ABSTRACT
Although there have been substantial advancements in the treatment of inflammatory arthritis, treatments for osteoarthritis (OA) have lagged and currently are primarily palliative until joints become totally dysfunctional and prosthetic replacement is needed. One obstacle for developing a preventive therapy for OA is the lack of good tools for efficiently diagnosing the disease and monitoring its progression during the early stages when the effect of therapeutic drugs or biologics is most likely to be effective. We have developed near infrared immunoliposomes conjugated with type II collagen antibody for diagnosis and treatment of early OA. These immunoliposomes bind to damaged but not normal cartilage. Utilizing these reagents, we can quantitate exposure of type II collagen during cartilage degradation in individual joints in vivo in a guinea pig. Immunoliposomes could be used to determine the effectiveness of therapeutic interventions in small animals as well as vehicles for localized drug delivery to OA chondrocytes. FROM THE CLINICAL EDITOR: This team of authors have developed near infrared immunoliposomes conjugated with type II collagen antibody for diagnosis and treatment of early OA, with promising results demonstrated in a guinea pig model.
Subject(s)
Immunoconjugates/therapeutic use , Liposomes/therapeutic use , Osteoarthritis/diagnosis , Osteoarthritis/therapy , Animals , Cartilage/immunology , Cartilage/pathology , Collagen Type II/analysis , Collagen Type II/immunology , Guinea Pigs , Immunoconjugates/immunology , Liposomes/immunology , Liposomes/ultrastructure , Osteoarthritis/immunologyABSTRACT
Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pull-down assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17ß-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an "activated" state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a "natural" ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.
Subject(s)
Breast Neoplasms/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Estrogen Receptor alpha/metabolism , Gene Expression Regulation, Neoplastic , Nucleosomes/metabolism , Proteomics , Response Elements/genetics , Breast Neoplasms/genetics , Chromatin Immunoprecipitation , DNA/genetics , DNA-Activated Protein Kinase/genetics , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/genetics , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism , Estrogen Receptor alpha/genetics , Estrogens/pharmacology , Female , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , HeLa Cells , Humans , MCF-7 Cells , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Receptor Coactivator 3/genetics , Nuclear Receptor Coactivator 3/metabolism , Nucleosomes/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation , Promoter Regions, Genetic , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trans-Activators , Transcription, Genetic , Transcriptional ActivationABSTRACT
The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.
