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E. Mejia-Ramirez and M.C. Florian et al.
might suggest that the methylation machinery in old HSC might access PRC2 target sites merely as a consequence of the reduced levels of PCR2 itself, which normally occupies these specific targets inducing their transcriptional down- regulation. However, this hypothesis has yet to be demonstrated.
A possible explanation for the limitation to a more glob- al and prominent hypomethylation on HSC during aging is linked to their quiescence during adult life. When murine young or old HSC are transplanted, and are there- fore subjected to an induced proliferative stress, global hypomethylation occurs.27 This observation agrees with reports for aging in human somatic cells and tissues (reviewed by Gonzalo81 and Jones et al.84). However, even in this case, the hypermethylation of PRC2 targets is still detectable in mice, suggesting that aging and exhaustion promote distinct alterations on the DNA methylation landscape.27
It is worth noting that differentially methylated genes in aged HSC compared to young HSC are expressed or high- ly expressed in progenitor cells downstream in murine hematopoietic differentiation.27 This observation suggests that the impact of alterations in DNA methylation during aging is not probably manifested until HSC differentiate, and never in HSC themselves. This aspect is also demon- strated by the work of Scadden’s team, who elegantly showed in mice that the differences in enhancer methyla- tion between distinct HSC clones in the same individual has no effect on gene expression until stem cells differen- tiate into subsequent hematopoietic sub-compartments.26
Histone post-translational modifications upon hematopoietic stem cell aging
A number of histone tail modifiers have been involved in HSC function and also have a role in chromatin remod- eling, which supports the concept that changes in the chromatin state within the HSC compartment are impor- tant for blood homeostasis in both mice and human sys- tems. Among them, we can find PRC2 subunits such as Ezh2, Suz12 and Eed,85-87 histone specific lysine-demethy- lases such as Jarid1b/KDM5b,88 UTX/KDM6a89 or Lsd1/KDM1a,90 or histone methyltransferases such as SUV39H1.91
Moreover, it is known that the expression levels of some of these proteins change upon aging,27,28,43,91 suggest- ing that histone modifications are different in old HSC when compared to young HSC.
Goodell’s group performed one of the most accurate studies of epigenomic modifications in murine HSC upon aging. Some of the principal regulatory histone marks were profiled by ChIP-seq in HSCs from 4-month old and 24- month old mice. Although the dataset revealed only mod- erate changes, some interesting unique features were reported. For example, regarding H3K4me3, there was only an increment of 6.3% in peak deposition in old versus young HSC, but most of the peaks were generally broader.
As for H3K27me3, peak counts remained similar but an increase in the length of coverage by 29% was detected upon HSC aging. Moreover, the intensity of the signal increased by 50%. In relation to H3K36me3, the peaks moved from the TSS to the transcription termination site, and H3K36me3 and H3K27me3 behaved in a mutually exclusive manner. A strong positive correlation between transcriptome changes and the three histone marks ana- lyzed in the study was also reported.28
Regarding other histone marks, H4K16ac levels and localization have been demonstrated to change with age in murine HSC. H4K16ac shows a prominent distribution in one pole of the nucleus in young HSC by single cell 3- dimensional (3D) immunofluorescence analysis. This characteristic is defined as “epigenetic polarity” or “epipo- larity”. Old HSC show reduced levels of H4K16ac, which also displays apolarity and localizes evenly all over the nucleus.14,15,29 The polarity and levels of several other his- tone marks such as H3K4me1, H3K4me3, H4K8ac, H3K27ac and H4K5ac showed no aged-dependent changes in murine HSC and ST-HSC.29 Interestingly, the loss of epipolarity and the reduction of global H4K16ac deposition upon aging in HSC did not primarily correlate with global changes in gene expression.15 H4K16ac was associated to the modulation of higher-order chromatin and protein interactions between chromatin and non-his- tone proteins,92 and was shown to be targeted by inhibit- ing Cdc42 activity and LaminA/C expression. H4K16ac role in chromatin architecture in HSC will be further dis- cussed below.
In the context of human AML, H4K16ac levels seem to be linked to the nuclear localization of PAK4, a target of the orphan gene C3orf54/INKA1, which promotes self- renewal by inhibiting differentiation of cord blood HSC.93 C3orf54/INKA1 seems to be up-regulated in leukemic stem cells in AML patients, while PAK4 is an effector of Cdc42 in humans.94
Altogether, the results obtained over the last decade sug- gest that aging has an effect on histone mark landscape in HSC. However, the specific alteration of histone marks upon aging not always correlates with global change on gene expression in HSC themselves, but it rather affects the expression of genes in downstream progeny during the differentiation program, as we stated above also for the methylation profile.15,26,27 Altogether, this makes epige- netic studies extremely cumbersome in HSC and subtle differences within this cell population might indeed make a functional impact visible only under specific challenges.
Chromatin architecture changes
Over recent years, chromatin architecture has become more important for gene expression, cell division, fate determination, growth and development, disease and even genome evolution (reviewed by Kong and Zhang95). Recent studies have proposed the existence of two differ- ent but related ways of 3D chromatin organization: the cohesin-dependent CTCF loops and the small compart- mental domains that are formed as a consequence of tran- scription and chromatin state (reviewed by Rowley and Corces96). In this regard, and in relation to HSC biology, some disease-associated mutations in humans that affect proteins involved in establishing or preserving chromatin architecture have been described, for example, proteins belonging to the cohesin complex, such as Stag2, Rad21 and Smc3, which have been associated with AML and MDS particularly in the elderly.97,98
Chromatin accessibility changes upon HSC aging have just started to be investigated. Recently, we performed sin- gle-cell ATAC-seq on HSC daughter pairs from young and old mice. The results showed that there was a greater dif- ference between young pairs, more “asymmetrical”, as based on quantity of ATAC-seq peaks, than those coming from aged pairs, which were, therefore, more “symmetri- cal”. This observation was correlated to H4K16ac distribu-
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haematologica | 2020; 105(1)