In this study we show that Lyl1 is a crucial player in the transcriptional network that regulates lymphoid specification of multi-potent BM progenitors and the maintenance of uncommitted T cell progenitors. Our data suggest that Lyl1 gains control over the survival and expansion of thymic progenitors during the critical stages of pro-T cell expansion.
Loss of Lyl1 in the hematopoietic system diminishes the capacity to generate LMPPs, most likely accounting for the profound deficiency in lymphoid engraftment seen after transplantation of Lyl1−/−
BM cells15, 17
. However, unlike loss of PU.17, 9, 19
or Ikaros11, 17
, ablation of Lyl1
still permits generation of LMPPs, although in reduced numbers. Consistent with this, we observed relatively mild changes in gene expression in Lyl1-deficient LMPPs.
These characteristics are reminiscent of LMPPs after loss of Tcfe2a
; both Lyl1 and E2A regulate the LMPP population in a dosage-dependent manner and loss of either is associated with reduced apoptosis of LMPPs and increased Bcl2
. Since E2a and Lyl1 are basic-HLH TFs, which heterodimerize in BM progenitors28
, they may interact during lymphoid priming of MPPs to regulate a set of common target genes. On the other hand, several known E2a target genes10, 29, 30
(e.g. Rag1, Dntt, Notch1
) were not differentially expressed in Lyl1−/−
, implying that the relationships of Lyl1 and E-proteins change during hematopoietic development31, 32
. Accordingly, loss of E2a, in contrast with loss of Lyl1, only in mild HSC defects, suggesting that Lyl1-E2a interactions are not critical in HSCs14, 15, 33
. Similarly, during lymphoid differentiation, Ikaros, Pu.1
are not dependent on E2a for their expression, signifying distinct roles for Lyl1 and E2a in early thymocyte progenitors8, 34
. Fully delineating these dynamic relationships would require further analyses.
Our data indicating that Lyl1 is critical to maintain thymopoiesis raises the question of how Lyl1 negatively impacts ETP development. Because ETP numbers are correlated with LMPP numbers35
, one simple explanation could be the reduced number of LMPPs, or a requirement of Lyl1 for thymic homing. However, our data demonstrate a key role for Lyl1 after thymic entry in part through activation of Gfi1. The overlap of the phenotypes observed after loss of either Gfi1 or Lyl1– reduced numbers of Flt3high
LSKs as well as lymphoid engraftment defects– supports this conclusion25, 26
. Moreover, T cell development in both Gfi1- or Lyl1-deficient mice is severely impaired due to increased apoptosis of c-kit+
thymocyte progenitors26, 35
. Nevertheless, Lyl1−/−
HSCs and MPPs exhibit normal Gfi1
expression and lack the defects seen in Gfi1−/−
mice 15, 17
, establishing that Lyl1 is not essential for Gfi1
expression before the LMPP stage. Therefore, control of Gfi1
expression in different progenitor populations is likely mediated by multiple transcription factors in addition to Lyl1 in a context-dependent manner.
Interestingly, Scl/Tal1 was also identified by ChIP-Seq to bind the Gfi1
35kb enhancer element24
. In BM HSCs, Scl and Lyl1 act redundantly to enable HSC survival14
and may be interchangeable in terms of regulating Gfi1
expression. The down regulation of Scl/Tal1 earlier than Lyl1 during thymocyte development may explain the specific sensitivity of T-progenitors to loss of Lyl1 and the non-redundant function of Lyl1. Other regulators such as Pu.1 and Gata2 that control Gfi1
during myeloid development are also not able to compensate for Lyl1 loss at the LMPP-ETP stage. Collectively, these findings support a model in which Lyl1 becomes increasingly important in lymphoid progenitor development and finally indispensible at the ETP-DN2 stage to maintain T progenitor survival and homeostasis via Gfi1.
Proliferation and survival of ETPs is also highly dependent on IL-7-IL-7R pathway activation of Jak-Stat via Bcl2
. During B-cell development, Gfi1 modulates IL-7 receptor signaling through Socs3, a negative Jak regulator, as well as through direct regulation of IL-7R expression38
. Therefore it is possible that similar to B-cell development, Lyl1 controls survival of early thymocytes through Gfi1-dependent regulation of IL-7-IL-7R signaling. This would explain our findings that over-expression of both Gfi1
partially rescued the Lyl1−/−
Since its initial description in human T-ALL, LYL1 has been linked to hematologic transformation, but the underlying mechanisms are elusive. Because loss of E2a leads to T cell lymphomas39
over-expression have been assumed to effect transformation primarily through disrupting E2a homodimers40
. However, our offering a distinct mechanism for the involvement of LYL1 in T-ALL, via control of the T-progenitor pool. Given that super-abundant levels of Lyl1
were fully compatible with T cell development also refutes the competitive inhibition model. The role of Lyl1 in expression of Gfi1
suggests a new potential mechanism for Lyl1-mediated malignant transformation. Activation of Rap1α promotes thymocyte proliferation and transformation41
, whereas Gfi1 inhibits apoptosis42
and enhances cell-cycle entrance43
. These data suggest that LYL1 contributes to transformation via deregulation of critical target genes rather than disruption of E protein function.
Collectively, our data demonstrate that pro-T cell expansion and survival is regulated through intrinsic control of thymic progenitors that employ a transcriptional program already established in hematopoietic stem and progenitor cells. Lyl1 is a critical component of this regulatory network, vital for the maintenance of T lineage homeostasis. Identification of downstream mediators of Lyl1 function illuminates molecular mechanisms underlying early T cell development and suggests previously unrecognized pathways likely to play a role in LYL1-mediated development of leukemia and lymphoma.