATA2和RUNX1来介导的。从这个模型中，我们假设这些转录因子的敲除将阻止干细胞程序的执行，并通过粘附素突变体观察到CD34表达细胞的增加。正如预测的那样，在粘连蛋白突变体存在的情况下，敲除ERG、GATA2或RUNX1，而不是GATA1或PU.1，完全阻止CD34表达细胞的增加。这些结果有力地支持了我们提出的模型，即突变的粘附素通过调节ERG、GATA2和RUNX1染色质的可及性、表达和活性来损害造血分化和执行干细胞计划。信息披露：47，Inc.：咨询、股权所有权、董事会或咨询委员会成员。

Recurrent mutations in the components of the cohesin complex (RAD21, SMC1A, SMC3, and STAG2) have been identified in human AML and other myeloid malignancies, and have been shown to occur as pre-leukemic mutations in HSC. Cohesin functions to hold chromatin strands within a ring-like structure composed of the four core components, and although its best-established role is to maintain the polarity of sister chromatids during mitosis, cohesin is also involved in double-stranded DNA damage repair and regulation of transcription. As little is known about their contributions to leukemogenesis, we sought to investigate the effects of cohesin mutants on human hematopoiesis, particularly hematopoietic stem and progenitor cells (HSPC). Introduction of mutant cohesin into AML cell lines and primary human HSPC resulted in a differentiation block with an increased frequency of CD34+ progenitor cells. A similar phenotype was observed with knockdown of core component RAD21 both in vitro and in vivo, indicating that mutant cohesin can act either through haploinsufficiency or dominant-negative mechanisms. Mutant cohesin increased the serial replating ability of HSPC in vitro and showed enrichment for HSC and leukemia stem cell gene expression programs, indicating an effect to enforce stem cell functions. Furthermore, we observed a skewing toward the myeloid lineage in cohesin mutant colonies cultured in methylcellulose, which was recapitulated by a strong myeloid skewing of human engrafted cells in vivo. Thus, mutant cohesin enforces stem cell programs and impairs human hematopoietic progenitor differentiation. Since cohesin complex mutations were identified in pre-leukemic HSC in many of the cases we investigated, we hypothesized that they may impart their phenotype in a cell context-dependent manner. To investigate this hypothesis, six human HSPC subpopulations (HSC, MPP, LMPP, CMP, GMP, and MEP) were isolated from cord blood, and these cells were transduced with cohesin WT, cohesin mutant, RAD21 shRNA, or control lentivirus. Transduced cells were then cultured in either myeloid differentiation or erythroid differentiation-promoting conditions. Strikingly, a strong myeloid differentiation block was only observed with cohesin mutant-transduced HSC and MPP, but not GMP. Similarly, a strong erythroid differentiation block was also observed in HSC and MPP, but not MEP. These results indicate that the effect of mutant cohesin is context dependent and restricted to the most immature HSPC. We next sought to elucidate the mechanism by which cohesin mutants exert their effects on human HSPC. Since the cohesin complex functions to establish and maintain DNA accessibility, and knockdown of cohesin can led to a decrease in chromatin accessibility at transcription factor (TF) clustered regions (Yan et al., 2013), we hypothesized that cohesin mutants impart their phenotypic effects through modulation of chromatin accessibility. To investigate this hypothesis, we used a newly developed method known as ATAC-Seq (Buenrostro et al., 2013) to assess genome-wide accessibility in cohesin WT and mutant HSPC. As expected, we found that cohesin mutants exhibited globally reduced chromatin accessibility at transcriptional regulatory elements. However, we detected increased chromatin accessibility at motifs for transcription factors known to be highly expressed in and critical regulators of HSPC including ERG, GATA2 and RUNX1. Further footprinting analysis, a proxy for ChIP-Seq experiments, showed a strong enrichment of binding of these factors in the mutant cells compared to WT cells. Based on these results, we developed a model in which the functional effects of mutant cohesin on human HSPC are mediated by transcription factors exhibiting increased chromatin accessibility such as ERG, GATA2 and RUNX1. From this model, we hypothesized that knockdown of these transcription factors would prevent the enforcement of stem cell programs and increase in CD34-expressing cells observed with cohesin mutants. As predicted, knockdown of ERG, GATA2, or RUNX1, but not GATA1 or PU.1, in the presence of cohesin mutants completely prevented the increase in CD34-expressing cells. These results strongly support our proposed model that mutant cohesin impairs hematopoietic differentiation and enforces stem cell programs through the modulation of ERG, GATA2, and RUNX1 chromatin accessibility, expression, and activity. Disclosures Majeti:Forty Seven, Inc.: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees.

Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation

文献摘要

被引文献

相似文献