ABSTRACT
Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting of H3K4me2 and -me1 marks when associated with CHD4. Our data suggest that GFI1/CHD4 complexes regulate histone modifications differentially to enable regulation of target genes affecting immune response, nucleosome organization or cellular metabolic processes and that both the target gene specificity and the activity of GFI1 during myeloid differentiation depends on the presence of chromatin remodeling complexes.
Subject(s)
Chromatin/metabolism , DNA-Binding Proteins/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Myeloid Progenitor Cells/metabolism , Transcription Factors/genetics , Transcription, Genetic , Animals , DNA-Binding Proteins/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Mice , Transcription Factors/metabolismABSTRACT
Targeted immunotherapy has improved the outcome of patients with high-risk neuroblastoma (NB). However, immune escape of tumor cells still occurs and about 40% of NB patients relapse and die from their disease. We previously showed that natural killer (NK) cell stimulation by Toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDC) increases the efficacy of dinutuximab-based immunotherapy against NB cell lines via the TRAIL death-receptor pathway. With the aim to translate our findings into a novel adoptive therapy of TLR-activated pDC, we investigated the pDC/NK cell axis in NB patients undergoing dinutuximab-based immunotherapy. We show that pDC counts were low at the beginning of immunotherapy but reached normal levels over time. Blood NK cell counts were normal in all patients, although a high proportion of CD56bright CD16low/- cells was observed. The stimulation of patient's blood cells with a TLR9 ligand led to IFN-α production by pDC, and TRAIL expression on NK cell surface. Patient's NK cells expressed high levels of CD69 and TRAIL after stimulation with activated pDC. Both CD56bright CD16low/- and CD56dim CD16+ NK cells degranulated against autologous target cells in the presence of dinutuximab. Importantly, pDC-induced NK cell activation increased the dinutuximab mediated autologous killing of patient-derived NB cells. Altogether, our study demonstrates that TLR-activated pDC strongly increase the cytotoxic functions of NK cells in high-risk NB patients undergoing immunotherapy. These results, therefore, support pDC adoptive immunotherapy as a novel approach to decrease the risk of relapse in patients with high-risk NB.
Subject(s)
Antibodies, Monoclonal/pharmacology , Dendritic Cells/immunology , Killer Cells, Natural/immunology , Neuroblastoma/drug therapy , Neuroblastoma/immunology , Adolescent , Antibodies, Monoclonal/immunology , Antigen Presentation/immunology , Child , Child, Preschool , Cytotoxicity, Immunologic/immunology , Female , Humans , Immunotherapy/methods , Immunotherapy, Adoptive/methods , Lymphocyte Activation/immunology , Male , Neoplasm Recurrence, Local/immunology , Toll-Like Receptors/immunologyABSTRACT
The alpha-crystallins are members of the small heat shock protein family of molecular chaperones that have evolved to minimize intracellular protein aggregation; however, they are also implicated in a number of protein deposition diseases. In this study, we employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We replicated the oligomeric rearrangements observed for post-translationally modified alpha-crystallins, without altering the protein sequence, by refolding the alpha-crystallins in vitro. This refolding resulted in a loss of dimeric substructure concomitant with an augmentation of substrate affinity. We show that packaging of small heat shock proteins into dimeric units is used to control the level of chaperone function by regulating the exposure of hydrophobic surfaces. We propose that a bias toward monomeric substructure is responsible for the aberrant chaperone behavior associated with the alpha-crystallins in protein deposition diseases.
