MMP-9 facilitates selective proteolysis of the histone H3 tail at genes necessary for proficient osteoclastogenesis

Abstract

Enhancer/promoter elements from two pancreas-specific genes, those encoding amylase and elastase, were ligated to the bacterial GPT gene. The resulting construct can be used to select for expression of gene products which activate these pancreas-specific promoters in hybrid cells. The selectable GPT construct was stably transferred into several cell lines either directly or by cotransfection with pSV2Neo. GPT hybrid regulator contains binding sites for several nuclear proteins, including two sites for PTF1 (9,19,38).In this paper, we demonstrate that the hybrid construct containing the amylase/elastase regulator fused to the selectable GPT gene is not expressed after transfection into mouse hepatoma or fibroblast cell lines but can be activated by fusion with pancreatic acinar cell lines. The nonexpressed construct is thus present in a state which permits activation by trans-acting factors. MATERIALS AND METHODSPlasmid construction. An amylase/elastase/CAT construct (AmyEICAT) (19) containing the Amy2.2 enhancer (bp -208 to -82) and the rat elastase I enhancer/promoter (bp -205 to +8) was digested with HindIll and BamHI to yield the hybrid enhancer/promoter fragment. This hybrid enhancer/ promoter element was used to replace the simian virus 40 (SV40) promoter region in pSV2GPT (27,28,37) by the following protocol. pSV2GPT was digested with AccI, blunt ended, ligated with HindIII linkers, and digested with BglII to remove the SV40 enhancer and promoter elements. AmyEIGPT was made by directional cloning in which the HindIII-BamHI fragment from AmyEICAT was ligated to the promoterless GPT construct (Fig. 1). EIGPT was made by subcloning the HindIII-BamHI fragment from EICAT (23) which contains the rat elastase I enhancer/promoter (bp -205 to +8) into the same promoterless GPT-containing vector. pG2EIGPT, used for the detection of GPT transcripts, was generated by subcloning the HindIII-KpnI fragment of EIGPT (bp -208

BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells in mice

“…The values were adjusted globally by matching count distributions at the 75th percentile and then adjusting counts to a uniform distribution between samples. Differential expression was estimated by selecting transcripts that displayed significant changes (P<0.05) after Benjamini and Hochberg correction using a null model constructed from 1% of transcripts showing the closest average level of observation to estimate experimental noise, as detailed previously (Kim et al, 2016) except that an additional replicate t-test was used (FDR P<0.05) that showed the consistency of gene expression across control and mutant groups. The gene list was further ranked using fold change criteria.…”

“…The raw data has been deposited with NCBI under accession number GSE79791. To investigate potential functional enrichment of various biological pathways in differentially expressed genes in RNA-seq, a ranked P-value was computed for each pathway from the Fisher exact test based on the binomial distribution and independence for probability of any gene belonging to any enriched set, as detailed previously (Kim et al, 2016).…”

Regulation of Breast Cancer-Induced Osteoclastogenesis by MacroH2A1.2 Involving EZH2-Mediated H3K27me3

“…In fact, previous studies including ours identified some of histone modifications as possible mechanisms underlying epigenetic regulation of osteoclastogenic transcription programs (Kim et al, 2016b; Zhang et al, 2015). Despite these advances, however, nothing is currently known about the effects of histone variants on the process of osteoclast differentiation.…”

“…CMs were prepared by culturing cells to ~80% confluence, changing media to α-minimum essential medium (α-MEM) containing 0.5% FBS, and collecting media after 24 hr incubation. For osteoclast differentiation assays, bone marrow cells were isolated from 6-week-old C57BL/6 mice (The Jackson Laboratory) as recently described (Kim et al, 2016b) and in accordance with the University of Southern California guidelines. After priming the harvested cells with a-MEM containing 10% FBS and macrophage colony stimulating factor (M-CSF, 5 ng/mL) for 16 hr, non-adherent mononuclear cells were collected and cultured for an additional 3 days with α-MEM containing M-CSF (30 ng/mL) to generate primary osteoclast precursor (OCP) cells.…”

Matrix metalloproteinases in bone development and pathology: current knowledge and potential clinical utility

“…27,38 Recently, MMP-9 has been reported to also be necessary for the regulation of gene pathways that are required for osteoclastogenesis, through its proteolytic interaction with the histone H3 N-terminal tail (H3NT). 39 Further studies must be conducted to better understand the mechanisms underlying these bone pathologies in MMP-9 null mice.…”