Defining the mechanisms regulating MLLT3 expression in human hematopoietic stem cells

定义人类造血干细胞中 MLLT3 表达的调节机制

• 批准号: 10113603
• 负责人: Hanna Katri Annikki Mikkola
• 金额: $ 28.08万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-16 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10113603
• 关键词: Binding; Bioinformatics; Biology; Blood Cells; CRISPR/Cas technology; Cell Culture Techniques; ChIP-seq; Chromatin; Clinical; Data; Development; EVI1 gene; Engineering; Engraftment; Enhancers; Epigenetic Process; Experimental Models; Fetal Liver; Future; Gene Expression; Generations; Genes; Genetic Enhancer Element; Goals; Hematological Disease; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; High-Throughput Nucleotide Sequencing; Histones; Human; Immunologics; In Vitro; Individual; KDM1A gene; Lead; Lentivirus Vector; Life; Lysine; MLLT3 gene; Maintenance; Malignant - descriptor; Mediating; Modification; Monitor; Mus; Names; Patients; Protein Isoforms; Proteins; Protocols documentation; Regenerative Medicine; Regulation; Regulator Genes; Regulatory Element; Reporter; Repression; Reverse Transcriptase Polymerase Chain Reaction; Role; Savings; Second Pregnancy Trimester; Testing; Tetanus Helper Peptide; Time; Transcript; Transcription Initiation Site; Transcriptional Elongation Factors; Umbilical Cord Blood; aptamer; base; blood formation; cell type; epigenome editing; gain of function; hematopoietic stem cell expansion; hematopoietic stem cell self-renewal; hematopoietic tissue; hemogenic endothelium; high throughput analysis; human RNA sequencing; human embryonic stem cell; improved; in vivo; insight; novel; novel strategies; overexpression; p300/CBP-Associated Factor; population based; programs; recruit; self-renewal; single-cell RNA sequencing; stem cell function; stem cell therapy; stemness; success; transplantation therapy

SUMMARY Hematopoietic stem cells (HSCs) sustain life-long blood formation due to their ability to self-renew and differentiate into all mature blood cell types (referred to as “stemness”). Transplantation of HSC-containing grafts is a life-saving therapy for multiple blood disorders; however, shortage of immunologically matched donors limits the number of patients that can be treated. Expansion/generation of human HSCs in culture would greatly improve transplantation therapy but has been unsuccessful due to poor understanding of the underlying biology of HSC stemness. We identified MLLT3 as HSC regulator that is highly enriched in human HSC at all stages once they have emerged from AGM and expand in the fetal liver (FL); however, MLLT3 expression declines in cultured HSC. Loss and gain of function studies on human FL and cord blood (CB) HSCs showed that MLLT3 is critical for their self-renewal, and when its expression is restored, it enables HSC expansion in culture by protecting their “stemness” program. Importantly, FL and CB HSC expanded in culture showed 10-30 fold increase in human engraftment in NSG mice, and ability to sustain the HSPC compartment and multilineage hematopoiesis without malignant transformation or differentiation block. This finding offers an unprecedented opportunity to understand how human HSC stemness is controlled, and harness MLLT3 for clinical use. So far, the regulatory mechanisms that govern MLLT3 expression in HSC are completely unknown. Using a combination of epigenetic studies and bioinformatic approaches, we identified several candidate enhancers in MLLT3 gene that may regulate its two isoforms. Our data suggests that these enhancers are epigenetically remodeled during differentiation and silenced during HSC culture, providing new avenues to understand why cultured HSC lose MLLT3 expression. To understand how MLLT3 expression is regulated in human HSCs, we propose two complementary approaches: 1) CRISPR/Cas9-mediated epigenome editing to dissect the role of MLLT3 enhancers, 2) combination of bioinformatic and experimental approaches using lentiviral overexpression to identify MLLT3 upstream regulators. Success in these approaches could help generate/expand human HSCs in culture and thereby increase the availability of HSCs for transplantation.

总结 造血干细胞（HSC）由于其自我更新和再生的能力而维持终生的血液形成。 分化成所有成熟的血细胞类型（称为“干细胞”）。含HSC移植物的移植 是多种血液疾病的救命疗法;然而，缺乏免疫匹配的供体限制了 可以治疗的患者数量。在培养物中扩增/产生人HSC将极大地 但由于对潜在生物学的理解不足， HSC的干性。我们将MLLT 3鉴定为HSC调节因子，其在所有阶段高度富集于人HSC中 一旦它们从AGM中出现并在胎肝（FL）中扩增，MLLT 3表达就会下降;然而， 培养的HSC。对人FL和脐带血（CB）HSC的功能丧失和获得研究表明，MLLT 3是 对于它们的自我更新至关重要，当其表达恢复时，它通过以下方式使HSC在培养中扩增： 保护他们的“sterness”计划重要的是，在培养中扩增的FL和CB HSC显示出10-30倍的扩增。 NSG小鼠中的人植入增加，以及维持HSPC隔室和多谱系的能力 造血无恶变或分化阻滞。这一发现提供了前所未有的 有机会了解人类HSC干性是如何控制的，并利用MLLT 3进行临床应用。到目前为止， 控制HSC中MLLT 3表达的调节机制完全未知。使用组合 通过表观遗传学研究和生物信息学方法，我们鉴定了MLLT 3基因的几个候选增强子 可以调节它的两种亚型。我们的数据表明，这些增强子是表观遗传重塑过程中， 在HSC培养过程中分化和沉默，提供了新的途径来了解为什么培养的HSC失去 MLLT 3表达。为了了解MLLT 3在人HSC中的表达是如何调节的，我们提出了两个 互补方法：1）CRISPR/Cas9介导的表观基因组编辑，以剖析MLLT 3的作用 增强子，2）使用慢病毒过表达的生物信息学和实验方法的组合， 识别MLLT 3上游调节子。这些方法的成功可以帮助在体外产生/扩增人HSC。 培养，从而增加用于移植的HSC的可用性。