In summary, our findings illustrate the dynamic nature of genome-scale epigenetic reprogramming in an experimental model of EMT, characterized by genome-wide reprogramming of large heterochromatin domains (LOCKs) to a state of reduced H3K9Me2, new LOCK-wide modifications of H3K4Me3 at specific GC-rich LOCKs, and enrichment of H3K36Me3 at LOCK boundaries and numerous EMT-related genes across the genome (). This reprogramming may be critical for proper execution of EMT functions, as inhibition of bulk chromatin changes with Lsd1 loss-of-function experiments had marked effects on cell migration and chemoresistance. Finally, it is probable that many other chromatin modifications not examined here are also reprogrammed during EMT. Indeed, repression of epithelial-specific genes during EMT may be mediated by H3K27Me3 via a Snai1-dependent process, and forced overexpression of Snai1
can also recruit Lsd1 to epithelial genes to assist in repression46
, suggesting that Lsd1 might play dual roles in repressing and activating areas of the genome important for EMT. In this regard, the balance of Lsd1-interacting factors identified in may play a crucial role in directing Lsd1 toward activation or repression.
It has been proposed that cells undergoing EMT acquire stem cell traits3,4
. Our findings of quantitative abatement of LOCKs and elevated levels of H3K36Me3 across the genome during EMT supports this notion at the level of chromatin, as previous studies have demonstrated similar results for cultured ESCs24,47
. In contrast, other studies have reported that the reverse process, mesenchymal to epithelial transition (MET), is required to produce induced pluripotent stem cells (iPSCs) from fibroblasts 48,49
. These findings raise the possibility that the reverse chromatin changes described here might occur during generation of iPSCs. Alternatively, the process described in the current work could be a general mechanism for increasing epigenetic plasticity and reprogramming, regardless of cell fate directionality.
EMT is also characterized by acquisition of malignant-type traits. Intriguingly, loss of LOCKs is seen in various malignant human cancer cell lines24
, similar to our EMT results. We further observed H3K36Me3 enrichment at numerous genes involved in cell motility during EMT, another trait common to both EMT and malignancy. Indeed, Lsd1 loss-of-function experiments interfered with both H3K36Me3 and cell migration during EMT. We also consider that a shift in balance from heterochromatic (H3K9Me2) to euchromatic (H3K4Me3) and transcription-coupled (H3K36Me3) modifications may increase susceptibility to DNA damaging agents during stress, injury, or malignancy. Other cells with euchromatic genomes appear to activate a surveillance DDR to cope with this threat43
, although this could generate chromosomal rearrangements at sites of hyperactive transcription50,51
. Thus, if a surveillance DDR is activated concomitant with elevated transcription across the genome during EMT, this might confer robust repair of DNA damage at the expense of generating mutations.
This latter possibility would be compatible with a surveillance role for H3K4Me3 in repair of DSBs during EMT, consistent with the effects of siLsd1 and pargyline on H3K4Me3 and chemoresistance. Our ChIP-chip results revealed that K4Me3 LOCKs are specifically situated in large gene and CG-rich regions of the genome flanked by genes with high levels of transcription, which are prone to acquire DSBs in response to genotoxic agents such as doxorubicin50–52
. Indeed, components of the DDR (such as γH2AX) must spread several hundred kilobases from a DSB to facilitate repair, and heterochromatin inhibits this process43
. We therefore hypothesize that one function of K4Me3 LOCKs might be to provide a euchromatic scaffold in gene-rich regions of the genome, that allows efficient spreading of the DDR through large heterochromatin domains in response to DSBs that might occur during EMT, thereby conferring robust repair of DSBs and enhanced chemoresistance.
Like stem cell differentiation7,27
, germ cell development53
, and malignant transformation5,8
, epigenetic reprogramming during EMT is mediated by widespread changes in chromatin modifications. Unlike reprogramming in these other cell types, DNA methylation patterns are faithfully preserved during EMT. Further studies in other cell systems are needed to determine whether our findings are a general property of EMT, and whether similar epigenetic reprogramming occurs in other physiological contexts.