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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

基本信息

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
项目类别：
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

项目摘要

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.

概括造血干细胞 (HSC) 因其自我更新和自我更新的能力而维持终生血液形成。分化为所有成熟的血细胞类型（称为“干细胞”）。含有 HSC 的移植物的移植是治疗多种血液疾病的救命疗法；然而，免疫匹配供体的短缺限制了可以治疗的患者数量。在培养物中扩增/产生人类 HSC 将极大地促进改善移植疗法，但由于对潜在生物学了解甚少而未能成功 HSC 干性。我们确定 MLLT3 是 HSC 调节因子，在人类 HSC 的各个阶段都高度富集一旦它们从 AGM 中出现并在胎儿肝脏 (FL) 中扩展；然而，MLLT3 表达下降培养的HSC。对人 FL 和脐带血 (CB) HSC 的功能丧失和增强研究表明 MLLT3 对于它们的自我更新至关重要，当其表达恢复时，它可以通过以下方式实现 HSC 在培养物中的扩增：保护他们的“干性”计划。重要的是，FL 和 CB HSC 在培养物中扩增显示 10-30 倍 NSG 小鼠中人类植入的增加以及维持 HSPC 区室和多谱系的能力造血无恶变或分化障碍。这一发现提供了前所未有的有机会了解如何控制人类 HSC 干性，并利用 MLLT3 进行临床应用。迄今为止， HSC 中控制 MLLT3 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 MLLT3 基因中的几个候选增强子可能调节其两种亚型。我们的数据表明这些增强子在 HSC 培养过程中的分化和沉默，为理解培养的 HSC 为何丢失提供了新途径 MLLT3 表达。为了了解人类 HSC 中 MLLT3 表达的调节方式，我们提出了两种补充方法：1) CRISPR/Cas9 介导的表观基因组编辑来剖析 MLLT3 的作用增强子，2) 结合生物信息学和实验方法，利用慢病毒过表达识别 MLLT3 上游调节器。这些方法的成功可能有助于在以下领域产生/扩展人类造血干细胞：培养，从而增加用于移植的 HSC 的可用性。