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“linc”ing Non-Coding RNAs with Pluripotency and Reprogramming

By Stuart P. Atkinson

Little by little, the processes behind cellular reprogramming of lineage-committed cells to induced pluripotent stem cells (iPSCs) are being discovered and more detailed comparative studies between iPSC and embryonic stem cells (ESCs) are being undertaken. The similarity of these two cell types is generally viewed as being essential if hiPSCs are to be taken towards the clinic and while this has led to detailed analyses of many cellular attributes, such as mRNA expression, miRNA expression and chromatin structure, other “avenues” remain relatively unstudied.

Now a study (Loewer et al), soon to be published in Nature Genetics from the laboratories of George Q Daley and John L Rinn, has begun the characterisation of large intergenic non-coding RNAs (lincRNAs) in hESC and hiPSC. Importantly, this study describes lincRNAs which may be important to the reprogramming process. LincRNAs have been previously linked to pluripotency and lineage commitment through their association with specific epigenetic modifiers and so may be necessary for the regulation of ESC-specific gene expression and chromatin regulation. In view of this, proper regulation of lincRNAs may be essential to pluripotency and may therefore also be important in the reprogramming process.

Initial experiments using an array to probe approximately 900 lincRNAs (Khalil et al), found that 133 lincRNAs were induced and 104 repressed in an independent and cell-type-specific manner, when comparing hiPSCs and hESCs with fibroblasts (from which the hiPSCs where generated). It was also observed that hiPSC and hESC were very similar with regards to their lincRNA expression, a vitally important note of interest. To highlight those lincRNAs that may be of importance to the reprogramming process, the authors identified those which showed higher expression in hiPSC. This gave a list of 28 hiPSC enriched lincRNAs and encouragingly, 10 of these were also found in another hiPSC line, this time reprogrammed from CD34+ cells. Although not completely conclusive, since only two hiPSC lines were used in the study, this is potentially important as it suggests that these 10 lincRNAs may be generally important to reprogramming and are not cell-of-origin specific. Previously generated data on OCT4 bindings sites (Marson et al) and enriched lincRNA loci (marked by domains of histone H3K4 and H3K36 methylation (Guttman et al and Khalil et al) in hESC were then utilised to compare against the data generated in this study which identified three overlapping lincRNAs, lincRNA-SFMBT2, lincRNA-VLDLR and lincRNA-ST8SIA3 (renamed by the authors as lincRNA-RoR), with the lincRNAs being named after their proximal 3’ gene. Such a striking overlap linking pluripotency and lincRNAs led the authors to study these in more detail.

RNAi-mediated knockdown of OCT4 and embryoid body-mediated differentiation in both hESC and hiPSC led to a decrease in the expression of all 3 lincRNAs, suggesting a link to pluripotency. When these lincRNAs where studied in relation to the reprogramming process, reduction of lincRNA-SFMBT2 surprisingly did not affect iPSC formation. However, a reduction in lincRNA-RoR did lead to a 2-8 fold decrease in colony formation while overexpression of lincRNA-RoR led to a 2-fold increase in iPSC colony formation, suggesting that lincRNA-RoR also has a positive role in the reprogramming process. RNAi-mediated knockdown of lincRNA-RoR led to an increase in the expression of genes involved in the p53 response, the response to oxidative-stress and DNA damage-inducing agents and cell death pathways. This then suggests a mechanism by which lincRNA-RoR functions, as bypass of the senescence pathways is understood to enhance hiPSC formation (Banito and Gil). Therefore, expression of lincRNA-RoR could dampen senescence-mediated pathways that are activated during reprogramming.

Of further interest, the authors note that there is very little overlap between this study (human) and a comparable study in mouse, with only lincRNA-VLDLR being linked to pluripotency in both studies, suggesting species-specific regulation by lincRNAs.

Overall, this study represents the first functional example of the involvement of lincRNAs in hiPSC generation and provides some interesting comparative data between hESCs and hiPSCs. How these lincRNAs function in this context is however unknown but the suggested role of lincRNAs in mediating large scale chromatin alterations through interactions with transcription factors and chromatin modifying complexes seems a likely mechanism of action. Further studies are therefore highly anticipated.



Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells.
Loewer S, Cabili MN, Guttman M, Loh YH, Thomas K, Park IH, Garber M, Curran M, Onder T, Agarwal S, Manos PD, Datta S, Lander ES, Schlaeger TM, Daley GQ, Rinn JL.
Nat Genet. 2010 Nov 7. [Epub]

Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.
Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A, Regev A, Lander ES, Rinn JL.
Proc Natl Acad Sci U S A. 2009 Jul 14;106(28):11667-72

Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.
Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, Johnstone S, Guenther MG, Johnston WK, Wernig M, Newman J, Calabrese JM, Dennis LM, Volkert TL, Gupta S, Love J, Hannett N, Sharp PA, Bartel DP, Jaenisch R, Young RA.
Cell. 2008 Aug 8;134(3):521-33.

Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.
Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, Cabili MN, Jaenisch R, Mikkelsen TS, Jacks T, Hacohen N, Bernstein BE, Kellis M, Regev A, Rinn JL, Lander ES.
Nature. 2009 Mar 12;458(7235):223-7

Induced pluripotent stem cells and senescence: learning the biology to improve the technology.
Banito A, Gil J.
EMBO Rep. 2010 May;11(5):353-9.