Pajerowski, Anthony G.

Abstract T cell development depends on the coordinated interplay between receptor signaling and transcriptional activation/repression. We performed a genetic complementation screen, and identified a novel transcriptional repressor, NKAP. NKAP associates with HDAC3 and is part of a DNA-binding complex, as shown by chromatin immunoprecipitation. NKAP also associates with CIR, a part of the Notch corepressor complex. The expression of NKAP during T cell development inversely correlates with the expression of Notch target genes, implying that NKAP may modulate Notch-mediated transcription. To examine the function of NKAP in T cell development, Lck-cre NKAP conditional knockout mice were generated. Interestingly, loss of NKAP blocks development of alpha/beta but not gamma/delta T cells. In addition, lck-cre NKAP cKO DP T cells express 8- to 20-fold higher levels of Hes1, Deltex1 and CD25, providing in vivo evidence that NKAP functions as a transcriptional repressor, acting at least in part, by repression of the Notch pathway.

Abstract During hematopoiesis, proper differentiation of the long-term, multi-potent, self-renewing hematopoietic stem cell (HSC) results in the cellular components of blood. The lineage fate decisions that occur during hematopoiesis result from the coordinated regulation of transcriptional activators, repressors, and chromatin modifiers. We have identified NKAP as a novel regulator of hematopoiesis, that functions at least in part as a transcriptional repressor. In this study we employ a combination of conditional and inducible mouse models of NKAP deletion to explore the role of NKAP in hematopoietic development starting at the HSC stage. Our models allow for both the pan-deletion of NKAP starting in the HSC stage by using a vav-cre transgenic, as well as temporally regulated deletion of NKAP in the hematopietic compartment through the use of the interferon-inducible mx1-cre model system. These complimentary approaches allow us to conclude that NKAP functions in a cell intrinsic manner, as a member of a unique group of transcriptional regulators, and is necessary in the earliest stage of definitive hematopoiesis.

Newly generated T cells are unable to respond to antigen/MHC. Rather, post-selection single-positive thymocytes must undergo T cell maturation to gain functional competency and enter the long-lived naive peripheral T cell pool. This process is poorly understood, as no gene specifically required for T cell maturation has been identified. Here, we demonstrate that loss of the transcriptional repressor NKAP results in a complete block in T cell maturation. In CD4-cre NKAP conditional knockout mice, thymic development including positive selection occurs normally, but there is a cell-intrinsic defect in the peripheral T cell pool. All peripheral naive CD4-cre NKAP conditional knockout T cells were found to be functionally immature recent thymic emigrants. This defect is not simply in cell survival, as the T cell maturation defect was not rescued by a Bcl-2 transgene. Thus, NKAP is required for T cell maturation and the acquisition of functional competency.

Abstract Steady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.