Search tips
Search criteria 


Logo of stemcrGuide for AuthorsAbout this journalExplore this journalStem Cell Reports
Stem Cell Reports. 2016 June 14; 6(6): 970–984.
Published online 2016 June 14. doi:  10.1016/j.stemcr.2016.05.007
PMCID: PMC4911502

Age-Associated Decrease of the Histone Methyltransferase SUV39H1 in HSC Perturbs Heterochromatin and B Lymphoid Differentiation


The capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.

Graphical Abstract

An external file that holds a picture, illustration, etc.
Object name is fx1.jpg


All myeloid and lymphoid blood cell lineages are continually replenished throughout adult life from a reservoir of rare multipotent hematopoietic stem cells (HSC) residing in the bone marrow. Studies in both humans and mice have shown that HSC are not constant throughout life (Chambers et al., 2007, Dykstra et al., 2011, Flach et al., 2014, Kuranda et al., 2011, Lescale et al., 2010, Pang et al., 2011, Rossi et al., 2005). There is an increase in the number of phenotypically defined HSC with age, but the stem cells that accumulate exhibit a diminished long-term reconstitution potential as well as a cell-intrinsic reduction in their capacity to generate immune-competent B lymphocytes, leading to a myeloid-biased differentiation output. This age-associated skewing of HSC differentiation potential from lymphoid to myeloid lineages, and the resultant decreased output of naive B cells, leads to a decline in antibody diversity and is believed to contribute to the general depletion of immune function observed in the elderly (reviewed in Denkinger et al., 2015). HSC aging is driven by both cell-extrinsic alterations in the stem cell niche and systemic signals, as well as changes intrinsic to the stem cells themselves (reviewed in Garrick et al., 2015, Geiger et al., 2013), including widespread changes in gene-expression patterns (Chambers et al., 2007, Flach et al., 2014, Pang et al., 2011, Rossi et al., 2005, Sun et al., 2014).

While the molecular triggers for these transcriptomic and functional changes are still incompletely understood, recent studies in mouse HSC have demonstrated that aging is associated with alterations in the DNA methylation and histone modification profiles (Beerman et al., 2013, Sun et al., 2014), suggesting that disruption of the normal epigenetic state is an important factor in the aging HSC phenotype. One key component of the epigenetic landscape is the formation of domains of heterochromatin. These regions of compacted and transcriptionally repressive chromatin are critical for diverse aspects of nuclear biology, including the regulation of gene-expression patterns, the transcriptional silencing of genomic repeats, and the maintenance of genome stability, as well as normal centromere and telomere function (Bulut-Karslioglu et al., 2014, Grewal and Jia, 2007, Peters et al., 2001, Schoeftner and Blasco, 2009). One of the principal enzymes involved in the formation of heterochromatin is SUV39H, a family of two histone methyltransferases (SUV39H1/KMT1A and SUV39H2/KMT1B) that catalyze tri-methylation of lysine 9 of histone H3 (H3K9me3) (Peters et al., 2001). The H3K9me3 histone modification is recognized and bound by members of the heterochromatin protein 1 (HP1) family (Lachner et al., 2001), critical adaptor proteins that coordinate chromatin compaction by undergoing self-association as well as recruiting histone deacetylases, DNA methyltransferases, and structural RNAs (reviewed in Maison and Almouzni, 2004). Consistent with a crucial role for heterochromatin during differentiation and development, it has been shown that SUV39H1-mediated H3K9me3 regulates lineage commitment during early mouse development by repressing lineage-inappropriate genes (Alder et al., 2010) and that depletion of SUV39H gives rise to pre- and postnatal developmental defects and lethality in mice (Peters et al., 2001). Accumulating evidence suggests that SUV39H may also regulate various aspects of hematopoiesis. The SUV39H1/HP1 regulatory axis is important to maintain cellular fate following commitment to the T helper 2 (TH2) lymphocyte lineage (Allan et al., 2012). Further, deletion of Suv39h in mice leads to the development of late-onset B cell lymphomas (Peters et al., 2001), while overexpression of SUV39H leads to impaired erythroid differentiation (Czvitkovich et al., 2001). However, at present the role of SUV39H and heterochromatin structure in HSC aging and the regulation of differentiation potential has not been investigated directly.

In this study we show that SUV39H1 plays an important role in the differentiation of human HSC toward the B cell lineage. Expression of SUV39H1 in HSC declines with age, leading to relaxation of heterochromatin and derepression of genomic repeat elements. The age-associated decrease in SUV39H1 in human HSC correlates with an increase in expression of miR-125b, a microRNA (miRNA) which has been shown previously to target SUV39H1 and is a key regulator of HSC self-renewal and differentiation potential. Both inhibition of miR-125 and overexpression of SUV39H1 improved the B cell output of HSC from elderly individuals. Our findings support previous studies implicating the loss of heterochromatin as a key hallmark of aging and suggest that this axis may be targeted to improve HSC function with age.


SUV39H1 Is Highly Expressed in Human HSC and Is Important during B Cell Generation

The SUV39H family comprises two members, SUV39H1 and SUV39H2. SUV39H1 is the predominant form expressed in both human cord blood and bone marrow CD34+CD38 HSC, as has previously been observed in mouse long-term repopulating HSC (LT-HSC) (Figures S1A and S1B; Sun et al., 2014). Analysis of SUV39H1 expression during human hematopoietic differentiation revealed that SUV39H1 is highly expressed in the primitive HSC compartment and that its expression decreases during myeloid differentiation, but is maintained in bone marrow B lymphoid cells (Figure 1A). To investigate the role of SUV39H1 during HSC differentiation, we treated bone marrow CD34+ progenitors with chaetocin, an inhibitor of SUV39H (Greiner et al., 2005), and analyzed the effects on B lymphoid and myeloid output in in vitro differentiation assays. Treatment with chaetocin induced a significant decrease in the global levels of H3K9me3 in CD34+CD38 HSC as assessed by fluorescence-activated cell sorting (FACS) analysis, while histone H3 acetylation (H3ac) levels remained unchanged, showing that SUV39H1 is important for the maintenance of H3K9me3 levels in HSC (Figures 1B and S2A). We found that chaetocin decreased the generation of B lineage cells following in vitro differentiation, whereas the production of myeloid colonies was not affected (Figure 1C). Treatment with chaetocin (10–100 μM) of CD34+CD38 HSC, CD34+CD33+ myeloid progenitors, or CD34+CD19+ B lymphoid progenitors did not lead to increased apoptosis as measured by Annexin V staining, indicating that loss of B cell generation observed in these differentiation assays was not due to treatment-related toxicity (data not shown). As chaetocin may also inhibit the activity of other histone lysine methyltransferases, to confirm that the decline in B cell differentiation was indeed due to an effect on SUV39H1, human CD34+ stem and progenitor cells were transfected with small interfering RNA (siRNA) targeting SUV39H1, or non-silencing control (Figures 1D and S2B–S2D). Knockdown of SUV39H1 mRNA and protein relative to control-treated cells was confirmed by qRT-PCR and FACS analysis, respectively (Figures S2C and S2D). In vitro differentiation assays performed on siRNA-transfected cells revealed that knockdown of SUV39H1 specifically decreased B cell differentiation, without affecting myeloid output (Figure 1D), similar to the result observed with chaetocin. In contrast, no effect on B cell differentiation was observed using the same concentration range of the control, non-targeting siRNA (Figure S2E). Taken together, these results indicate that SUV39H1 is highly expressed in HSC and is an important factor for human B lymphopoiesis.

Figure 1
The Histone Methyltransferase SUV39H1 Is Critical for B Lymphoid but Not Myeloid Differentiation

Decreased SUV39H1 and Global Levels of H3K9me3 in HSC with Age

Aging is associated with functional changes in the HSC compartment and, in particular, the capacity of HSC to generate B cells is decreased with age in both mice and humans (Dykstra et al., 2011, Kuranda et al., 2011, Lescale et al., 2010, Pang et al., 2011, Rossi et al., 2005). Our observations that SUV39H1 is highly expressed in the HSC compartment and is required for B cell differentiation prompted us to investigate whether expression of this factor is altered with age. We found that expression of SUV39H1 is significantly decreased in human CD34+CD38 HSCs isolated from the bone marrow of elderly individuals (>70 years) relative to the level observed in the same population from young individuals (<35 years) (Figures 2A, S3A, and S3B). In contrast to SUV39H1, expression of SETDB1/KMT1E, a methyltransferase which also catalyzes H3K9me3 (Schultz et al., 2002) did not change with age in HSC (Figure S3B). In order to investigate whether the observed decrease of SUV39H1 affected global levels of H3K9me3, we measured this modification by intracellular FACS in CD34+38 HSC isolated from young and old individuals. We found that aging was associated with a reduction in the levels of H3K9me3, while no change was observed in the global levels of H3ac (Figure 2B). Consistent with these findings in human HSCs, we observed a similar decline with age in the expression of Suv39h1 in both the mouse LinSCA1+c-KIT+ (LSK) population of hematopoietic stem and progenitor cells, as well as in the more primitive LT-HSC (LinSCA1+c-KIT+FLT3CD34CD150+) (Figures 2C and S3C). Within the murine HSC compartment, reduced expression of Suv39h1 was associated with a decrease in the global levels of H3K9me3 as assessed by immunofluorescence in LT-HSC and short-term repopulating HSC (ST-HSC; LinSCA1+c-KIT+FLT3CD34+) (Figure 2D). In ST-HSC, where a higher proportion of cells have exited quiescence (Wilson et al., 2008), we also observed a decrease with age in the level of the heterochromatin-binding protein HP1α, suggesting that downstream changes in chromatin are more strongly detected once the stem cells start cycling (Figure 2E). These results demonstrate that aging is associated with reduced SUV39H1 expression and decreased levels of H3K9me3 in the HSC compartment, indicative of perturbed heterochromatin.

Figure 2
Age-Related Decrease in SUV39H1 Expression and Histone H3K9me3 Levels in Human and Mouse HSC

Decreased Expression of SUV39H1 in HSC Results in Derepression of Genomic Repeats with Age

To investigate the effects of these age-associated changes on heterochromatin function in HSC, we analyzed the expression of a number of genomic repetitive elements which are normally repressed by the H3K9me3/HP1 heterochromatic axis (Sharma et al., 2012). In human HSC, aging was associated with increased expression of several of these repeat elements, including Satellite II tandem pericentromeric repeats as well as certain dispersed human endogenous retrovirus elements (HERV-K/env102 and HERV-H/env62) (Figure 3A). Consistent with these results, we found that several mouse endogenous retroviruses and interspersed repeats previously shown to be controlled by Suv39h-mediated H3K9me3 in embryonic stem cells (particularly active LINE1 family members L1_A and L1_Tf as well as intracisternal A-type particles [IAP]) (Bulut-Karslioglu et al., 2014) were also upregulated with age in murine LSK cells (Figure 3B). The transcriptional derepression of interspersed repeats has also been reported previously in LT-HSC of old mice (Sun et al., 2014). Similar to these changes observed with age, expression of LINE1 and IAP elements was also upregulated in LSK cells of Suv39h1null mice (Figures 3C and S4A), indicating that SUV39H1-mediated H3K9me3 is indeed required for the normal transcriptional repression of these repeats in the HSC compartment. Consistent with previous observations, no significant differences were observed between the B cell compartments of Suv39h1null and wild-type (WT) mice under homeostatic conditions (data not shown and Allan et al., 2012, Braig et al., 2005, Peters et al., 2001). However, competitive transplantation experiments revealed that Suv39h1null bone marrow progenitors exhibited a myeloid-biased engraftment potential, a characteristic of aged HSC (Figure S4B). Interestingly, Suv39h1null LSK cells also showed elevated expression of the Selp gene encoding P-SELECTIN, a hallmark gene whose expression is increased with age in both human (Pang et al., 2011) and mouse HSC (Flach et al., 2014, Rossi et al., 2005, Sun et al., 2014) (Figure S4C). These results support the idea that the age-associated decrease in expression of SUV39H1 in HSC leads to relaxation of heterochromatin structure and to transcriptional derepression, including upregulation of certain genomic repeat elements.

Figure 3
Expression of Genomic Repeat Elements in HSC Is Upregulated with Age and in the Absence of SUV39H1

miR125b Increases with Age and Targets SUV39H1 in HSC

We next investigated the mechanism responsible for the age-associated decline in expression of SUV39H1. miRNAs are known to play important roles in controlling hematopoietic differentiation programs (Baltimore et al., 2008) and have more recently been implicated in HSC aging (Mehta et al., 2015). We used miRWalk2.0 (Dweep et al., 2011) to search for miRNAs that are predicted to target the human SUV39H1 transcript. Among the most significant hits were members of the miR-125 family (miR-125a and miR-125b), which are predicted to bind to a perfect 8-mer target site that has been evolutionarily conserved within the 3′ UTR of the SUV39H1 transcript (Figure 4A). This was of particular interest, since members of this family have been shown to be critical regulators of HSC self-renewal and differentiation potential in mice (Guo et al., 2010, O'Connell et al., 2010, Ooi et al., 2010). Notably, overexpression of miR-125 has been reported to lead to a myeloid-biased differentiation of HSC, similar to that observed with age (Gerrits et al., 2012, Guo et al., 2010, O'Connell et al., 2010, Wojtowicz et al., 2014). The miR-125 family consists of three mature miRNAs (miR-125b-1, miR-125b-2, and miR-125a), which share an identical seed sequence and exert similar effects on HSC function (Wojtowicz et al., 2014). We found that the level of mature miR-125b is significantly increased in CD34+CD38 HSC from elderly (>70 years) as compared with young (<35 years) individuals, a result that was confirmed with two independent real-time PCR assays (Figures 4B and S5A). We also observed a significant increase in the immature primary miRNA transcript pri-miR-125b-2, but not pri-miR-125b-1 (Figures 4B and S5B). An increase was also observed in the levels of mature miR-99a, which is part of a tricistronic cassette with miR-125b-2 and let-7c on human chromosome 21 (Emmrich et al., 2014), suggesting that the entire miR-125b-2 tricistronic cluster is upregulated with age in human HSC (Figure 4B). We also observed an age-associated increase in the level of mature miR-125a, although this miRNA appears to be expressed at lower levels in CD34+CD38 HSC (Figures S5C and S5D). These findings, therefore, reveal an age-associated increase in the level of miR-125 miRNA in human HSC.

Figure 4
Expression of miR-125b Increases with Age in Human HSC

To test whether SUV39H1 is indeed a target of miR-125, we initially ectopically expressed miR-125b in HeLa cells. Transfection of HeLa cells with a miR-125b mimic, a double-stranded RNA oligonucleotide mimicking the mature miRNA, resulted in downregulation of SUV39H1 mRNA and protein (Figures 5A–5C). We also observed reduced SUV39H1 expression and decreased enrichment of H3K9me3 at pericentromeric (SAT II) and dispersed (HERV/env102) genomic repeats following lentiviral-mediated expression of miR-125b in HeLa cells (Figures 5D–5F). Consistent with these findings, SUV39H1 has also been shown to be regulated by miR-125b in murine muscle cells (Villeneuve et al., 2010) and human hepatocellular carcinomas (Fan et al., 2013). To investigate whether miR-125b also regulates SUV39H1 in HSC, CD34+CD38 HSC from elderly individuals were treated with a miR-125b inhibitor, a chemically modified single-stranded oligonucleotide that interacts with mature miR-125b. Treatment of aged HSC with the inhibitor resulted in upregulation of SUV39H1 transcripts indicating that SUV39H1 is a target of miR-125b in human HSC (Figure 5G). This was confirmed in a complementary set of experiments where cord blood CD34+CD38 HSC, which express high levels of SUV39H1 (Figure S1A), were transfected with the miR-125b mimic. After 24 hr with the miR-125b mimic, expression of SUV39H1 was significantly depleted (Figure 5H). Furthermore, overexpression of miR-125b in CD34+CD38 HSC led to a decrease in levels of H3K9me3 (Figures 5I and 5J), as well as increased expression of a subset of genomic repeats (Figure 5K), indicating a disruption of the normally repressive heterochromatin structure at these elements. Interestingly, the repeat elements that responded to the miR-125b mimic (HERV-H/env62 and HERV-K/env102 and Satellite II) were the same as those whose expression increased with age in human HSC (Figure 3A). These results demonstrate that SUV39H1 is targeted by miR-125b in HSC and that the levels of this miRNA impact upon HSC heterochromatin.

Figure 5
SUV39H1 Is a Target of miR-125b

miR125b Regulates the B Cell Potential of Human HSC

The above experiments indicate that overexpression of mir-125b causes similar changes in heterochromatin as those occurring in aged HSC. To examine the effects of miR-125b on the differentiation potential of human HSC, we initially transduced cord blood CD34+CD38 HSC with control or miR-125b-expressing lentiviruses, and the cells were plated in in vitro differentiation cultures to assess B lymphoid and myeloid output. Overexpression of miR-125b resulted in a significant decrease in the generation of CD19+ B cells and an increase in CD33+ myeloid cells (Figures 6A and 6B), resulting in an increased ratio of myeloid to B lymphoid progeny in the assay (Figure 6C). No changes in differentiation were observed in the untransduced population (Figures S6A and S6B). These experiments indicate that, as with age, overexpression of miR-125b in human HSC leads to decreased B lymphopoiesis and a myeloid-biased differentiation output.

Figure 6
Expression of miR-125b Decreases B Cell Output in Human HSC

Inhibition of miR-125 or Overexpression of SUV39H1 Enhances B Lymphoid Differentiation Capacity of Aged HSC

Finally we investigated whether inhibition of miR-125b or overexpression of SUV39H1 in HSC from elderly individuals improved their capacity to generate B lymphocytes. We first transduced CD34+CD38 HSC purified from the bone marrow of elderly individuals (>70 years) with a miR-125 “sponge” lentivirus, which expresses a transcript containing four target sites for miR-125, or a control lentivirus containing four scrambled target sites. In vitro differentiation analysis of transduced cells revealed that expression of the miR-125 sponge in aged HSC resulted in a significant increase in the output of CD19+ B cells and a reduction in the myeloid:lymphoid ratio (Figures 7A and 7B). No changes were observed in the untransduced population (Figures S6C and S6D). We next examined the effect of SUV39H1 overexpression on the differentiation potential of aged HSC. For these experiments, we generated a lentiviral construct expressing human SUV39H1 together with GFP. This lentivirus restored the WT concentration of H3K9me3 at pericentromeric heterochromatin when transduced into mouse embryonic fibroblasts lacking SUV39H1/2 (Figure S7). CD34+CD38 HSC isolated from the bone marrow of elderly individuals were transduced with the empty control (expressing GFP alone) or SUV39H1 lentivirus, then differentiated in vitro on MS5 stromal cells as above. Similar to the results obtained following inhibition of miR-125, expression of SUV39H1 in aged HSC increased the generation of CD19+ B cells and resulted in an overall decrease in the myeloid:lymphoid differentiation output (Figures 7C and 7D). Again, no changes were observed in untransduced cells (Figures S6E and S6F). These experiments show that increasing the expression of SUV39H1 or inhibition of miR-125 enhances the capacity of HSC from elderly individuals to generate B cells and attenuates the age-associated myeloid bias. These findings support the idea that repression of SUV39H1 by miR-125 and perturbed heterochromatin regulation are important factors contributing to the decline in B cell potential of HSC with age.

Figure 7
Expression of a miR-125 Sponge and SUV39H1 in Aged HSC Increases B Cell Output and Alleviates Age-Associated Myeloid Bias


Loss of heterochromatin structure and its associated histone mark H3K9me3 have been observed during aging in many cell types and diverse organisms, leading to the proposal that a decline in heterochromatin integrity may be a common feature of aging (Tsurumi and Li, 2012). That age-associated decline of heterochromatin is likely to be functionally important in driving the aging phenotype in mammals is strongly supported by the observation that global loss of heterochromatin markers is a characteristic feature of the human premature aging disorders, Hutchinson-Gilford progeria and Werner syndromes (Shumaker et al., 2006, Zhang et al., 2015). Furthermore, in model organisms, it has been shown that the heterochromatin protein HP1α and the regulation of heterochromatin formation control life span (Larson et al., 2012). Heterochromatin has recently been shown to play a key role in the regulation of HSC differentiation (Ugarte et al., 2015); however, little was known about the contribution of H3K9me3 and heterochromatin structure to HSC aging. In this study, we show that expression of SUV39H1, a critical factor in heterochromatin formation, decreases with age in both human and mouse HSC, leading to widespread changes in HSC heterochromatin state, and contributing to the decline in B cell potential observed with advancing age. Consistent with our observations in HSC, reduced expression of SUV39H1 and a decrease in global H3K9me3 have also been observed recently during physiological aging both in human mesenchymal stem cells and in immune organs of the rat (Sidler et al., 2013, Zhang et al., 2015). Overall, our observations of decreased expression of SUV39H1 and perturbed HSC heterochromatin with age support recent studies implicating epigenetic change as an important driver of the decline in HSC function during aging (Beerman et al., 2013, Sun et al., 2014).

At present the molecular pathways affected by the decrease in SUV39H1 and heterochromatin, which contribute to the age-associated changes in HSC function, are not known. As a critical factor in the establishment of heterochromatin, SUV39H1 plays an important role in the maintenance of genome stability. Indeed, hotspots of chromosomal translocation breakpoints associated with hematological malignancy are characterized by depleted levels of H3K9me3 in human CD34+ cells, and SUV39H1-mediated deposition of this histone mark has been shown to directly protect against the formation of DNA double-strand breaks (Burman et al., 2015). Thus, the age-associated decline in SUV39H1 shown here could contribute to the accumulation of double-strand breaks observed in HSC from elderly individuals and predispose hematopoietic progenitors to increased translocations and malignant transformation (Rube et al., 2011, Yahata et al., 2011). In this study, we also observed a derepression of repeat and retroviral elements, which could also contribute to genomic instability (Belgnaoui et al., 2006, Kines et al., 2014), and indeed a breakdown in the normal transcriptional repression of retrotransposable elements is emerging as a common characteristic of aging (Gorbunova et al., 2014). Interestingly, recent evidence suggests that B cell differentiation is particularly sensitive to transcriptional derepression of endogenous retroviruses (Collins et al., 2015). Activated genomic repeats can impact upon the expression of neighboring genes through the formation of chimeric transcripts (Cruickshanks and Tufarelli, 2009, Nigumann et al., 2002). Indeed enhanced expression of genes proximal to transcriptionally reactivated repeat elements have been reported both in murine HSC (Sun et al., 2014) and proB (Collins et al., 2015) cells, indicating that derepression of repeat elements can lead to dysregulation of gene-expression programs. Finally, reduced expression of SUV39H1 could also contribute directly to the widespread transcriptomic changes that have been observed in HSC with age (Pang et al., 2011, Rossi et al., 2005, Sun et al., 2014) by the upregulation of genes normally silenced by facultative heterochromatin. This idea is supported by our findings that the expression of Selp, one of the most strongly upregulated genes with age in HSC, is also increased in the hematopoietic progenitors of Suv39h1null mice.

In this study, we further show that expression of SUV39H1 in HSC is controlled by the highly conserved miR-125 miRNA family. While previously shown to be important regulators of HSC self-renewal and differentiation potential (O'Connell et al., 2010, Ooi et al., 2010), the contribution of miR-125 members to age-associated changes in the hematopoietic system had not previously been investigated. We found that the expression of miR-125b is increased with age in HSC and that inhibition of miR-125b leads to increased SUV39H1 expression and attenuates the myeloid-biased differentiation potential of HSC from elderly individuals. miRNAs play an important role in the regulation of lifespan and aging in model organisms and a growing body of evidence indicates that they may also act as regulators of aging pathways in mammalian tissues (reviewed in Smith-Vikos and Slack, 2012). Indeed a recent study has shown that the miR-212/132 cluster is upregulated with age in mouse HSC and that this miRNA can affect HSC function with age via the regulation of the transcription factor FOXO3 (Mehta et al., 2015). As well as impacting on expression of SUV39H1, overexpression of the miR-125b-2 tricistron in HSC has been shown to suppress signaling through the transforming growth factor β (TGFβ) pathway (Emmrich et al., 2014). TGFβ signaling is a critical regulator of HSC homeostasis and quiescence (Blank and Karlsson, 2015) and, interestingly, reduced TGFβ signaling is a prominent feature of aging in mouse HSC (Sun et al., 2014). As well as playing an important role during normal hematopoiesis, miR-125 has also been implicated as an oncomir in a number of myeloid and lymphoid leukemias (Shaham et al., 2012). Thus, the age-associated increase in miR-125 observed in this study may also contribute to the enhanced susceptibility to hematological malignancies observed in the elderly (Sant et al., 2010). At present, the mechanism driving upregulation of miR-125 with age in HSC is not known and will require further investigation. Interestingly, HSC aging is associated with increased levels of both homeobox factors (Pang et al., 2011, Sun et al., 2014) and nuclear NF-κB (Chambers et al., 2007), two transcription factors that have been shown to be activators of miR-125b expression (Emmrich et al., 2014, Tan et al., 2012).

In summary, our study has characterized an axis in which age-associated increase in expression of miR-125b contributes to a decline in the B cell potential of human HSC via an effect on SUV39H1 and HSC heterochromatin. These findings add to a growing body of evidence demonstrating that epigenetic change, and in particular a disruption of heterochromatin integrity, are a general feature of age-associated tissue decline. The implication of miR-125 and SUV39H1 in the aging HSC phenotype presents the possibility of using miR antagonists and epigenetic modifiers as potential therapeutic approaches to address the decline in immune function observed in the elderly.

Experimental Procedures


Cord blood and human bone marrow samples from young (18–35 years) and elderly (70–98 years) individuals were obtained from Hôpital Saint Louis and Hôpital Lariboisière, Paris. Young (2–3 months) and old (20–24 months) C57BL/6 male mice were purchased from Janvier. Suv39h1null mice (Peters et al., 2001) were obtained from S. Amigorena (Institute Curie). Details of cell labeling, FACS sorting, and analysis are described in the Supplemental Experimental Procedures.

In Vitro Differentiation Assays

B lymphoid and myeloid differentiation assays were performed as described in Kuranda et al. (2011) and in the Supplemental Experimental Procedures.

Immunofluorescence Staining

Immunofluorescence staining of human and murine HSC was performed with anti-H3K9me3 (07-523, Millipore) or anti-HP1α (MAB3584, Millipore) antibodies as described in the Supplemental Experimental Procedures.


All data are expressed as means and SD except where stated otherwise. All statistical significance of differences was measured by unpaired two-tailed Student's t test (*p < 0.05; **p < 0.01). Unless stated otherwise, n indicates the number of independent experiments performed on HSC isolated from different pools of two to four individual mouse or human bone marrow samples.

Study Approval

Ethical approval for the use of human samples was granted by Institutional Review Board number 00006477 of Paris North Hospitals (approval no. 13-052) and samples were obtained after informed consent. All animal experiments were performed at the animal facility of the Institut Universitaire d’Hématologie (no. B75-10-08) in accordance with the guidelines of the Institutional Animal Care Committee.

Author Contributions

D.D. and D.G. designed and conducted the experiments, analyzed the data, and wrote the manuscript. K.K., I.K., D.B., C.C., and M.P. conducted the experiments. I.N. and F.P. provided reagents, and assisted in the study design and the writing of the paper. J.C.B. and C.D. provided scientific advice and support. V.V. and G.S. provided advice and clinical samples. M.G. designed the study and wrote the manuscript.


We thank Drs T. Jenuwein and S. Amigorena for Suv39h1null mice, Dr C. Muchardt for iMEFs, and Drs H. Raslova and D. Bluteau for the pRRL-EF1α-MCS-PGK-GFP lentiviral vector. We are grateful to Dr D. Bonnet for critical reading of the manuscript. The authors also wish to thank clinicians of the Service Hématologie-Greffe of Hôpital Saint Louis and Chirurgie Orthopédique of Hôpital Lariboisière as well as the Fondation Générale de Santé and members of the Unité de Thérapie Cellulaire, Hôpital Saint Louis for human samples. We are also grateful to the Pole Cytométrie of the IUH for help with FACS sorting, to A. Biffi for help with analysis of Suv39h1null mice, and to L. Renou for preparation of miR-125b lentiviral vectors. M.G. was supported by grants from INSERM, the Agence de la Biomedecine, and the Fondation de France. D.G. was supported by a Marie-Curie Intra-European Fellowship. F.P. was supported by grants from INSERM and the Ligue Contre le Cancer Comité d’Ile de France. D.D. is recipient of a fellowship from the Fondation de France and D.B. is recipient of a fellowship from the Ligue Nationale Contre le Cancer.


Published: June 14, 2016


Supplemental Information includes Supplemental Experimental Procedures, seven figures and one table and can be found with this article online at

Supplemental Information

Document S1. Supplemental Experimental Procedures, Figures S1–S7, and Table S1:
Document S2. Article plus Supplemental Information:


  • Alder O., Lavial F., Helness A., Brookes E., Pinho S., Chandrashekran A., Arnaud P., Pombo A., O'Neill L., Azuara V. Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment. Development. 2010;137:2483–2492. [PubMed]
  • Allan R.S., Zueva E., Cammas F., Schreiber H.A., Masson V., Belz G.T., Roche D., Maison C., Quivy J.P., Almouzni G., Amigorena S. An epigenetic silencing pathway controlling T helper 2 cell lineage commitment. Nature. 2012;487:249–253. [PubMed]
  • Baltimore D., Boldin M.P., O'Connell R.M., Rao D.S., Taganov K.D. MicroRNAs: new regulators of immune cell development and function. Nat. Immunol. 2008;9:839–845. [PubMed]
  • Beerman I., Bock C., Garrison B.S., Smith Z.D., Gu H., Meissner A., Rossi D.J. Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging. Cell Stem Cell. 2013;12:413–425. [PubMed]
  • Belgnaoui S.M., Gosden R.G., Semmes O.J., Haoudi A. Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells. Cancer Cell Int. 2006;6:13. [PubMed]
  • Blank U., Karlsson S. TGF-beta signaling in the control of hematopoietic stem cells. Blood. 2015;125:3542–3550. [PubMed]
  • Braig M., Lee S., Loddenkemper C., Rudolph C., Peters A.H., Schlegelberger B., Stein H., Dorken B., Jenuwein T., Schmitt C.A. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature. 2005;436:660–665. [PubMed]
  • Bulut-Karslioglu A., De La Rosa-Velazquez I.A., Ramirez F., Barenboim M., Onishi-Seebacher M., Arand J., Galan C., Winter G.E., Engist B., Gerle B. Suv39h-dependent H3K9me3 marks intact retrotransposons and silences LINE elements in mouse embryonic stem cells. Mol. Cell. 2014;55:277–290. [PubMed]
  • Burman B., Zhang Z.Z., Pegoraro G., Lieb J.D., Misteli T. Histone modifications predispose genome regions to breakage and translocation. Genes Dev. 2015;29:1393–1402. [PubMed]
  • Chambers S.M., Shaw C.A., Gatza C., Fisk C.J., Donehower L.A., Goodell M.A. Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation. PLoS Biol. 2007;5:e201. [PubMed]
  • Collins P.L., Kyle K.E., Egawa T., Shinkai Y., Oltz E.M. The histone methyltransferase SETDB1 represses endogenous and exogenous retroviruses in B lymphocytes. Proc. Natl. Acad. Sci. USA. 2015;112:8367–8372. [PubMed]
  • Cruickshanks H.A., Tufarelli C. Isolation of cancer-specific chimeric transcripts induced by hypomethylation of the LINE-1 antisense promoter. Genomics. 2009;94:397–406. [PubMed]
  • Czvitkovich S., Sauer S., Peters A.H., Deiner E., Wolf A., Laible G., Opravil S., Beug H., Jenuwein T. Over-expression of the SUV39H1 histone methyltransferase induces altered proliferation and differentiation in transgenic mice. Mech. Dev. 2001;107:141–153. [PubMed]
  • Denkinger M.D., Leins H., Schirmbeck R., Florian M.C., Geiger H. HSC aging and senescent immune remodeling. Trends Immunol. 2015;36:815–824. [PubMed]
  • Dweep H., Sticht C., Pandey P., Gretz N. miRWalk–database: prediction of possible miRNA binding sites by “walking” the genes of three genomes. J. Biomed. Inform. 2011;44:839–847. [PubMed]
  • Dykstra B., Olthof S., Schreuder J., Ritsema M., de Haan G. Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells. J. Exp. Med. 2011;208:2691–2703. [PubMed]
  • Emmrich S., Rasche M., Schoning J., Reimer C., Keihani S., Maroz A., Xie Y., Li Z., Schambach A., Reinhardt D., Klusmann J.H. miR-99a/100~125b tricistrons regulate hematopoietic stem and progenitor cell homeostasis by shifting the balance between TGFbeta and Wnt signaling. Genes Dev. 2014;28:858–874. [PubMed]
  • Fan D.N., Tsang F.H., Tam A.H., Au S.L., Wong C.C., Wei L., Lee J.M., He X., Ng I.O., Wong C.M. Histone lysine methyltransferase, suppressor of variegation 3-9 homolog 1, promotes hepatocellular carcinoma progression and is negatively regulated by microRNA-125b. Hepatology. 2013;57:637–647. [PubMed]
  • Flach J., Bakker S.T., Mohrin M., Conroy P.C., Pietras E.M., Reynaud D., Alvarez S., Diolaiti M.E., Ugarte F., Forsberg E.C. Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells. Nature. 2014;512:198–202. [PubMed]
  • Garrick D., Djeghloul D., Kuranda K., Goodhardt M. Aging of human haematopoietic stem cells. In: Geiger H., Jasper H., Florian M.-C., editors. Stem Cell Aging: Mechanisms, Consequences, Rejuvenation. Springer; 2015. pp. 127–148.
  • Geiger H., de Haan G., Florian M.C. The ageing haematopoietic stem cell compartment. Nat. Rev. Immunol. 2013;13:376–389. [PubMed]
  • Gerrits A., Walasek M.A., Olthof S., Weersing E., Ritsema M., Zwart E., van Os R., Bystrykh L.V., de Haan G. Genetic screen identifies microRNA cluster 99b/let-7e/125a as a regulator of primitive hematopoietic cells. Blood. 2012;119:377–387. [PubMed]
  • Gorbunova V., Boeke J.D., Helfand S.L., Sedivy J.M. Human genomics. Sleeping dogs of the genome. Science. 2014;346:1187–1188. [PubMed]
  • Greiner D., Bonaldi T., Eskeland R., Roemer E., Imhof A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9. Nat. Chem. Biol. 2005;1:143–145. [PubMed]
  • Grewal S.I., Jia S. Heterochromatin revisited. Nat. Rev. Genet. 2007;8:35–46. [PubMed]
  • Guo S., Lu J., Schlanger R., Zhang H., Wang J.Y., Fox M.C., Purton L.E., Fleming H.H., Cobb B., Merkenschlager M. MicroRNA miR-125a controls hematopoietic stem cell number. Proc. Natl. Acad. Sci. USA. 2010;107:14229–14234. [PubMed]
  • Kines K.J., Sokolowski M., deHaro D.L., Christian C.M., Belancio V.P. Potential for genomic instability associated with retrotranspositionally-incompetent L1 loci. Nucleic Acids Res. 2014;42:10488–10502. [PubMed]
  • Kuranda K., Vargaftig J., de la Rochere P., Dosquet C., Charron D., Bardin F., Tonnelle C., Bonnet D., Goodhardt M. Age-related changes in human hematopoietic stem/progenitor cells. Aging Cell. 2011;10:542–546. [PubMed]
  • Lachner M., O'Carroll D., Rea S., Mechtler K., Jenuwein T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature. 2001;410:116–120. [PubMed]
  • Larson K., Yan S.J., Tsurumi A., Liu J., Zhou J., Gaur K., Guo D., Eickbush T.H., Li W.X. Heterochromatin formation promotes longevity and represses ribosomal RNA synthesis. PLoS Genet. 2012;8:e1002473. [PubMed]
  • Lescale C., Dias S., Maes J., Cumano A., Szabo P., Charron D., Weksler M.E., Dosquet C., Vieira P., Goodhardt M. Reduced EBF expression underlies loss of B-cell potential of hematopoietic progenitors with age. Aging Cell. 2010;9:410–419. [PubMed]
  • Maison C., Almouzni G. HP1 and the dynamics of heterochromatin maintenance. Nat. Rev. Mol. Cell Biol. 2004;5:296–304. [PubMed]
  • Mehta A., Zhao J.L., Sinha N., Marinov G.K., Mann M., Kowalczyk M.S., Galimidi R.P., Du X., Erikci E., Regev A. The MicroRNA-132 and MicroRNA-212 cluster regulates hematopoietic stem cell maintenance and survival with age by buffering FOXO3 expression. Immunity. 2015;42:1021–1032. [PubMed]
  • Nigumann P., Redik K., Matlik K., Speek M. Many human genes are transcribed from the antisense promoter of L1 retrotransposon. Genomics. 2002;79:628–634. [PubMed]
  • O'Connell R.M., Chaudhuri A.A., Rao D.S., Gibson W.S., Balazs A.B., Baltimore D. MicroRNAs enriched in hematopoietic stem cells differentially regulate long-term hematopoietic output. Proc. Natl. Acad. Sci. USA. 2010;107:14235–14240. [PubMed]
  • Ooi A.G., Sahoo D., Adorno M., Wang Y., Weissman I.L., Park C.Y. MicroRNA-125b expands hematopoietic stem cells and enriches for the lymphoid-balanced and lymphoid-biased subsets. Proc. Natl. Acad. Sci. USA. 2010;107:21505–21510. [PubMed]
  • Pang W.W., Price E.A., Sahoo D., Beerman I., Maloney W.J., Rossi D.J., Schrier S.L., Weissman I.L. Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age. Proc. Natl. Acad. Sci. USA. 2011;108:20012–20017. [PubMed]
  • Peters A.H., O'Carroll D., Scherthan H., Mechtler K., Sauer S., Schofer C., Weipoltshammer K., Pagani M., Lachner M., Kohlmaier A. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell. 2001;107:323–337. [PubMed]
  • Rossi D.J., Bryder D., Zahn J.M., Ahlenius H., Sonu R., Wagers A.J., Weissman I.L. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc. Natl. Acad. Sci. USA. 2005;102:9194–9199. [PubMed]
  • Rube C.E., Fricke A., Widmann T.A., Furst T., Madry H., Pfreundschuh M., Rube C. Accumulation of DNA damage in hematopoietic stem and progenitor cells during human aging. PLoS One. 2011;6:e17487. [PubMed]
  • Sant M., Allemani C., Tereanu C., De Angelis R., Capocaccia R., Visser O., Marcos-Gragera R., Maynadie M., Simonetti A., Lutz J.M., Berrino F. Incidence of hematologic malignancies in Europe by morphologic subtype: results of the HAEMACARE project. Blood. 2010;116:3724–3734. [PubMed]
  • Schoeftner S., Blasco M.A. A 'higher order' of telomere regulation: telomere heterochromatin and telomeric RNAs. EMBO J. 2009;28:2323–2336. [PubMed]
  • Schultz D.C., Ayyanathan K., Negorev D., Maul G.G., Rauscher F.J., 3rd SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev. 2002;16:919–932. [PubMed]
  • Shaham L., Binder V., Gefen N., Borkhardt A., Izraeli S. MiR-125 in normal and malignant hematopoiesis. Leukemia. 2012;26:2011–2018. [PubMed]
  • Sharma P., Azebi S., England P., Christensen T., Moller-Larsen A., Petersen T., Batsche E., Muchardt C. Citrullination of histone H3 interferes with HP1-mediated transcriptional repression. PLoS Genet. 2012;8:e1002934. [PubMed]
  • Shumaker D.K., Dechat T., Kohlmaier A., Adam S.A., Bozovsky M.R., Erdos M.R., Eriksson M., Goldman A.E., Khuon S., Collins F.S. Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging. Proc. Natl. Acad. Sci. USA. 2006;103:8703–8708. [PubMed]
  • Sidler C., Woycicki R., Ilnytskyy Y., Metz G., Kovalchuk I., Kovalchuk O. Immunosenescence is associated with altered gene expression and epigenetic regulation in primary and secondary immune organs. Front Genet. 2013;4:211. [PubMed]
  • Smith-Vikos T., Slack F.J. MicroRNAs and their roles in aging. J. Cell Sci. 2012;125:7–17. [PubMed]
  • Sun D., Luo M., Jeong M., Rodriguez B., Xia Z., Hannah R., Wang H., Le T., Faull K.F., Chen R. Epigenomic profiling of young and aged HSCs reveals concerted changes during aging that reinforce self-renewal. Cell Stem Cell. 2014;14:673–688. [PubMed]
  • Tan G., Niu J., Shi Y., Ouyang H., Wu Z.H. NF-kappaB-dependent microRNA-125b up-regulation promotes cell survival by targeting p38alpha upon ultraviolet radiation. J. Biol. Chem. 2012;287:33036–33047. [PubMed]
  • Tsurumi A., Li W.X. Global heterochromatin loss: a unifying theory of aging? Epigenetics. 2012;7:680–688. [PubMed]
  • Ugarte F., Sousae R., Cinquin B., Martin E.W., Krietsch J., Sanchez G., Inman M., Tsang H., Warr M., Passegue E. Progressive chromatin condensation and H3K9 methylation regulate the differentiation of embryonic and hematopoietic stem cells. Stem Cell Rep. 2015;5:728–740. [PMC free article] [PubMed]
  • Villeneuve L.M., Kato M., Reddy M.A., Wang M., Lanting L., Natarajan R. Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1. Diabetes. 2010;59:2904–2915. [PubMed]
  • Wilson A., Laurenti E., Oser G., van der Wath R.C., Blanco-Bose W., Jaworski M., Offner S., Dunant C.F., Eshkind L., Bockamp E. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. 2008;135:1118–1129. [PubMed]
  • Wojtowicz E.E., Walasek M.A., Broekhuis M.J., Weersing E., Ritsema M., Ausema A., Bystrykh L.V., de Haan G. MicroRNA-125 family members exert a similar role in the regulation of murine hematopoiesis. Exp. Hematol. 2014;42:909–918.e1. [PubMed]
  • Yahata T., Takanashi T., Muguruma Y., Ibrahim A.A., Matsuzawa H., Uno T., Sheng Y., Onizuka M., Ito M., Kato S., Ando K. Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells. Blood. 2011;118:2941–2950. [PubMed]
  • Zhang W., Li J., Suzuki K., Qu J., Wang P., Zhou J., Liu X., Ren R., Xu X., Ocampo A. Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science. 2015;348:1160–1163. [PubMed]

Articles from Stem Cell Reports are provided here courtesy of Elsevier