ABSTRACT
An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Epigenesis, Genetic , HMGN Proteins/physiology , Histones/metabolism , Nerve Tissue Proteins/genetics , Oligodendroglia/cytology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line , Embryonic Stem Cells/metabolism , Enhancer of Zeste Homolog 2 Protein/metabolism , Female , HMGN1 Protein/genetics , HMGN1 Protein/physiology , HMGN2 Protein/genetics , HMGN2 Protein/physiology , Male , Mice , Mice, Knockout , Nerve Tissue Proteins/metabolism , Oligodendrocyte Transcription Factor 2ABSTRACT
The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling.
Subject(s)
Breast Neoplasms/metabolism , HMGN2 Protein/physiology , Histones/physiology , Prolactin/pharmacology , STAT5 Transcription Factor/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Female , Humans , Protein Processing, Post-Translational , STAT5 Transcription Factor/antagonists & inhibitors , Transcription, Genetic/physiologyABSTRACT
The high-mobility group N (HMGN) proteins are abundant nonhistone chromosomal proteins that bind specifically to nucleosomes at two high-affinity sites. Here we report that purified recombinant human HMGN1 (HMG14) and HMGN2 (HMG17) potently repress ATP-dependent chromatin remodeling by four different molecular motor proteins. In contrast, mutant HMGN proteins with double Ser-to-Glu mutations in their nucleosome-binding domains are unable to inhibit chromatin remodeling. The HMGN-mediated repression of chromatin remodeling is reversible and dynamic. With the ACF chromatin remodeling factor, HMGN2 does not directly inhibit the ATPase activity but rather appears to reduce the affinity of the factor to chromatin. These findings suggest that HMGN proteins serve as a counterbalance to the action of the many ATP-dependent chromatin remodeling activities in the nucleus.
Subject(s)
Chromatin Assembly and Disassembly/physiology , HMGN1 Protein/physiology , HMGN2 Protein/physiology , Nucleosomes/metabolism , Recombinant Proteins/metabolism , Adenosine Triphosphate/metabolism , Chromatin/metabolism , DNA Helicases/metabolism , HMGN1 Protein/genetics , HMGN1 Protein/metabolism , HMGN2 Protein/genetics , HMGN2 Protein/metabolism , Humans , Molecular Motor Proteins/physiology , Mutation , Nuclear Proteins/metabolism , RNA-Binding Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Due to the predominance of multiple-antibiotic-resistant Klebsiella pneumoniae strains, the search for new approaches for the prevention of K. pneumoniae infections is now under intensive investigation. The objective of the present study was to investigate the effects of high mobility group nucleosomal binding domain 2 (HMGN2) protein, which acts on the bladder epithelial cells T 24, on the invasion of K. pneumoniae 03183 and explore its possible mechanisms. Pretreatment with HMGN2 significantly reduced K. pneumoniae 03183 uptake by T 24 cells. In T 24 cells, there were no detectable cytotoxic effects of HMGN2 at any concentration between 32 and 256 µg/ml after 2 h incubation. HMGN2 exhibited no appreciable antibacterial activity against K. pneumoniae 03183. Fluorescence microscopy and flow cytometry analysis revealed that HMGN2 blocked K. pneumoniae 03183-induced actin polymerization. K. pneumoniae 03183-induced phosphorylation of extracellular signal-regulated kinase (ERK) and cofilin were prevented by pretreatment with HMGN2. These results indicated that pretreatment with HMGN2 inhibited cofilin phosphorylation and then induced actin disruption which may block ERK phosphorylation. These changes led to inhibition of K. pneumoniae 03183 invasion of T 24 bladder epithelial cells.
Subject(s)
Epithelial Cells/microbiology , HMGN2 Protein/physiology , Klebsiella pneumoniae/pathogenicity , Urinary Bladder/microbiology , Actins/metabolism , Blotting, Western , Cell Survival/physiology , Cells, Cultured , Flow Cytometry , Humans , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/growth & development , Microbial Sensitivity Tests , Microscopy, Fluorescence , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Signal Transduction , Urinary Bladder/cytologyABSTRACT
The urinary tract is vulnerable to frequent challenges from environmental microflora. Uropathogenic Escherichia coli (UPEC) makes a major contribution to urinary tract infection (UTI). Previous studies have characterized positive roles of non-histone nuclear protein HMGN2 in lung epithelial innate immune response. In the study presented here, we found HMGN2 expression was up-regulated in UPEC J96-infected urothelium. Surprisingly, over-expression of HMGN2 promoted disruption of BECs 5637 cells' intercellular junctions by down-regulating tight junction (TJs) components' expression and physical structure under J96 infection. Further investigation showed that BECs 5637 monolayer, in which HMGN2 was over-expressed, had significantly increased permeability to J96. Our study systemically explored the regulatory roles of HMGN2 in BECs barrier function during UPEC infection and suggested different modulations of intracellular and paracellular routes through which UPEC invades the bladder epithelium.