Specification and Maintenance of the Scl Induced Hematopoietic Stem Cell Fate.
Authors: Gereige Laurraine-Marcelle , Ferrari Roberto , Montel-Hagen Amelie , Pellegrini Matteo , Li Xinmin , Kurdistani Siavash , Mikkola Hanna 
Published: October 2019

Abstract
Abstract 1504
Poster Board I-527
The hematopoietic program is initiated in the embryo by the basic helix-loop-helix (bHLH) transcription factor Scl/Tal1 (stem cell leukemia gene). In the absence of Scl, mesodermal precursors are unable to commit to the hematopoietic fate and the embryo dies due to lack of blood formation. However, Scl becomes dispensable for hematopoietic stem cell(HSC) function shortly after hematopoietic specification, suggesting that once specified by Scl, the hematopoietic fate is maintained by alternative regulatory mechanisms. Scl is required again later for the proper maturation of erythrocytes and megakaryocytes. Despite Scl's pivotal role in initiating hematopoiesis, how Scl dictates HSC development at a molecular level remains unknown. To define how the hematopoietic fate is established by Scl, we performed genome-wide gene expression and ChIP-chip Scl binding analysis on in vitro differentiated hemangioblasts isolated from Scl−/− and control ES cells. Analysis of the 655 Scl-dependent genes and Scl binding revealed that Scl acts both as an activator and a repressor. The group of genes activated by Scl included major hematopoietic transcription factors such as Tel/Etv6, Gfi1, Fli1, cMyb, Gata2, Hhex, Sox17, Lyl1, and JunB, as well as regulators of embryonic vasculogenesis/angiogenesis and/or arterio-venous specification such as Ets1, Ets2, Elk3, Foxo1, Hoxb5, Smarca2, and Sox18. The group of genes repressed by Scl included regulators of alternative mesodermal fates such as Gata4, Tbx20 and Isl1, Foxf1a, and Snail1. These data show that Scl induces the hematopoietic program both by directly activating the major transcriptional networks required for the formation of the hemogenic endothelium and the emergence, self-renewal and survival of HSCs, as well as repressing alternative mesodermal fates. Considering the large number of transcription factors that Scl regulates during specification, we sought to determine at the molecular level how the hematopoietic fate is maintained independently of Scl. Gene expression analysis on Lin−cKit+ HSCs/progenitors isolated from Sclfl/flVavCre+ mice revealed that only 41 genes were Scl dependent, none of which were major hematopoietic factors. Notably, the key hematopoietic transcription factors remained expressed in both control and Scl deficient HSCs/progenitors indicating that the Scl induced program is maintained. Regulators of alternative mesodermal fates remained silenced during adult hematopoiesis. These molecular data were in agreement with the functional data showing that the bone marrow HSC/progenitor pool is maintained stably in the absence of Scl. To test whether Lyl1, a bHLH family member and Scl target gene, has an active role in maintaining the Scl induced hematopoietic fate, we generated HSCs/progenitors that do not express either Scl, or Lyl1, or both, by knocking down Lyl1 in Scl-deficient and control HSCs/progenitors via lentiviral shRNA. Analysis of colony forming capacity of transduced Lin−cKit+ HSCs/progenitors revealed that removing either Lyl1 or Scl alone did not have major impact on the clonogenic progenitor pool, whereas loss of both Lyl1 and Scl abrogated colony formation completely. Furthermore, ChIP-chip data revealed that Lyl1 is recruited to the promoters of the majority of Scl's target genes, and can maintain the hematopoietic program in the absence of Scl. In summary, these data show that Scl is critical in inducing the major transcriptional network of HSC genes while repressing alternative mesodermal fates during hematopoietic specification, after which the program is stabilized by Scl's target gene Lyl1. During adult hematopoiesis, relative redundancy between Scl and Lyl1 ensures stability of the HSC fate, while both factors retain unique functions in lineage differentiation.

Disclosures:
No relevant conflicts of interest to declare.