Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing

Abstract

Epigenetic mechanisms control phenotypic commitment of mesenchymal stromal/stem cells (MSCs) into osteogenic, chondrogenic or adipogenic lineages. To investigate enzymes and chromatin binding proteins controlling the epigenome, we developed a hybrid expression screening strategy that combines semi-automatic real-time qPCR (RT-qPCR), next generation RNA sequencing (RNA-seq), and a novel data management application (FileMerge). This strategy was used to interrogate expression of a large cohort (n>300) of human epigenetic regulators (EpiRegs) that generate, interpret and/or edit the histone code. We find that EpiRegs with similar enzymatic functions are variably expressed and specific isoforms dominate over others in human MSCs. This principle is exemplified by analysis of key histone acetyl transferases (HATs) and deacetylases (HDACs), H3 lysine methyl transferases (e.g., EHMTs) and demethylases (KDMs), as well as bromodomain (BRDs) and chromobox (CBX) proteins. Our results show gender-specific expression of H3 lysine 9 [H3K9] demethylases (e.g., KDM5D and UTY) as expected and upregulation of distinct EpiRegs (n>30) during osteogenic differentiation of MSCs (e.g., HDAC5 and HDAC7). The functional significance of HDACs in osteogenic lineage commitment of MSCs was functionally validated using panobinostat (LBH-589). This pan-deacetylase inhibitor suppresses osteoblastic differentiation as evidenced by reductions in bone-specific mRNA markers (e.g., ALPL), alkaline phosphatase activity and calcium deposition (i.e., Alizarin Red staining). Thus, our RT-qPCR platform identifies candidate EpiRegs by expression screening, predicts biological outcomes of their corresponding inhibitors, and enables manipulation of the human epigenome using molecular or pharmacological approaches to control stem cell differentiation.

Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice

“…RNA was then isolated utilizing Ultra Turrax T25 (IKA) and the Direct-zol TM RNA kit (Zymo Research). High-throughput read mapping and bioinformatic analyses for RNA-Seq were performed as reported previously (26,27,29,62). Gene expression is expressed in RPKM.…”

“…Several recent studies from our group have demonstrated that Ezh2 and other epigenetic regulators play a critical role during mesenchymal lineage commitment and osteoblast differentiation (18 -25). It has been established that Ezh2 expression is significantly down-regulated during osteogenic commitment of human adipose-derived stem cells (AMSCs) (26,27). Furthermore, loss or inhibition of EZH2 was shown to enhance osteogenic and inhibit adipogenic differentiation of AMSCs (26), human bone marrow-derived mesenchymal stem cells (BMSCs) (28), and MC3T3 mouse pre-osteoblasts (29).…”

Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affect cartilage development

“…Despite mechanistic differences in chromatin regulation, Ezh1 was shown to regulate a subset of Ezh2 target genes, reinforcing the idea that these proteins may potentially compensate for each other. In addition to mechanism-related differences, Ezh1 and Ezh2 may be expressed at different stages of mesenchymal differentiation (2,19). Previous studies suggest that Ezh2 is expressed in pro-…”

“…transforming growth factor ␤ (TGF-␤), bone morphogenic protein (BMP), wingless/integrated (WNT) (16). Recent studies have also identified epigenetic processes, including histone acetylation and methylation, that contribute to the regulation of differentiation and maturation of mesenchymal progenitors, chondrocytes, and osteoblasts (17)(18)(19)(20)(21)(22)(23)(24).…”

The epigenetic reader Brd4 is required for osteoblast differentiation

“…Several studies have demonstrated that HDAC loss or inhibition promotes hyperacetylation of histones and accelerates osteoblast differentiation (Di Bernardo et al, ; Dudakovic et al, ; Iwami & Moriyama, ; Jensen, Schroeder, Bailey, Gopalakrishnan, & Westendorf, ; H. W. Lee et al, ; S. Lee et al, ; Schroeder, Kahler, Li, & Westendorf, ; Xu et al, ). However, it must be noted that these pro‐osteogenic effects are context dependent as other studies have reported inhibitory effects on osteoblast differentiation and bone formation upon HDAC loss and inhibition (Dudakovic, Camilleri, Lewallen, et al, ; Dudakovic et al, ; McGee‐Lawrence et al, ; Nissen‐Meyer et al, ; Pratap et al, ; Razidlo et al, ; Senn et al, ). Considering the biological complexities of these previous results, it is appreciated that hyperacetylation of bone‐related loci does not suffice to alter gene expression levels and that multiple factors contribute to epigenetic control of the osteogenic gene expression program.…”