Barbara L. Kee

Mature natural killer cell and lymphoid tissue–inducing cell development requires Id2-mediated suppression of E protein activity

The Id2 transcriptional repressor is essential for development of natural killer (NK) cells, lymphoid tissue–inducing (LTi) cells, and secondary lymphoid tissues. Id2 was proposed to regulate NK and LTi lineage specification from multipotent progenitors through suppression of E proteins. We report that NK cell progenitors are not reduced in the bone marrow (BM) of Id2−/− mice, demonstrating that Id2 is not essential for NK lineage specification. Rather, Id2 is required for development of mature (m) NK cells. We define the mechanism by which Id2 functions by showing that a reduction in E protein activity, through deletion of E2A, overcomes the need for Id2 in development of BM mNK cells, LTi cells, and secondary lymphoid tissues. However, mNK cells are not restored in the blood or spleen of Id2−/−E2A−/− mice, suggesting a role for Id2 in suppression of alternative E proteins after maturation. Interestingly, the few splenic mNK cells in Id2−/− and Id2−/−E2A−/− mice have characteristics of thymus-derived NK cells, which develop in the absence of Id2, implying a differential requirement for Id2 in BM and thymic mNK development. Our findings redefine the essential functions of Id2 in lymphoid development and provide insight into the dynamic regulation of E and Id proteins during this process.

Targeting the NF-κB signaling pathway in Notch1-induced T-cell leukemia

T-cell acute lymphoblastic leukemia (T-ALL), unlike other ALL types, is only infrequently associated with chromosomal aberrations, but it was recently shown that most individuals with T-ALL carry activating mutations in the NOTCH1 gene. However, the signaling pathways and target genes responsible for Notch1-induced neoplastic transformation remain undefined. We report here that constitutively active Notch1 activates the NF-κB pathway transcriptionally and via the IκB kinase (IKK) complex, thereby causing increased expression of several well characterized target genes of NF-κB in bone marrow hematopoietic stem cells and progenitors. Our observations demonstrate that the NF-κB pathway is highly active in established human T-ALL and that inhibition of the pathway can efficiently restrict tumor growth both in vitro and in vivo. These findings identify NF-κB as one of the major mediators of Notch1-induced transformation and suggest that the NF-κB pathway is a potential target of future therapies of T-ALL.

E and ID proteins branch out

E and inhibitor of DNA binding (ID) proteins are transcriptional regulators that function in many developmental processes in vertebrates and invertebrates. One subset of E proteins, the E2A proteins, have a central role in the transcriptional regulatory networks that promote commitment to and differentiation of the B- and T-cell lineages, and their function in these lineages is modulated by ID proteins. In this Review, I discuss recent studies that reveal a more extensive role for E and ID proteins in the transcriptional networks that drive the differentiation of many lymphoid lineages, as well as new functions for these proteins in haematopoietic stem cells and their multipotent, but lymphoid-primed, progeny.

Adaptor-mediated Recruitment of RNA Polymerase II to a Signal-dependent Activator

The second messenger cAMP stimulates the expression of a number of target genes via the protein kinase A-mediated phosphorylation of CREB at Ser-133 (Gonzalez, G. A., and Montminy, M. R.(1989) Cell 59, 675-680). Ser-133 phosphorylation enhances CREB activity by promoting interaction with a 265-kDa CREB binding protein referred to as CBP (Arias, J., Alberts, A., Brindle, P., Claret, F., Smeal, T., Karin, M., Feramisco, J., and Montminy, M.(1994) Nature 370, 226-228; Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H.(1993) Nature 365, 855-859). The mechanism by which CBP in turn mediates induction of cAMP-responsive genes is unknown but is thought to involve recruitment of basal transcription factors to the promoter. Here we demonstrate that CBP associates specifically with RNA polymerase II in HeLa nuclear extracts. This association in turn permits RNA polymerase II to be recruited to CREB in a phospho-(Ser-133)-dependent manner. As anti-CBP antiserum, which inhibits recruitment of CBP and RNA polymerase II to phospho-(Ser-133) CREB, attenuates transcriptional induction by protein kinase A in vitro, our results demonstrate that the CBP•RNA polymerase II complex is critical for expression of cAMP-responsive genes.

E2A proteins promote development of lymphoid-primed multipotent progenitors

The first lymphoid-restricted progeny of hematopoietic stem cells (HSCs) are lymphoid-primed multipotent progenitors (LMPPs), which have little erythromyeloid potential but retain lymphoid, granulocyte, and macrophage differentiation capacity. Despite recent advances in the identification of LMPPs, the transcription factors essential for their generation remain to be identified. Here, we demonstrated that the E2A transcription factors were required for proper development of LMPPs. Within HSCs and LMPPs, E2A proteins primed expression of a subset of lymphoid-associated genes and prevented expression of genes that are not normally prevalent in these cells, including HSC-associated and nonlymphoid genes. E2A proteins also restricted proliferation of HSCs, MPPs, and LMPPs and antagonized differentiation of LMPPs toward the myeloid fate. Our results reveal that E2A proteins play a critical role in supporting lymphoid specification from HSCs and that the reduced generation of LMPPs underlies the severe lymphocyte deficiencies observed in E2A-deficient mice.

Early B Cell Factor Promotes B Lymphopoiesis with Reduced Interleukin 7 Responsiveness in the Absence of E2A

The basic helix-loop-helix transcription factors encoded by the E2A gene function at the apex of a transcriptional hierarchy involving E2A, early B cell factor (EBF), and Pax5, which is essential for B lymphopoiesis. In committed B lineage progenitors, E2A proteins have also been shown to regulate many lineage-associated genes. Herein, we demonstrate that the block in B lymphopoiesis imposed by the absence of E2A can be overcome by expression of EBF, but not Pax5, indicating that EBF is the essential target of E2A required for development of B lineage progenitors. Our data demonstrate that EBF, in synergy with low levels of alternative E2A-related proteins (E proteins), is sufficient to promote expression of most B lineage genes. Remarkably, however, we find that E2A proteins are required for interleukin 7–dependent proliferation due, in part, to a role for E2A in optimal expression of N-myc. Therefore, high levels of E protein activity are essential for the activation of EBF and N-myc, whereas lower levels of E protein activity, in synergy with other B lineage transcription factors, are sufficient for expression of most B lineage genes.

Id3 inhibits B lymphocyte progenitor growth and survival in response to TGF-beta.

E proteins function in many developmental processes and are essential for the formation of lymphocyte progenitors. However, it is not known whether E proteins regulate lymphocyte survival, proliferation or differentiation or how their activity is regulated during lymphocyte development. We show here a role for Id3, an inhibitor of E protein activity, in the induction of apoptosis and growth arrest. Id3 is induced in response to transforming growth factor β (TGF-β), a pleiotropic cytokine that inhibits the growth and survival of normal and transformed lymphocyte progenitors. In the absence of Id3, the response of lymphocyte progenitors to TGF-β is perturbed, which indicates that Id3 is a mediator of this response. Our data show a key role for E proteins in lymphocyte survival and link the activity of E proteins, and their antagonists, to members of the TGF-β family of cytokines.

Epigenetic repression of the Igk locus by STAT5-mediated recruitment of the histone methyltransferase Ezh2

During B lymphopoiesis, Igk recombination requires pre-B cell receptor (pre-BCR) expression and escape from interleukin 7 receptor (IL-7R) signaling. By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that STAT5 tetramer bound the Igk intronic enhancer (Eκi), leading to recruitment of the histone methyltransferase Ezh2. Ezh2 marked H3K27me3 throughout Jκ to Cκ. In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R–STAT5 signaling and E2A bound Eκi, resulting in acquisition of H3K4me1 and H4Ac. Genome-wide analyses revealed a STAT5 tetrameric binding motif associated with transcriptional repression. These data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.During B lymphopoiesis, recombination of the locus encoding the immunoglobulin κ-chain complex (Igk) requires expression of the precursor to the B cell antigen receptor (pre-BCR) and escape from signaling via the interleukin 7 receptor (IL-7R). By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that a STAT5 tetramer bound the Igk intronic enhancer (Eκi), which led to recruitment of the histone methyltransferase Ezh2. Ezh2 marked trimethylation of histone H3 at Lys27 (H3K27me3) throughout the κ-chain joining region (Jκ) to the κ-chain constant region (Cκ). In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R–STAT5 signaling, and the transcription factor E2A bound Eκi, which resulted in acquisition of H3K4me1 and acetylated histone H4 (H4Ac). Genome-wide analyses showed a STAT5 tetrameric binding motif associated with transcriptional repression. Our data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.

The TLX1 oncogene drives aneuploidy in T cell transformation

The TLX1 oncogene (encoding the transcription factor T cell leukemia homeobox protein-1) has a major role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). However, the specific mechanisms of T cell transformation downstream of TLX1 remain to be elucidated. Here we show that transgenic expression of human TLX1 in mice induces T-ALL with frequent deletions and mutations in Bcl11b (encoding B cell leukemia/lymphoma-11B) and identify the presence of recurrent mutations and deletions in BCL11B in 16% of human T-ALLs. Most notably, mouse TLX1 tumors were typically aneuploid and showed a marked defect in the activation of the mitotic checkpoint. Mechanistically, TLX1 directly downregulates the expression of CHEK1 (encoding CHK1 checkpoint homolog) and additional mitotic control genes and induces loss of the mitotic checkpoint in nontransformed preleukemic thymocytes. These results identify a previously unrecognized mechanism contributing to chromosomal missegregation and aneuploidy active at the earliest stages of tumor development in the pathogenesis of cancer.

E2A proteins: essential regulators at multiple stages of B-cell development.

Summary: The development of mature B lymphocytes from multipotent progenitor cells requires the co‐ordinated activities of a number of transcriptional regulatory proteins. The transcription factors encoded by the E2A gene are required for the development of committed B‐lineage cells and regulate the expression of essential B‐lineage genes at multiple stages of differentiation and activation. In this review we discuss the evidence that the E2A gene products function in the regulation of 1) transcription factors required for B‐lineage determination, 2) essential proteins involved in pro‐B and pre‐B‐cell development, 3) accessibilty and recombination of the IgH and IgL chain loci, and 4) isotype switching in activated, mature B lymphocytes.