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：
9894797
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 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9894797
关键词：
Binding Bioinformatics Biology Blood Cells CRISPR/Cas technology Cell Culture Techniques Cell physiology 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 Transplantation 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 therapy stemness success

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）由于自我更新和分化为所有成熟的血细胞类型（称为“茎”）。含HSC的移植物的移植是一种用于多种血液疾病的救生疗法；但是，缺乏免疫学匹配的捐助者限制可以治疗的患者数量。文化中人类HSC的扩展/产生将很棒改善移植疗法，但由于对潜在生物学的了解不足，但没有成功 HSC茎。我们将MLLT3确定为HSC调节剂，在各个阶段都高度富集了人类HSC 一旦它们从AGM出来并在胎儿肝脏中膨胀（FL）；但是，mllt3表达在培养的HSC。关于人FL和脐带血（CB）HSC的功能研究的丧失和获取研究表明MLLT3为对他们的自我更新至关重要，当恢复表达时，它可以通过保护他们的“ Stemness”计划。重要的是，FL和CB HSC在培养中扩展了10-30倍 NSG小鼠中人类植入的增加，以及维持HSPC室和多轨道的能力没有恶性转化或分化阻滞的造血。这一发现提供了前所未有的了解人类HSC茎的控制方式，并利用MLLT3进行临床使用。迄今为止，在HSC中控制MLLT3表达的调节机制是完全未知的。使用组合在表观遗传学研究和生物信息学方法中，我们确定了MLLT3基因中的几个候选增强子这可能会调节其两种同工型。我们的数据表明，在在HSC文化期间的分化和沉默 mllt3表达。为了了解如何在人类HSC中调节MLLT3的表达，我们提出了两个完全方法：1）CRISPR/CAS9介导的表观基因组编辑以剖析MLLT3的作用增强剂，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MLLT3上游调节器。这些方法的成功可以帮助产生/扩展人类HSC 培养，从而增加HSC进行移植的可用性。