NAMPT/NAD+/SIRT1-Triggered Deacetylation Of Hematopoietic Cell-Specific Lyn-Substrate 1 (HCLS1) In Congenital Neutropenia
Authors: Samareh-Abolhasani Bardia , Welte Karl , Skokowa Julia 
Published: October 2019

Hematopoietic-specific lyn-substrate 1 (HCLS1) protein was found to play an important role in the G-CSFR-triggered granulocytic differentiation of hematopoietic cells in vitro and in vivo (Skokowa J. et al., Nature Medicine 2012). Treatment with G-CSF resulted in the phosphorylation and activation of HCLS1 protein. HCLS1 together with its binding partner HAX1 (HCLS1-associated protein X 1) interacts with the LEF-1 transcription factor, inducing nuclear translocation of LEF-1 and activation of LEF-1 target genes (e.g. LEF-1, C/EBPα, HCLS1). These events are essential for myeloid differentiation. In patients with severe congenital neutropenia (CN) HCLS1 expression and functions are severely downregulated leading to severe defects in LEF-1 and LEF-1 target genes expression followed by defective granulopoiesis. We aimed to identify the mechanisms of HCLS1 downregulation in myeloid cells of CN patients. Recently, we described hyperactivation of Nicotinamide phosphoribosyltransferase (NAMPT) / NAD+/Sirtuins pathway upon G-CSF treatment in CN patients. Sirtuins are NAD+-dependent protein deacetylases playing an important role in the regulation of gene transcription. Therefore, we investigated whether HCLS1 is regulated by NAMPT/NAD+/Sirtuin pathway via deacetylation. We identified four putative acetyl-lysine sites in HCLS1 protein and using immunoprecipitation with anti-acetyl-lysine antibody we found that HCLS1 is acetylated in HL60 and NB4 myeloid cells upon pre-treatment with histone deacetylase inhibitors. We generated rabbit polyclonal antibodies specifically recognized each of these acetylated lysines and showed that treatment of HL60 and NB4 cells with high doses of Nicotinamide but not Trichostatin A enhanced HCLS1 acetylation on two lysines, suggesting that class III histone deacetylases of Sirtuin family deacetylate HCLS1 protein.
We further found that NAD+ and NAMPT treatment or overexpression of NAMPT in HL60 and NB4 cells led to deacetylation of HCLS1 protein. At the same time, inhibition of NAMPT using specific inhibitor FK866 increased HCLS1 acetylation. To evaluate the mechanism of NAMPT-dependent deacetylation of HCLS1, we performed immunoprecipitation experiments in cell lysates of HL60 cells and of 293T cells over-expressing HCLS1 and SIRT1 cDNAs using anti-SIRT1 antibody. We found interaction between endogenous HCLS1 and SIRT1 proteins in HL60 cells and between overexpressed HCLS1 and SIRT1 proteins in 293T cells.
Using Duolink in situ proximity ligation assay as well as Western blotting we found that HCLS1 is deacetylated upon G-CSF treatment in CD34+ cells of healthy individuals via NAMPT/NAD+/SIRT1 pathway.
HCLS1 is known to regulate F-actin rearrangement, which is severely affected in myeloid cells of patients with severe congenital neutropenia. Therefore, we evaluated whether deacetylation of HCLS1 affects F-actin rearrangement and cell migration in scratch assay. We transfected 293T cells with different acetyl-lysine mutants of HCLS1 and found significantly less migration of 293T cells transfected with three HCLS1 acetyl-lysine mutants in comparison to cells transfected with wild type HCLS1 protein. These data suggested that deacetylated HCLS1 failed to regulate F-actin rearrangement.
Knowing that HCLS1 plays an important role in the activation of LEF-1 transcription factor we further aimed to evaluate whether deacetylation of HCLS1 has any effects on LEF-1 transcriptional activity. We first analysed the effects of HCLS1 deacetylation on HCLS1:LEF-1 interaction. Using immunoprecipitation we found that mutation at three acetyl-lysines of HCLS1 protein resulted in a weaker HCLS1:LEF1 interaction, in comparison to the interaction between LEF-1 and wild type HCLS1 proteins. Moreover, we demonstrated that in the presence of acetyl-lysine mutants of HCLS1, LEF-1 was not able to activate TOP reporter construct consisting of six LEF1/TCFs binding sites and luciferase promoter, as compared to wild-type HCLS1. Thus, deacetylation of HCLS1 protein by NAMPT/NAD+/SIRT1 pathway disturbs its interaction with LEF-1 protein leading to diminished transcriptional activation of LEF-1. Based on these data, we suggest that in patients with severe congenital neutropenia diminished activation of HCLS1 by deacetylation and a lack of LEF-1-triggered granulocytic differentiation is caused by hyperactivation of NAMPT/NAD+/SIRT1 pathway.

Disclosures:
No relevant conflicts of interest to declare.