Wednesday, September 22, 2010

Increase Height with DNA Methylation?

Previously, in the blog I reported the function of osteoclasts in growing taller.  The use of alendronate inhibited osteoclasts reobsorption capacity and that reduced overall height growth.  What's interesting is that the growth plate was actually larger when osteoclasts were inhibitied.  The authors suggested that this was a result of failure of VEGF to induce chondrocyte apoptosis.  It was also reported previously that growth plate senescence is a result of a decrease in DNA Methylation(Senescence being when a growth plate is inactive).

DNA Methylation is the addition of a Methyl Group to DNA and affects DNA regulation(so transcriptional proteins that stop growth can no longer bind to the gene). DNA Methylation also play a role in cell memory(so cells that were once chondrocytes no longer remember that they're supposed to be chondrocytes).

How does DNA Methylation affect height growth and how does it relate to osteoclasts? Are there any chemicals or substances we can use to alter DNA Methylation within the body?

Expression profile of genes related to osteoclastogenesis in mouse growth plate and articular cartilage.

"Cartilage tissue is broadly classified into transient cartilage (e.g. growth plate, GP) and permanent cartilage (e.g. articular cartilage, AC). The former eventually disappears and is replaced by bone during the endochondral ossification process, whereas the latter retains its permanency. Osteo(chondro)clasts, multinucleated giant cells of the monocyte/macrophage lineage, are selectively induced in the GP during endochondral ossification and play central roles in the resorption of cartilagenous matrices. The aim of this study was to investigate the factors determining the GP-specific recruitment of osteo(chondro)clasts. We especially focused on the expression pattern of the receptor activator of NF-kappaB ligand (RANKL), an essential factor for osteo(chondro)clast differentiation, and on that of epigenetic and transcriptional factors affecting RANKL gene expression. Knee joints of male BALB/c mice aged 8 weeks were dissected and subjected to immunohistochemical analysis using anti-RANKL, Runx2, Dlx5 and Msx2 antibodies. The methylation status of the mouse RANKL gene promoter in both the GP and the AC was analyzed. The expression of BMP-2, -3, -4, -6 and type X collagen mRNA was examined. At the boundary between the calcifying cartilage and the hypertrophic chondrocytes of the GP, RANKL-expressing chondrocytes overlapped those expressing Runx2, Dlx5 and Msx2, near numerous osteo(chondro)clasts. Although similar BMP-2 and -4 expression was observed in chondrocytes in both the GP and the AC as well as in maturing osteoblasts, a rather restricted BMP-6 expression pattern was observed in resting and proliferating chondrocytes in the GP. Mostly non-CpG methylation was scattered in a non-specific manner in chondrocytes in the GP and the AC. Putative Runx2 binding elements are located in the RANKL promoter. Runx2, an essential transcription factor for skeletal development, is a key regulator of RANKL expression in chondrocytes in the GP."

DNA Methylation in growth plates is linked to osteoclasts by RANKL which activates osteoclasts.  BMP-6 is a difference between that chondrocytes of the Articular Cartilage and Growth Plate but if endochondral ossification can be performed safely and effectively within the articular cartilage is unknown.  Osteoclasts may absorb catiligenous matrices which prevent cartilage cells from reaching their full potential.

Growth plate senescence is associated with loss of DNA methylation

"One mechanism that has been proposed to explain replicative senescence involves epigenetic changes in methylation of genomic DNA. Some CG sequences in mammalian genomic DNA are methylated on the cytosine moiety. When DNA is replicated, the new strand is initially not methylated. DNA methyltransferase 1, a maintenance methylase, recognizes the hemimethylated CGs and adds the missing methyl groups. If maintenance methylation is incomplete, methylation levels may gradually decrease with repeated cell replication.  The level of DNA methylation could serve as a cell-cycle counter. The level of DNA methylation can then affect gene expression, in part by altering interaction of transacting elements with the promoter region and in part by altering histone modification, and thus chromatin structure. Such epigenetic changes may contribute to replicative senescence and terminal differentiation in some cell types. Growth plate chondrocytes cultured at high density and exposed to a demethylating agent (5-azacytidine), undergo hypertrophic differentiation. Disruption of PASG (proliferation-associated SNF2-like gene), PASG, which is required for normal maintainance of DNA methylation, results in growth retardation and premature aging. "

So if DNA Methylation does not occur then there is growth retardation but DNA Methylation may serve as a cell-cycle counter to tell growth plates when to stop. So what we have to do is set back the cell-cycle counter some steps to trick it into thinking the number of divisions it's done is less than it currently is.

"Growth plate chondrocytes cultured at high density and briefly exposed to 5-azacytidine, a demethylating agent, differentiate and start expressing markers specific for hypertrophic chondrocytes"<-Since methylation markers increase with differentiation, demethylating chondrocytes may trick them into thinking that they're at an earlier differentiation state.

"We first tested the prediction that cultured growth plate chondrocytes from older animals will undergo fewer cell divisions before undergoing replicative senescence than will chondrocytes from younger animals. Resting zone chondrocytes extracted from fetal, 4-, and 16-week-old male rabbits proliferated for approximately 50 days and underwent three passages (with increasing intervals) before reaching senescence. During this time, chondrocytes from animals of different ages underwent a similar number of population doublings (13.1 ± 1.1 vs 14.6 ± 0.6 vs 14.3 ± 0.8; fetal vs 4 weeks vs 16 weeks respectively). Chondrocytes in primary culture were small, had a round or polygonal shape, and stained for alcian blue and alkaline phosphatase activity, but not for senescence related ß-galactosidase. In contrast, chondrocytes that were becoming senescent were larger, only stained faintly or not at all for alcian blue and alkaline phosphatase activity, but showed an increased senescence-related ß-galactosidase activity"<-Senesent chondrocytes had low alkaline phosphatase level so that may be key for growth. A Senesence B-Galactosidase inhibitor might also increase growth.

"[The] loss of DNA methylation could occur as these resting zone chondrocytes gradually replicate if there is incomplete maintenance methylation of the newly synthesized strand of DNA.  Growth plate senescence is associated with a loss of DNA methylation in rib resting zone chondrocytes. Loss of DNA methylation was observed in the resting zone chondrocytes of the distal ulna. In the distal ulna, loss of methylation with age was observed in the proliferative and hypertrophic zone chondrocytes which are thought to be progeny of the resting zone chondrocytes. Within each age, there was no significant difference in the level of DNA methylation between the different zones of the growth plate. Loss of methylation appears to occur specifically during replication of resting zone chondrocytes but not during the more rapid proliferation of proliferative zone chondrocytes.  There may be complete maintenance methylation in the proliferative zone, but not in the resting zone. Loss of methylation might be responsible for the temporal limits that cause chondrocyte replication to slow with age but not the spatial limits that cause chondrocyte proliferation to slow as the cells descend farther down the chondrocyte columns. The spatial limitation on proliferation may not be controlled by a cell-cycle counter, but instead it may be controlled by a chemical gradient, e.g. parathyroid hormone-related protein."

So you have to make sure those resting zone chondrocytes(stem cells) stay methylated.  I don't know of any good DNA Methylation stimulators.

According to SOX trio decrease in the articular cartilage with the advancement of osteoarthritis, there was no association between age and a reduction of SOX9 methylation promoter regions.

Epigenetic modifiers influence lineage commitment of human bone marrow stromal cells: Differential effects of 5-aza-deoxycytidine and trichostatin A.

"We investigated the effects of the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) or the histone deacetylase inhibitor trichostatin A (TSA) on osteogenic and chondrogenic differentiation. Monolayer cultures of HBMSCs were treated for 3 days with the 5-aza-dC or TSA, followed by culture in the absence of modifiers. Cells were subsequently grown in pellet culture to determine matrix production. 5-aza-dC stimulated osteogenic differentiation as evidenced by enhanced alkaline phosphatase activity, increased Runx-2 expression in monolayer, and increased osteoid formation in 3D cell pellets. In pellets cultured in chondrogenic media, TSA enhanced cartilage matrix formation and chondrogenic structure. epigenetic modifiers, as agents, possibly in combination with other factors,  enhance the ability of HBMSCs to form functional bone or cartilage with significant therapeutic implications therein."

In the study TSA increased COL2A1 and Aggrecan levels which are Sox related genes.

So the supplement that encourage demethylation enhanced osteogenic differentiation.  So this indicates that perhaps supplements that encourage DNA methylation may have the reverse effect(chondrogenic differentiation).

Epigenetic regulation in chondrogenesis.

"DNA methylation in CpG-rich promoters correlates with gene silencing. Histone modification including histone acetylation and deacetylation determines the stability of the chromatin structure. Condensed chromatin (heterochromatin), which has a higher-order histone-DNA structure, prevents the access of transcriptional activators to their target genes. The fundamental unit of eukaryotic chromatin consists of 146 bp of DNA wrapped around a histone octamer. Posttranslational modifications of the histone tail and the chromatin remodeling complex disrupt histone-DNA contacts and induce nucleosome mobilization. Histone acetylation of specific lysine residues in the histone tail plays a crucial role in epigenetic regulation. Histone acetylation is a dynamic process regulated by the antagonistic actions of 2 families of enzymes - the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). The balance between histone acetylation and deacetylation serves as a key epigenetic mechanism for transcription factor-dependent gene expression and the developmental process. DNA methylation, histone acetylation modified by HAT and/or HDAC, and transcription factor-associated molecules contribute to a mechanism that can alter chromatin structure, gene expression, and cellular differentiation during chondrogenesis."

"In chondrocyte differentiation, TGF-β stimulation is necessary for MSC-derived primary chondrogenesis. On the other hand, chondrocyte maturation is inhibited by TGF-β. These conflicting effects of TGF-β during chondrogenesis might depend on chromatin structure and/or the epigenetics of each differentiated stage."

"TGF-β-regulated Smad3 activates the Sox9-dependent transcription on the chromatin structure"

"CpG-rich promoters of chondrogenic-related genes, such as Sox9, Runx2, chondromodulin-I, and fibroblast growth factor receptor 3, are hypomethylated during synovium-derived chondrogenesis"<-Hypomethylated means undermethylated so perhaps too much methylation is bad for chondrogenesis.

"The Col2a1 gene is less methylated in chondrocytes than in fibroblasts"

"The demethylation of the Col10a1 promoter correlates with Col10a1 induction during MSC-derived chondrogenesis"

"In chondrogenesis, p300 stimulates transcription factor-mediated chromatin disruption. Coactivator p300 directly associates with the master chondrogenic factor Sox9, and activates Sox9-dependent transcription. Sox9-dependent transactivation is induced by p300-mediated histone acetylation of chromatin"

"p300 potentiates Sox9-dependent transcription on a chromatinized DNA template and is associated with hyperacetylated histones"

"Coactivator Tip60, which mainly acetylates H4, increases Sox9/Sox5-dependent Col2a1 transcription by associating with Sox9 on chromatin"

"HDAC4, which is expressed in prehypertrophic chondrocytes, regulates chondrocyte hypertrophy and endochondral bone formation by interacting with and inhibiting the activity of Runx2"

"Growth factors, cytokines, and nonproteinaceous chemical compounds including dexamethasone, vitamin D3, prostaglandin E2, and ascorbic acid influence gene expression and cellular differentiation during chondrogenesis"

"BMP-2 induces histone hyperacetylation and methylation at the Sox9 gene on chromatin"

So perhaps you actually want less methylation in genes related to chondrogenesis so a supplement like Sam-e wouldn't help increase height.

Genetic and non-genetic influences during pregnancy on infant global and site specific DNA methylation: role for folate gene variants and vitamin B12.

"[Folate metabolism has a] central role in provision of methyl groups for DNA methylation. Global (LUMA) and gene specific (IGF2, ZNT5, IGFBP3) DNA methylation were quantified in 430 infants. Seven polymorphisms in 6 genes (MTHFR, MTRR, FOLH1, CβS, RFC1, SHMT) involved in folate absorption and metabolism were analysed in DNA from both infants and mothers. Red blood cell folate and serum vitamin B(12) concentrations were measured as indices of vitamin status. Relationships between DNA methylation patterns and several covariates viz. sex, gestation length, maternal and infant red cell folate, maternal and infant serum vitamin B(12), maternal age, smoking and genotype were tested. Length of gestation correlated positively with IGF2 methylation[length of gestation means time it took for growth to occur, IGF2 methylation made growth take longer, larger animals have a larger gestation period] and inversely with ZNT5 methylation.  Methylation of the IGFBP3 locus correlated inversely with infant vitamin B(12) concentration[IGFBP3 decreases height], whilst global DNA methylation correlated inversely with maternal vitamin B(12) concentrations. Analysis of common genetic variants in folate pathway genes highlighted several associations including infant MTRR 66G>A genotype with DNA methylation and maternal MTHFR 677C>T genotype with IGF2 methylation. Both environmental and genetic factors involved in one-carbon metabolism influence DNA methylation in infants. Vitamin B(12) status, infant MTRR genotype and maternal MTHFR genotype, may influence the supply of methyl groups for DNA methylation. Gestational length [may determine] infant DNA methylation patterns."

"Reduced methylation at the IGF2 differentially methylated region, H19 DMR, in cord blood DNA has been associated with increased folic acid intake during pregnancy"<-IGF2 methylation could increase height and therefore folic acid intake during pregnancy may be contraindicated for height growth.

"maternal peripheral blood DNA methylation at the IGF2 locus was associated with maternal serum vitamin B12 levels"<-instead you'd want to take vitamin B12.

Bone morphogenetic protein-2 induces chromatin remodeling and modification at the proximal promoter of Sox9 gene.

"BMP-2 [induces] alterations in chromatin organization around the Sox9 core promoter. Nuclease hypersensitive site mapping following BMP-2 stimulation showed an inducible hypersensitive site in the Sox9 proximal promoter. BMP-2 increased the association of the transcription factor NF-Y with histone acetyltransferase p300/CBP.  The binding of the NF-Y-p300 complex to the Sox9 gene proximal promoter along with PCAF and RNA polymerase II. BMP-2 stimulation caused histone hyperacetylation and methylation at the Sox9 gene{But is Sox9 methylation a way to induce height growth or is it a marker that no further stimulation should be applied to Sox9?}. The activation of Sox9 gene transcription by BMP-2 is associated with chromatin remodeling and histone modification."

"BMP-2 regulates the chromatin structure of the Sox9 promoter through the p38 pathway, independent of the Smad pathway"

Epigenetic regulation of mesenchymal stem cells: a focus on osteogenic and adipogenic differentiation.

"The H3K27me3 mark is thought to be critical to the “stemness” of stem cells, as H3K27 demethylation triggers cellular differentiation"

"the ability of HMTs to methylate H3K9 in order to silence transcription often depends on the methylation status of adjacent lysine residues on H3"

"Acetylation of H3K9 (H3K9ac) and acetylation of H4K16 (H4K16ac) are common marks found on euchromatin near genes that are actively being transcribed"

"histone modification-mediated epigenetic alterations in late-passage MSCs may be responsible for a deceased ability to differentiate as cultured MSCs age."

"strong methylation of lineage specification and developmental promoters may restrict MSC differentiation capacity"

Epigenetic Regulation during Fetal Femur Development: DNA Methylation Matters.

" Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1(+) marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels. During fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1{up}, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development."

"COL9A1 expression levels were significantly correlated with fetal foot length"  Degree of Col9a1 methylation inversely correlated with foot length.

" the degree of [iNos] methylation was inversely correlated to foot length"

"[MMP13] expression significantly correlated with femur length"  Methylation of MMP13 is slightly inversely correlation with foot length.

Foot length is correlated with IL1B Methylation.

DNMT1 expression levels decrease with chondrocyte age.  There is much less of a correlation with age of TET1.


  1. SAMe folinic acid. what do you think of them?

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