NKAP Must Associate with HDAC3 to Regulate Hematopoietic Stem Cell Maintenance and Survival

Abstract

NKAP is a multifunctional nuclear protein that associates with the histone deacetylase HDAC3. Although both NKAP and HDAC3 are critical for hematopoietic stem cell (HSC) maintenance and survival, it was not known whether these two proteins work together. To assess the importance of their association in vivo, serial truncation and alanine scanning was performed on NKAP to identify the minimal binding site for HDAC3. Mutation of either Y352 or F347 to alanine abrogated the association of NKAP with HDAC3, but did not alter NKAP localization or expression. Using a linked conditional deletion/re-expression system in vivo, we demonstrated that re-expression of the Y352A NKAP mutant failed to restore HSC maintenance and survival in mice when endogenous NKAP expression was eliminated using Mx1-cre and poly-IC, whereas re-expression of wild type NKAP maintained the HSC pool. However, Y352A NKAP did restore proliferation in murine embryonic fibroblasts when endogenous NKAP expression was eliminated using ER-cre and tamoxifen. Therefore, Y352 in NKAP is critical for association with HDAC3 and for HSC maintenance and survival but is not important for proliferation of murine embryonic fibroblasts, demonstrating that NKAP functions in different complexes in different cell types.

The Interaction between NKAP and HDAC3 Is Critical for T Cell Maturation

“…Mice YFP-NKAP(WT) and YFP-NKAP(Y352A) knock-in mice were previously described (16). These were crossed to mice with a loxneo-lox tetracycline transactivator [lnl-tTA; The Jackson Laboratory (19)] expression cassette to allow cre-mediated induction of YFP-NKAP when subsequently crossed with CD4-cre NKAP cKO mice (5), CD4-cre mice (20), Foxp3-YFP-cre NKAP cKO mice (7), and Foxp3-YFP-cre mice (21).…”

“…Similar maturation defects in CD4-cre NKAP cKO and CD4-cre HDAC3 cKO T cells raise the possibility that NKAP and HDAC3 work together to regulate T cell maturation. A single-point mutation, NKAP(Y352A), abrogates NKAP-HDAC3 binding (16). Here, the impact of this mutation on NKAP's roles in T cell maturation was examined.…”

“…Here, the impact of this mutation on NKAP's roles in T cell maturation was examined. We used a mouse model for cre-mediated deletion of endogenous NKAP coupled with cremediated expression of YFP-NKAP(WT) or YFP-NKAP(Y352A) (16). Mice were engineered to express YFP-NKAP(WT) or YFP-NKAP(Y352A) upon CD4-cre-mediated endogenous NKAP gene deletion [i.e., CD4-cre NKAP cKO LNL-tTA YFP-NKAP(WT) and CD4-cre NKAP cKO LNL-tTA YFP-NKAP(Y352A) mice].…”

“…Given the phenocopy between mouse models with cKO of NKAP or HDAC3, and their known interaction, the importance of NKAP association with HDAC3 was recently examined in hematopoietic stem cells (HSCs) (16). Truncation analysis coupled with alanine scanning identified a single point mutation (Y352A) sufficient to abrogate the association of NKAP with HDAC3.…”

“…Induction of YFP-NKAP(WT) but not YFP-NKAP(Y352A) rescued the defects in HSC maintenance and survival resulting from NKAP deficiency, showing that the Y352A mutation impairs the function of NKAP in vivo. Although HSCs required NKAP association with HDAC3, proliferation of mouse embryonic fibroblasts was restored when YFP-NKAP(Y352A) was expressed (16). Therefore, the necessity for NKAP to associate with HDAC3 is context dependent.…”