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Blood, 1 February 2005, Vol. 105, No. 3, pp. 1365-1366.
CORRESPONDENCE To the editor:SCL/Tal1 and lymphoid versus myeloid lineage assignmentIn their recent paper, Kunisato et al1 describe the role of stem cell leukemia gene (SCL) in regulating lineage fate in hematopoietic stem cells. Their experiments involve retroviral expression of SCL and a "dominant-negative" mutant of SCL (DN-SCL) in hematopoietic stem cells and their progeny. They propose that levels of SCL regulate lineage commitment: enforced expression of SCL favored myeloid differentiation, while expression of the DN-SCL favored lymphoid differentiation. We query the interpretation of the results obtained with the DN-SCL mutant, as its design and effects are not suggestive of a specific dominant-negative function. The authors cite Aplan et al2 and Krosl et al3 for the design of the dominant-negative SCL. In these papers the basic domain of SCL was deleted. This mutant is unable to bind to DNA, however, heterodimerization with E2A proteins remains intact through the presence of the helix-loop-helix (HLH) domain. The DN-SCL mutant used by Kunisato et al1 lacks both the basic and HLH domains. Such a mutant would be predicted to abrogate not only DNA binding, but also the ability to interact with E2A proteins. The remaining N- and C-terminal portions of SCL have no known function—indeed, a truncation mutant comprising only the basic and HLH domains could rescue hematopoiesis of SCL-null embryonic stem cells,4 suggesting that the N- and C-terminal amino acids are not essential. Since a dominant-negative mutant usually relies on deletion of specific functional domains while retaining vital protein interactions, it is difficult to understand how this mutant could act as a dominant negative. Moreover, enforced expression of the DN-SCL only mildly affects erythroid cell production in vitro or in vivo (Figures 3 and 7), whereas loss of SCL by conditional deletion has demonstrated that SCL is essential for erythroid burst-forming units (BFU-E) and production of red cells in vivo.5-7 Thus, there is no available data to positively suggest that the DN-SCL used by Kunisato and colleagues1 inhibits the function of SCL. Nonetheless, it is possible that the N- and C-terminal portions of SCL have an unknown function that causes the observed effects on lineage specification. However, without the correct controls, such as rescue of the DN-SCL effect with wild-type SCL, it is impossible to discriminate specific from nonspecific effects. In light of this and since the effects on myeloid and lymphoid lineage output are subtle and transient, it is important to regard with caution the assertion that the effects are due to a dominant-negative effect on SCL.
Correspondence: Mark Hall, Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia; e-mail: hall{at}wehi.edu.au. References
Response:Dominant-negative activity of stem cell leukemia (SCL) lacking bHLH domainQueries from Hall and Curtis on our paper1 in Blood include some important issues. As they argue, the construct of interest ( bHLH SCL) may not have an ability to interact with E2A proteins. Indeed, our experiment showed that it does not interact with wild-type (WT) stem cell leukemia (SCL) (data not shown). However, this does not imply that bHLH SCL consisting only of the N- and C-terminal portions of SCL does not have any function. Contrary to the argument by Porcher et al,2 their results could indicate that the N- and C-terminal portions of SCL have some roles, since it appears that the bHLH domain alone does not completely rescue the SCL-null phenotype. In addition, as was described in our paper (Figure 7), we found maturation arrest in the erythroid progenitors by introducing bHLH SCL. This observation is considered to be biologic evidence of dominant-negative effect of bHLH SCL on wild-type SCL, given the phenotype of SCL conditional knockout mice.3 In this regard, we are afraid that the questioners may misunderstand our description in the paper.
To explore the proteins that interact with
We disagree with the comment by the questioners that we should be able to show the rescue of the effect of
Although the biochemical mechanisms need to be further disclosed, clear are our findings on the distinct biologic functions of WT SCL and
Correspondence: Shigeru Chiba, Department of Cell Therapy and Transplantation Medicine, University of Tokyo Hospital 7-3-1, Hongo, Bunkyo-ku, Tokyo; e-mail: schiba-tky{at}umin.ac.jp.
References
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