In some previous posts I have commented on the pivotal role that epigenetic regulation may have on regeneration. It is known that in mammalian embryonic stem cells chromatin regulation is fundamental to control self-renewal, pluripotency and differentiation. Freshwater planarians are truly champions of regeneration and, remarkably, they carry out it through a population of adult pluripotent stem cells, the neoblasts. The neoblasts are the only proliferative cells in these animals and upon amputation they give rise to all the cell types needed to restore the missing parts. An important question is how similar are neoblasts and mammalian stem cells at the level of genetic programs and epigenetic regulators. Previous works have, in fact, highlighted a significant conservation between some of these factors as well as their functional relevance during planarian regeneration (http://www.ncbi.nlm.nih.gov/pubmed/23235145 , http://www.ncbi.nlm.nih.gov/pubmed/22543868).
Now, a paper from the laboratory of Qing Jing further expands our knowledge on the role of chromatin regulators during planarian regeneration (http://www.ncbi.nlm.nih.gov/pubmed/23629965). From the planarian draft genome they first identified about 210 genes that contained motifs common to conserved chromatin regulators. Upon RNAi silencing 12 of them gave a regeneration phenotype meaning that regeneration was, in fact, severely impaired. These genes belonged to at least six chromatin complexes: CAF1, BAF, NuRD, FACT, Cdk-activating kinase and MCm2-7 complex. All of them were enriched in the neoblast population and their silencing decreased the expression of typical neoblast markers such as smedwi-1.
Most of the paper is focussed on the characterization of a homologue of the HP1 (heterochromatin protein 1) family. This is a conserved family whose function in stem cells is not well characterized yet. Planarians possess two HP1 genes, but only one of them, HP1-1 seems required for neoblast function. When ectopically expressed in NIH3T3 cells HP1-1 is localized in the nucleus. Different experiments show that HP1-1 is expressed in neoblasts that also co-express smedwi-1. Upon its silencing a normal blastema seems to be initially formed at 1 day of regeneration but after that it does not grow and in fact it regresses, the animals curl and finally die in the typical pattern associated to neoblast depletion. The function of HP1-1 is equally required in intact non-regenerating planarians to sustain homeostatic cell turnover. Through BrdU labelling and double stainings with early and late neoblast progeny markers the authors conclude that HP1-1 is important for neoblast self-renewal and its silencing leads to a failed neoblast proliferative response that results in their premature differentiation.
In order to better characterize the mechanism through which HP1-1 functions the authors followed two strategies: i) they did microarray analyses to identify genes up- and down-regulated after HP1-1 silencing, and ii) they functionally characterized candidate genes that can bind HP1-1 and whose silencing gives similar defective regeneration phenotypes. By doing it, they found two genes of the FACT complex, SSRP1 and Spt16 that co-localize with HP1-1 in neoblasts. In fact, SSRP1 coimmunoprecipitates with HP1-1. As SSRP1 seems to be important for transcription, the authors suggest that HP1-1 and SSRP1 may cooperate to activate gene transcription during regeneration. Finally the authors performed additional microarray analyses to characterize genes missregulated after silencing SSRP1. When comparing the profiles obtained after HP1-1 and SSRP1 RNAi they identified 85 genes shared in both lists that are decreased upon their silencing. One of them is Mcm5 and chromatin immunoprecipitation (ChIP) suggest that HP1-1 protein binds the promoter region of Mcm5. Overall these results allow the authors to suggest that HP1-1 and SSRP1 activate Mcm5 during transcription and that is necessary to support proliferation and self-renewal of planarian stem cells.
These results will be important to understand how adult pluripotent stem cells are maintained and how respond to amputation. This work highlights also how planarian stem cells are regulated by conserved chromatin factors found also in mammalian ES cells and how studying their function in these flatworms can provide novel information on their function on stem cell biology.