The common theme emerging from recent work in this field is that many chromatin modifiers involved in EMT play dual roles in the repression and activation of EMT marker, motility, or EMT-related genes. However, no single one has been shown to be a “master regulator” that governs all the gene expression patterns regulating EMT, suggesting certain EMT pathways require more than one chromatin modifier to execute both the repression and activation of EMT marker and EMT-related genes. This is unsurprising given that EMT involves so many different aspects and changes in gene expression. The dependence on a network of chromatin modifications and post-transcriptional mechanisms provides a measure of built-in control for this critical cellular process, such that multiple levels of regulation (including EMT transcriptional regulators, chromatin modifiers, alternative splicing, and microRNA) are all required to achieve the full spectrum of EMT. This coordinated action is beneficial because relying on a single level of control may be disastrous as EMT is tightly linked to cell migration and metastasis, which may have
detrimental effects on the organism.
In going forward, it will be important to identify specific chromatin modifiers or a unique histone mark or sets of marks associated with EMT. These could then be
used to search for novel genes with the same signature, leading to a more complete understanding of the gene expression patterns involved in EMT. It will also be important to search for differential post-transcriptional modifications that determine the expression fate of EMT marker or EMT-related genes. The epigenetic mechanisms and chromatin modifiers identified from these researches will provide important targets for future treatment because chromatin modifiers have proven to be targetable by small molecules and mutations or changes in the levels of chromatin
modifiers are constantly linked to human diseases [99].
Finally, the results from the literature support the hypothesis that chromatin modifications are essential mediators of the activity of numerous EMT transcriptional regulators and play an indispensable role in this process. Information obtained from these mechanistic approaches should help guide target-tailored therapy (e.g. HDAC3-specific inhibitors in hypoxic tumors) using the different epigenetic targets identified under various signaling pathways or post-transcriptional mechanisms.
Acknowledgments
We apologize to the authors whose work could not be cited due to the space constraint of reference citation. The authors declare no competing financial interests. This work was supported in part to K.J.W. by National Science Council Frontier grant (NSC100-2321-B-010-011), National Research Program for Biopharmaceuticals (NSC101-2325-B-010-004), a grant from Ministry of Education, Aim for the Top University Plan (101AC-T505), center of excellence for cancer research at Taipei Veterans General Hospital (DOH101-TD-C-111-007), Taichung Veterans General Hospital (TCVGH-YM1000301), and National Health Research Institutes (NHRI-EX101-9931BI).
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Figure 1. A model of the epigenetic changes during hypoxia-induced EMT. (a) For epithelial genes, HDAC3 deacetylates H3K4Ac and interacts with transcription factors (TF) to cause gene repression. The H3K27me3 and H3K4me bivalent domain is observed on the promoters. The question marks indicate the possible deacetylation of H3K27Ac by HDAC3. (b) For mesenchymal genes, HDAC3 deacetylates H3K4Ac and interacts with transcription factors and histone methyltransferase (HMT) complexes to cause gene activation. The activation mark H3K4me is observed on the promoters. Red arrows: indicate removal of the acetyl group of H3K4Ac or H3K27Ac by HDAC3. The labeled marks are on histone 3.
Figure 2. A model depicting TGF--induced EMT. (a) The H3K9me2 labeled chromatin state before TGF- treatment. (b) TGF- treatment causes LSD1 (together with EMT transcriptional regulators) to demethylate H3K9me2. This is followed by increased H3K4me and H3K36me, leading to derepression of LOCK regions and activation of motility genes. The question mark indicates the unidentified chromatin modifier(s) that regulate(s) EMT marker and motility gene expression (vimentin and E-cadherin). Arrows indicate the activation or repression of genes. Motility genes are located in the boundary regions. The empty arrowheads indicate the contribution of
multiple different processes (e.g. regulation of EMT marker genes, widespread chromatin changes, activation of motility genes) to the final EMT phenotypes.
Figure 3. A summary of four microRNA regulatory hubs. (a) The regulatory hub of miR-200 family, miR-205, and miR-34 as well as their associated regulations. (b) The regulatory hub of let-7i and miR-10b as well as their associated regulations. (c) The regulatory hub of various microRNAs regulated by c-Myc and/or TGF-. (d) The regulatory hub of miR-21 and its associated regulations. Pathways in blue indicate activation and pathways in red indicate repression.
Glossary
Bivalent domains: the simultaneous presence of a repressive histone mark (e.g.
H3K27me3) and an activation histone mark (e.g. H3K4me2/3) on the same promoter region of genes, usually indicating an inactive but poised state that allows rapid activation of the genes in response to a signaling cue.
Epithelial-mesenchymal transition (EMT): the process mediating the cell-type switch from epithelial to mesenchymal. It is characterized by down-regulation of epithelial markers (e.g. E-cadherin) and upregulation of mesenchymal markers (e.g.
vimentin and N-cadherin) as well as loss of apical-basal polarity, loss of cell-cell adhesion, and reorganization of the actin cytoskeleton.
EMT marker genes: epithelial and mesenchymal genes whose expression changes during EMT (i.e. repression of epithelial genes and activation of mesenchymal genes).
EMT transcriptional regulators: transcription factors (e.g. Twist1, Snail, and Slug) that are induced by different signaling pathways to mediate EMT.
Epithelial splicing regulatory proteins (ESRPs): are epithelial-specific RNA binding proteins that promote splicing of the epithelial variant of the FGFR2, ENAH, and CD44 transcripts.
EZH: EZH1 and EZH2 are polycomb group silencers and lysine methyltransferases.
EZH2 is a critical component of polycomb repressive complex 2 (PRC2), which methylates H3K27, leading to gene repression.
G9a (EHMT2): a HMT that catalyzes mono- and di-methylation of H3K9, leading to gene repression. G9a also methylates non-histone proteins to modulate gene expression.
H3K4 acetylation (H3K4Ac): a mark commonly found at promoter regions of EMT marker genes.
H3K4 di- or tri-methylation (H3K4me2/3): marks commonly found at gene promoters of activated genes.
H3K9 di- or tri-methylation (H3K9me2/3): marks commonly found at promoter regions of repressed genes.
H3K18 acetylation (H3K18Ac): a mark commonly found at promoter regions of transcriptionally active genes. Coupled with H3K9me3, this mark constitutes a bivalent domain and serves as a platform for activation of downstream targets of Nodal, including Gsc and Mixl1.
H3K27 trimethylation (H3K27me3): a mark commonly found at promoter regions of repressed genes, (e.g. increased H3K27me3 levels at epithelial gene promoters during EMT). Loss of H3K27me3 usually indicates that the gene has become activated (e.g. decreased H3K27me3 levels at the mesenchymal gene promoters during EMT).
H3K36 trimethylation (H3K36me3): a mark commonly found at promoter regions of transcriptionally active genes. However, this mark also induces histone deacetylation to restore normal chromatin structure in the wake of elongating RNA polymerase II to prevent inappropriate initiation.
HMT (histone methyltransferase): a generic term for proteins/complexes that add methyl groups to residues (typically lysines).
JMJDs (KDMs): Jumonji-domain-containing proteins with lysine histone demethylase catalytic activity.
LOCKs: large (100 kb-5 Mb) non-repetitive heterochromatin domains that are enriched for H3K9me2 and overlap with nuclear lamina-associated domains.
LSD1 (KDM1): is the first histone demethylase identified. It removes methyl groups of H3K4 and H3K9. Although removal of H3K4me by LSD1 induces gene repression, LSD1 is required for estrogen/androgen receptor-dependent gene transcription through removal of H3K9me, indicating that LSD1 has different roles depending on the context in which it functions.
Protein arginine methyltransferase (PRMT): a generic term for proteins/complexes that add methyl groups to arginine residues of histones and other proteins.
Reprogramming: changes in gene activity that shifts cells to a different cell type (e.g. epithelial to mesenchymal, somatic cell to pluripotent stem cell).