ROLE OF THE CHROMATIN REGULATOR, MLL, IN T CELL DEVELOPMENT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 为了开发可以评估MLL在成人造血和淋巴生成过程中作用的模型系统，我们开发了小鼠MLL基因的loxP侧翼版本，回交该菌株，并将其与Mx1-Cre转基因菌株杂交。Mx1转基因是通过注射PolyI：PolyC诱导的，PolyI：PolyC是一种双链RNA类似物，可以注射到成年动物的腹膜腔中。这种方法使我们能够研究MLL缺失在急性环境中的影响。利用这个模型，我们证明了造血干和祖细胞种群的维持依赖于MLL的同源凝集。我们证明，这可能是由于MLL在这些原始细胞类型中的两个不同的细胞上下文依赖角色。首先，造血干细胞中MLL的缺失导致它们摆脱静止状态，增加增殖，并伴随着子代细胞的对称分化。这导致了骨髓中干细胞活性的迅速耗尽。第二，我们证明在骨髓红系祖细胞中，MLL缺乏导致增殖减少，导致祖细胞池大小减小。总之，这些改变导致MLL缺失后骨髓细胞数量迅速下降，最终导致骨髓衰竭和动物死亡。令人惊讶的是，当我们将MLL loxP侧翼株与仅在分化谱系(T细胞[Lck-Cre]、B细胞[CD19-Cre]或骨髓单核细胞前体[Lysozyme M-Cre])中表达Cre的转基因动物杂交时，我们发现对稳定状态的细胞数量没有影响，表明MLL只在早期干祖细胞群中是必需的。 在淋巴生成方面，朝着该项目主要目标的方向取得了很大进展。我们目前正在完成的实验将形成一份手稿的大部分，该手稿定义了在没有MLL的情况下B淋巴细胞生成的阻断。为了进行这些研究，我们从T.Graf博士(通过H.Mikkola博士)那里获得了Vav-Cre品系的小鼠。该菌株从胚胎第13天开始在胎肝的造血系统中表达Cre重组酶。使用这种方法，我们能够研究向淋巴系(T和B细胞)承诺的早期步骤，而在上述研究中使用Mx1-Cre株是不可能的，因为PolyI：PolyC注射的混杂效应。在Vav-cre模型中，我们观察到在B淋巴细胞生成中对MLL有严格的要求，但在T淋巴细胞生成中不是。这很有趣，因为MLL易位的患者，特别是婴儿，有早期B细胞表型的急性淋巴细胞白血病(ALL)，而T细胞ALL非常罕见。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To develop model systems in which the role of MLL can be assessed during adult hematopoiesis and lymphopoiesis, we have developed a loxP-flanked version of the murine Mll gene, backcrossed this strain, and crossed it to the Mx1-Cre transgenic strain. The Mx1 transgene is induced by the injection of polyI:polyC, a double stranded RNA analog that can be injected into the peritoneal cavity of adult animals. This approach allowed us to study the effect of Mll deletion in an acute setting. Using this model, we demonstrated that the maintenance of hematopoietic stem and progenitor populations depend on Mll for their homoestasis. We demonstrated that this was likely due to two different cell-context dependent roles of Mll in these primitive cell types. First, Mll deletion in hematopoietic stem cells resulted in their escape from quiescence and an increase in proliferation, coupled to symmetric differentiation of progeny cells. This resulted in the rapid exhaustion of stem cell activity from the bone marrow. Second, we demonstrated that in myelo-erythroid progenitors, Mll deficiency resulted in the reduction in proliferation resulting in reduced progenitor pool sizes. Together, these alterations resulted in the rapid decline in bone marrow cell numbers upon Mll deletion and ultimately bone marrow failure and animal death. Surprisingly, when we crossed the Mll loxP flanked strain to Cre transgenic animals that express Cre only in differentiating lineages (T cells [lck-Cre], B cells [CD19-Cre] or myelomonocytic precursors [lysozyme M-Cre]), we found that there was no effect on steady state cell numbers, demonstrating that Mll is only essential in the early stem and progenitor populations. Progress toward the main goal of this Project has been great in the area of lymphopoiesis. We are currently finishing experiments that will form the bulk of a manuscript that defines a block in B lymphopoiesis in the absence of Mll. To perform these studies, we obtained a Vav-Cre strain of mice from Dr. T. Graf (via Dr. H. Mikkola). This strain expresses the Cre recombinase within the hematopoietic system starting at embryonic day 13 in the fetal liver. Using this approach, we are able to study the early steps in commitment to the lymphoid lineages (T and B cells) which was not possible using the Mx1-Cre strain used in the studies described above, due to the confounding effects of polyI:polyC injection. With the Vav-cre model, we observe a stringent requirement for MLL in B lymphopoiesis but not T lymphopoiesis. This is interesting since patients with MLL translocations, particularly infants, have acute lymphocytic leukemia (ALL) of an early B cell phenotype and T cell ALL is very rare.