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Splitting hairs: Follicking about with mir-302

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By Carla Mellough

Continuing on with our recent focus on microRNA (miRNA) and induced pluripotency (see ‘Reprogramming the methods that induce pluripotency’), a recent article1 published in the September advance issue of Nucleic Acids Research from David Wu’s group (WJWU and LYNN Institute for Stem Cell Research) in California may shed light on the mechanisms that govern somatic cell reprogramming. In this article Lin et al.1 uncover a regulatory mechanism that controls global DNA demethylation and histone methylation, a requirement for the reprogramming of somatic cells. Global demethylation is an event largely associated with early zygote development and is key for the establishment of stem cell pluripotency, but how cell stemness is reset in somatic cells during reprogramming is not yet clearly understood. Of the four Yamanaka factors, two (Oct3/4 and Sox2) are essential for reprogramming to occur and both of these were recently shown to be crucial for expression of the miRNA Mir-3022,3. MiRNAs are a class of non-coding RNAs that suppress the translation of target messenger RNAs. Mir-302 expression, encoded for within a region of chromosome 4 that is associated with longevity, is high in both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) with mir-302 downregulation accompanying cell differentiation4. This suggests a role for mir-302 in the regulation of stemness and pluripotency (alongside other stem cell-associated miRNAs) and therefore also the reversion of lineage restricted cells to a pluripotent state.

Using high throughput analysis with online miRNA-target prediction programs, the authors identified two particular groups of epigenetic regulators that are targeted by mir-302; lysine specific histone demethylases (AOF, KDM and LSD) and methyl CpG-binding proteins (MECP). Loss or inhibition of AOF family members (AOF1,2) increases methylation of histone 3 on lysine 4 (H3K4), stimulates Oct3/4 expression in embryonal carcinoma (EC) cells and causes a loss of de novo genomic DNA methylation capability. The targeted silencing of AOF would thus likely induce global DNA demethylation and, indeed, ectopic expression of mir-302 has previously been shown both by Lin et al.5 and others6 to do just that (by silencing MECP1-p66 and MECP2) as well as activate expression of Oct3/4, Sox2 and nanog and reprogram human skin cells to pluripotency. However the direct link between mir-302 and AOF until now had not yet been established.

The authors set out to determine the functional role of mir-302 on AOF silencing and somatic cell reprogramming in human hair follicle cells (hHFCs) from the dermal papillary region. They transfected hHFCs via electroporation with a doxycycline (Dox) inducible vector encoding all four native mir-302 family members (mir-302a-d) and following microarray analysis identified the efficient inducible expression of mir-302a,c and d in transfected cells. Their results show that induced expression of mir-302 in hHFCs reduced the expression of melanocytic markers and stimulated the expression of pluripotency factors Oct3/4, Sox2 and nanog in a dose-dependant manner (≥7.5μM and <12μM Dox required for reprogramming to occur). Treatment of hHFCs with Dox within this range elevated mir-302 to levels above that found in hESCs and induced sufficient co-expression of pluripotency factors for reprogramming to ensue, thus establishing a threshold concentration of this miRNA for somatic cell reprogramming. Dox-treatment gave rise to mir-302 induced pluripotent stem cells (mirPS) that varied in appearance between two-dimensional (2D) hESC-like flat colonies to 3D embryonic body (EB) like structures following low (7.5μM) or high (10μM) Dox-induction of mir-302 expression respectively, with no difference in hESC-specific gene expression between the two colony types. Further, alongside pluripotency factor reactivation western blot analysis revealed a mir-302 concentration-dependant loss of AOF2, MECP1-p66 and MECP2, indicating that suppression of AOF2 and silencing of MECP1/2 play an important role in causing the global demethylation that accompanies Oct3/4, Sox2 and nanog co-activation during reprogramming. Using a luciferase reporter assay and western blot analysis the interaction between mir-302 and these epigenetic targets was confirmed, revealing that under 10μM Dox induction mir-302 silenced over 80% of target gene expression. AOF2 expression normally stabilises DNA methyl transferase 1 (DNMT1) to maintain global DNA methylation. Mir-302 suppression of AOF2 also led to a significant decrease in DNMT1, causing global demethylation that was augmented by MECP1/2 suppression and resulting in the induction of pluripotency factors. Bisulphite DNA sequencing confirmed the demethylation status of Oct3/4 and nanog promoters to be akin to that of hESCs, and microarray analysis of gene expression patterns showed mirPS to be 91% similar to H1/H9 hESCs. Lin et al. also demonstrate the teratoma-forming ability of mirPS in immunocompromised mice and their multilineage potential. Interestingly, they found that blocking mir-302 in mirPS cells with anti-mir-302 LNA-DNA oligonucleotides induced neuronal differentiation, whilst nuclear microinjection of recombinant AOF2 into differentiated cells prohibited mir-302 induced reprogramming; the latter failing to complete demethylation. Further experiments localised the effects of mir-302 to the cytoplasm and ruled out the presence of a nuclear effector in mir-302-mediated global genomic DNA demethylation.

This work demonstrates that mir-302-targeted AOF/DNMT1 silencing and MECP1/2 co-suppression is required for global demethylation and H3K4 modification during somatic cell reprogramming. The DNA demethylation and histone methylation which occurs during somatic cell reprogramming alters chromatin structure and gene activity on a genome-wide scale. Tapping into the mechanisms governing this event will no doubt enhance the efficiency of somatic cell reprogramming and bring us closer to achieving clinically applicable patient-specific cell types that may help ameliorate human disease. The key factors described in this paper and that are required in somatic cell reprogramming are not new; induced expression of mir-302 induced co-expression of the core reprogramming factors Oct3/4, Sox2 and nanog, consistent with methods which generate iPSC by ectopic transfection of these factors. However, what this study now shines light on are some of the mechanisms which govern the epigenetic reprogramming of genomic methylation patterns, and that cell stemness can be activated in somatic cells using mir-302. The cytoplasmic effects of mir-302 may also implicate this miRNA in nuclear reprogramming.

 

References

  1. Lin et al. (2010) Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Research 1-12. doi:10.1093/nar/gkq850.
  2. Marson et al. (2008) Connecting microRNA genes to the core transcriptional regulatory circuit of embryonic stem cells. Cell 134, 521-533.
  3. Card et al. (2008) Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells. Mol. Cell. Biol. 28, 642-6438.
  4. Suh et al. (2004) Human embryonic stem cells express a unique set of microRNAs. Dev. Biol. 270, 488-498.
  5. Lin et al. (2008) Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like-state. RNA, 14, 2115-2124.
  6. Rosa et al. (2009) The miR-430/427/302 family controls mesendodermal fate specification via species-specific target selection. Dev. Cell. 16, 517-527.