Epigenomics Study for Osteoporosis


Research focuses:

Epigenetics is the new paradigm on the horizon for basic and translational research for complex diseases including osteoporosis. Epigenetic factors refer to reversible, heritable changes in gene regulation that occur without a change in DNA sequences. Two of the most extensively investigated epigenetic factors are DNA methylation and histone modifications. Other epigenetic mechanisms include regulation by non-coding RNAs, such as microRNAs, and mechanisms that control the higher-level organization of chromatin within the nucleus. The constitution of epigenetic marks at a locus for a given time point and cell type forms the epigenotype. Because genomic DNA must exist in a particular chromatin configuration, the genotype can only give rise to phenotype through the prism of the epigenotype, making it an excellent candidate to modify the effects of the genotype and play a role in mediating penetrance and variation in expression.


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The epigenotype shows far greater plasticity than the genotype in the normal development of an individual, and it is reasonable to speculate that epigenetic errors could be a major contributor to human diseases. Emerging evidence suggests that dysregulation of epigenetic factors is an important regulatory and heritable mechanisms in the pathophysiology of human complex diseases, especially for common late-onset disorders. Moreover, given the reversible and relatively easily modifiable nature of epigenetic changes, understanding and manipulating the epigenetic mechanisms hold enormous imminent promise to significantly improve the intervention and treatment of human complex diseases.

A number of molecular epigenetic studies have suggested that epigenetic factors play significant roles in osteogenic cell differentiation and bone metabolism. However, the comprehensive role of epigenetic factors in the pathophysiology of osteoporosis in humans is largely unknown at this time. Such knowledge is necessary and essential in order to identify additional and novel heritable factors contributing to BMD variation and osteoporosis risks, and holds great potential to cross-link basic and clinical research.


We have recently initiated research projects to comprehensively investigate potential epigenetic changes associated with risk to osteoporosis, using state-of-the-art technologies, such as MeDIP-seq and ChIP-seq. These projects are focused on three major epigenetic mechanisms (DNA methylation, histone modification, and microRNA expression), respectively, at the whole epigenome level.  




DNA Methylome Study for Osteoporosis Risk

DNA methylation refers to addition of a methyl group at the carbon-5 site of cytosine residues of the CpG dinucleotide by DNA methyltransferases (DNMTs). CpG dinucleotide accounts for ~1% of human genome and 60%-90% of all CpGs are methylated. Unmethylated CpGs are grouped in clusters called "CpG islands" that are present in the 5' regulatory regions of many genes. DNA methylation can repress gene expression by blocking the binding of transcription factors or modifying chromatin structure to a repressive state.

It has become increasingly evident that alteration in DNA methylatioin has a profound role in the pathogenesis of human diseases, such as cancer and neurodegenerative disorders. The role of DNA methylation in cancer has been relatively well studied. While wide spread DNA hypomethylation can lead to chromosomal instability and inappropriate gene activation, tumor suppressor genes can show promoter hypermethylation and gene inactivation, which contribute to tumour development.


By Barros & Offenbacher J Dent Res 2009; 88: 400

Several studies have suggested that DNA methylation plays a significant role in osteogenic cell differentiation and bone metabolism. For example, Kitazawa et al. (Mol.Endocrinol., 2007. 21: 148-158) analyzed the effect of DNA methylation on the cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. Higher level of CpG methylation at the RNAKL promoters was detected in various tissues expressing no/lower level of RANKL, and in subpopulations of stromal/osteoblastic cells that barely supports osteoclastogenesis. In vitro methylation of the RANKL gene promoter construct in stromal/osteoblastic cells resulted in reduced transcriptional activity and poor response to vitamin D3. In contrast, treatment with DNA methyltransferase inhibitor significantly restored RANKL expression and osteoclastogenesis. These results suggested that CpG methylation of the RANKL gene promoter reversibly suppresses RANKL gene expression, and the heterogeneity of stromal/osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter. Additionally, previous studies have demonstrated that CpG methylation at promoters of the osteocalcin (OC) gene (Villagra A, et al., J Cell Biochem., 2002. 85: 112-122) and the estrogen receptor alpha (ESR1) gene (Penolazzi L., et al., J Steroid Biochem Mol Biol., 2004. 91: 1-9) may influence their gene expression in human osteoblastic cells and osteoblast differentiation.

Therefore, it is imperative to ascertain genomic DNA methylation patterns in osteogenic cells, identify their targeted genes, and determine their relationship to BMD variation. This project holds a great promise of award to yield ground breaking outcomes in the osteoporosis research field. The results will lead to identification of novel heritable factors contributing to human BMD variation and osteoporosis risks. The study may thus lead to a major paradigm shift to greatly expand human genetic studies of osteoporosis from classical DNA sequence variants to novel epigenetics/epigenomics mechanisms of heritable risk to osteoporosis in vivo in humans.

Despite these pioneering DNA methylation studies in the bone field, genome-wide DNA methylation patterns – the methylome – in primary human osteogenic cells is poorly understood. More importantly, the role of DNA methylation in the pathophysiology of osteoporosis in humans is largely unknown at this time.  To address these questions, we recently carried out a pilot comparative methylome profiling analysis in peripheral blood monocytes (PBMs, potential precursors of bone-resorbing osteoclasts), using the MeDIP-Seq method (methylated DNA immunoprecipitation coupled with the next-generation sequencing).  


                                           Workflow of MeDIP-chip /-Seq

Differential methylated regions (DMRs) related to BMD variation will be identified by comparing methylome pattern in PBMs from subjects with low BMD vs. high BMD. Furthermore, by integrating the methylome data with transcriptome data and conducting molecular functional studies, we will identify the DMR targeted genes and determine how these genes contribute to human BMD variation and osteoporosis risks.

Analysis of Histone Modification for Osteoporosis Risk


                                                           By Kishimoto M, Endocrine J, 2006; 53: 157

Histone modification (e.g., acetylation and methylation) is another major epigenetic mechanism in multicellular organisms that regulates gene expression and relates to disease etiology.

The basic structure unit of chromatin is nucleosomes. A single nucleosome contains a histone octamer core, which is formed by two molecules of each of the four core histones (H2A, H2B, H3 and H4)  and ~146 bp DNA wrapped around this octamer core.

N-terminal histone tails are protruded from the nuclesome core and can be post-translationally modified by a number of processes, such as acetylatioin, methylaton, phosporylation. The covalent modification of histones constitutes a potential "histone code" that can be stably transmitted from parent cell to daughter cells.

The histone acetylation (Kac) has been extensively studied and shown to be closedly linked to gene transcriptional regulation through modified chromatin configuration and transcription factor binding. For example, histone acetylation at H3K9 (H3K9ac) and H4K12 (H4K12ac) have been demonstrated to be a predominant signal for gene activation by enhancing the accessibility of the transcription machinery, with the level of acetylation correlating with the rate of transcription. In contrast, histone deacetylases (HDACs) remove acetyl groups, which results in condensed chromatin and gene inactivation. H3K9ac and H4K12ac have been implicated as a key regulatory mechanism in the etiology of a variety of human complex diseases.

Histone acetylation status of several bone candidate genes has been studied in terms of their effects on gene expression and subsequent bone cell differentiation. For example, in vitro experiments indicated that the HDAC inhibitor, Trichostatin A (TSA), has a significant effect on the endogenous expression of RANKL (well known for its significance in bone metabolism) through enhanced acetylation of histones H3 and H4 on the proximal RANKL promoter sequence in bone stromal cells (Fan X, et al., J Cell Biochem., 2004. 93: 807-18). In addition, H3K9ac and H4ac were greatly enhanced at the promoters of osterix and osteocalcin (OC) genes in differentiated osteoblasts, and HDAC inhibitors can enhance osteoblast differentiation and osteogenic gene expression (Lee HW et al. 2006 Mol Endocrinol., 20:2432-43).

                                            Workflow of ChIP-Seq

Despite these pioneering studies on cultured cell lines, the global histone modification profiles in primary human osteogenic cells related to risk of osteoporosis are largely unknown. We are initiating a pilot epigenome-wide H3K9ac and H4K12ac profiling analysis in human PBMs (potential precursors of bone-resorbing osteoclasts), by using the ChIP-Seq technique (chromatin immunoprecipitation followed by next-generation sequencing). Moreover, we will compare the PBM histone modification profiles between subjects with high vs. low BMD values in order to identify human genomic locations with differential intensity of histone modifications, termed differential histone modification site (DHMSs), that are associated with BMD variations. Characterizing DHMSs and thus the associated genes in relation to osteoporosis will provide novel insights into the etiologic and pathogenic mechanisms of osteoporosis.

Epigenomic microRNA Profiling Study for Osteoporosis

Recent discoveries of microRNAs -- small, noncoding RNAs of average 22 nucleotides in length --have revealed a new class of epigenetic mechanisms of gene regulation. microRNAs can regulate gene expression at the post-transcriptional level by inducing RNA degradation through non-perfect base-pairing with their target mRNAs or by inhibiting protein translation.

Mature functional microRNAs are generated from long primary microRNA (pri-microRNA) transcripts. The pri-microRNAs, which usually contain a few hundred to a few thousand base pairs, are first processed into 70-nucleotide pre-miRNAs by Drosha and Pasha inside the nucleus. Pre-microRNAs are then transported into the cytoplasm by Exportin 5 and Ran-GTP and are further processed into small RNA duplexes by Dicer. The functional strand of the microRNA duplex is then assembled into the RNA-induced silencing complex (RISC). Finally, the microRNA guides the RISC to its   mRNA target for translational repression or mRNA degradation.

microRNAs have been implicated to play a profound role in an increasing number of human complex diseases such as cancer, Alzheimer's disease and cardiovascular diseases.  The role of miRNAs in the pathophysiology of osteoporosis in humans is largely unknown at this time. Several studies have suggested that miRNAs play a significant role in osteogenic cell differentiation and bone metabolism.

                       By Chen CZ, N Engl J Med. 2005; 353: 1768

For example, Sugatani et al. (J Cell Biochem. 2007; 101:996-9) demonstrated miR-223 isexpressed in mouse osteoclast precursor RAW264.7 cell line, and plays a key factor in osteoclast differentiation. A recent study by Li et al. (J Clin Invest. 2009; 119:3666-77) identified a new microRNA (miR-2861) in primary mouse osteoblasts that promotes osteoblast differentiation by repressing histone deacetylase 5 (HDAC5) expression at the post-transcriptional level. Importantly, miR-2861 was found to be conserved in humans, and a homozygous mutation in pre-miR-2861 that blocked expression of miR-2861 was shown to cause primary osteoporosis in 2 related adolescents. These results provided compelling evidence that microRNAs play important physiological roles in bone metabolism and pathogenesis of osteoporosis.

Recently we performed a pioneering microRNA profiling study of human PBMs and circulating B cells (another important osteoclastogenic cell) toidentify key microRNAs involved in BMD variation. Using ABI TaqManâLow Density Array that includes 365 human microRNA probes, we performed microRNA profiling of PBMs and B cells in 10 postmenopausal Caucasian females with high BMD vs. 10 with low BMD. In the PBMs, we identified a microRNA, miR-151, that was upregulated in the low vs. the high BMD subjects. By integrating the microRNA profiling data with mRNA expression profiling data of the same set of PBMs, we identified three genes that have expression levels correlated with that of miR-151, suggesting that they may be potential target genes of miR-151. The three genes are TNFSR11, LRCH1 and FZD5. In particular, the LRCH1 and FZD5 genes were also predicted as targets of miR-151 according to online microRNA bioinformatics analysis ( Our findings suggested that miR-151 may contribute to osteoporosis risk through regulating LRCH1 and FZD5. Similarly, we revealed that miR-181b is upregulated in B cells, in low vs. high BMD subjects. Further analysis on the genome-wide gene expression data in the same set of B cells suggested two potential target genes, FGFR1 and MECP2. Currently, we are performing a more comprehensive global microRNA profiling study for osteoporosis using high-density microRNA array and microRNA-Seq techniques, and are expected to identify additional novel microRNAs and their novel target genes for osteoporosis risk.


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