The roles of retinoic acid and retinoic acid receptors in inducing epigenetic changes

Abstract

Epigenetics is "the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being" as defined by Conrad Waddington in 1942 in a discussion of the mechanisms of cell differentiation. More than seven decades later we know that these mechanisms include histone tail post-translational modifications, DNA methylation, ATP-dependent chromatin remodeling, and non-coding RNA pathways. Epigenetic modifications are powerful drugs targets, and combined targeting of multiple pathways is expected to significantly advance cancer therapy.

Figure 7.1. The Epigenetic Landscape

Conrad Waddington's Epigenetic Landscape illustrates the causal mechanisms by which the genotype brings about phenotypic effects. The progressive cellular differentiation (represented by purple, blue, red, and green) into specific cell types (illustrated by the different valleys) is associated with distinct epigenetic modifications. Polycomb repression and promoter hypermethylation are key mechanisms in the regulation of cellular differentiation. The corresponding epigenetic status of the various differentiation states is depicted on the right. (Hochedlinger and Plath, 2009).

Figure 7.2. The Nucleosome and the Histone Tails

The basic functional unit of chromatin is the nucleosome (Panel A), which is composed of a histone octamer around which DNA is wrapped. Octamers are separated by linker DNA. The histone octamer is assembled from a histone H3:H4 tetramer and two H2A:H2B dimers. The histone tails of all four core histones are subject to a variety of post-translational modifications (Panel B). These include methylation (Me), acetylation (Ac), phosphorylation (Ph), ubiquitylation (Ub), and proline isomerization (Iso), all of which occur at the site of a specific amino acid, such as K4 and K9 on the histone H3 tail. The same histone amino acid may be subject to different post-translational modifications, which may facilitate different biologic outcomes. (Dawson et al., 2012).

Figure 7.3. Epigenetic changes induced along the Hoxa cluster in response to RA

The epigenetic changes of the RA responsive Hoxa gene cluster are shown, with the locations of the Hoxa1 proximal promoter (PP) and RA responsive element (RARE) indicated by arrows. The levels of acH3, H3K4me3, and H3K27me3 determined by ChIP-chip are presented as heatmaps, with rows representing individual timepoints for each genotype, and columns indicating specific genomic regions. The genotypes of thestem cell lines are as follows: WT, RARE-KO (E-), and RARγ-KO (γ-). The cells were cultured in RA for 1, 8, and 24 h, as indicated. The color scale representing log2-transformed ChIP enrichment is indicated at the top of the figure. Note the reduced levels of H3ac and H3K4me3 at Hoxa1 PP and RARE in the RARγ-KO cell line Models for RA mediated transcription of RA target genes Hoxa1 and Nr2F1. In the absence of RA, RARγ-RXRαheterodimers associated with Hoxa1 RAREs presumably associate with co-repressors, thereby generating a SUZ12-rich environment which represses transcription. Binding of the RA ligand causes a conformational change in the RARγ-RXRα heterodimer bound to the Hoxa1 RARE. This results in the recruitment of pCIP/p300, which generates an euchromatic environment, presumably by acetylating the histone tails. This allows pol II to initiate transcription of Hoxa1. The Nr2F1 promoter region (PP) is bound by SUZ12 in the absence of RA. Upon exposure to RA the increase in activating marks is initially counteracted by a concomitant increase in SUZ12, which attenuates the transcription of Nr2F1. Eventually, the SUZ12 levels decline, allowing the increased transcriptional activation of Nr2F1. (modified from Kashyap et al., 2011 and Gillespie and Gudas, 2007b).

Figure 7.4. Epigenetic Signatures Associated with RA and RAR regulated transcription

Hoxa1 represents a groupof direct target genes induced by RA (upper panel). The induction is characterized by dissociation of PRCs (ovals) and depletion of the H3K27me3 repressive mark (hexagons), and by increased levels of transcriptionally permissive marks, H3K4me3 (circle), H3K9ac (triangle), and H3K14ac (diamond). CoupTF1 representsa group of target genes with delayed transcriptional induction by RA (middle panel). The induction is characterized by an initial increase of PRCs (ovals) and of the H3K27me3 repressive mark (hexagons), concurrent with increased levels of transcriptionally permissive marks; H3K4me3, H3K9ac, and H3K14ac. The imprinted gene Mest is transcribed in the presence of RARα, but is silenced by DNA methylation upon knockout of RARα (lower panel). The transcriptional silencing of Mest is associated with increased DNA methylation, increased levels of the H3K9me3 repressive mark, and with decreased levels of transcriptionally permissive marks; H3K4me3, H3K9ac, and H3K14ac. Note that for Hoxa1 and CoupTF1 the transcriptionally active state is shown to the right, whereas for Mest the transcriptionally active state is shown to the left, Published in parts (CoupTF1) in Nucleic Acid Research (modified from Laursen, et al., 2013).

Figure. 7.5. Current drugs targeting various epigenetic machinery

Histone acetyltransferase inhibitors (HATi) may be effective treatments since HAT proteins are sometimes considered to be tumor promoters. Histone deacetylase inhibitors (HDACi) such as vorinostat (SAHA) and romidepsin have been FDA approved for treatments of certain hematological cancers. SIRT inhibitors (SIRTi) specifically target the SIRT family of histone deacetylases. Histone demethyltransferase inhibitors (HDMi) and histone methylasetransferase (HMTi) have importantly become more selective towards specific marks, since methylation can act as both a repressive and an activating mark. DNA methyltransferase inhibitors (DNMTi) may serve as promising drugs for re-sensitizing certain cancer cells to chemotherapy, and two (vidaza and decitabine) are FDA approved for cancer treatment. (Rodríguez-Paredes et al., 2011)

Figure. 7.6. Representation of RA and DZNep effects on apoptosis regulation in human colon cancer cells

Retinoic acid treatment promotes TRAIL-related apoptosis in RARβ/RARγ–positive HT29 cells, but not in SW480 cells which express only low levels of RARβ/RARγ. The functional depletion of PRC2 by either inhibition with DZNep or by knockdown of SUZ12 increases TRAIL-mediated apoptosis in both HT29 and SW480 cell lines. In this scenario the PRC2-mediated repression is alleviated, thereby activating TNFRSF10 even in the absence of RARβ/RARγ.