Histone Epigenetic Signatures in Embryonic Limb Interdigital Cells Fated to Die

Abstract

During limb formation in vertebrates with free digits, the interdigital mesoderm is eliminated by a massive degeneration process that involves apoptosis and cell senescence. The degradation process is preceded by intense DNA damage in zones located close to methylated DNA, accompanied by the activation of the DNA repair response. In this study, we show that trimethylated histone 3 (H3K4me3, H3K9me3, and H3K27me3) overlaps with zones positive for 5mC in the nuclei of interdigital cells. This pattern contrasts with the widespread distribution of acetylated histones (H3K9ac and H4ac) and the histone variant H3.3 throughout the nucleoplasm. Consistent with the intense labeling of acetylated histones, the histone deacetylase genes Hdac1, Hdac2, Hdac3, and Hdac8, and at a more reduced level, Hdac10, are expressed in the interdigits. Furthermore, local treatments with the histone deacetylase inhibitor trichostatin A, which promotes an open chromatin state, induces massive cell death and transcriptional changes reminiscent of, but preceding, the physiological process of interdigit remodeling. Together, these findings suggest that the epigenetic profile of the interdigital mesoderm contributes to the sensitivity to DNA damage that precedes apoptosis during tissue regression.

Regulation of Developmental Cell Death in the Animal Kingdom: A Critical Analysis of Epigenetic versus Genetic Factors

“…In the course of interdigit remodeling, γH2AXfoci associate with zones of intense DNA methylation and histone 3 trimethylation at lysines 4, 9, and 27 (H3K4me [3]; H3K9me [3]; and H3K27me [3]) [56,64] suggesting that regions of elevated DNA fragility depend on chromatin architectural cues. Consistent with this interpretation, major epigenetic regulators responsible for DNA methylation, such as UHRF1 (ubiquitin-like containing plant homeodomain and RING finger domain), DNA methyltransferases (Dnmt1, Dnmt 3a, and Dnmt 3b), and different histone deacetylase genes (Hdac1, Hdac2, Hdac3, and Hdac8), show regulated expression domains in the interdigital regions [56,65,66]. Experimental analysis designed to unravel the relevance of those expression domains on cell death revealed a dramatic increase in interdigital cell death in vivo following local inhibition of histone deacetylases with trichostatin A [65,67].…”

“…Consistent with this interpretation, major epigenetic regulators responsible for DNA methylation, such as UHRF1 (ubiquitin-like containing plant homeodomain and RING finger domain), DNA methyltransferases (Dnmt1, Dnmt 3a, and Dnmt 3b), and different histone deacetylase genes (Hdac1, Hdac2, Hdac3, and Hdac8), show regulated expression domains in the interdigital regions [56,65,66]. Experimental analysis designed to unravel the relevance of those expression domains on cell death revealed a dramatic increase in interdigital cell death in vivo following local inhibition of histone deacetylases with trichostatin A [65,67]. In addition, cell death was increased and decreased in primary cultures of limb skeletal progenitors after overexpression or silencing of the Dnmt3b gene, respectively [56].…”

Influence of Global Dna-Methylation on Chondrogenic Fate During Digit Development

“…These mechanisms ultimately determine the accessibility of the chromatin to transcription factors [26,40]. As a result, a DNA hypomethylated state can modify cellular fate by inducing or repressing gene expression [28, 30,41,42].…”

“…DNA methylation also regulates proapoptotic genes to maintain their expression and promote interdigital cell death. The DNMT's and HDAC's family proteins are also widely expressed in the interdigital tissue compared to DC/PFR [28, 30,43], suggesting they mediate genomic instability to induce cell death and inhibit chondrogenic differentiation. In concordance, Sox9 expression is higher in TGF-β and 5-Aza-C simultaneous treatment in the interdigital tissue.…”

“…In the same micromass cultures, continuous administration of four-day 5-azacytidine (5-Aza-C), a hypomethylating agent, can induce chondrogenesis [28,29]. During digit elongation, chromatin-remodeling and DNA methylating molecules are also present [28,30]. Protein arginine methyltransferase 5 (PRMT5), a family of arginine methyltransferase enzymes, plays a vital role in maintaining chondrocyte progenitor cells by inhibiting apoptosis via repressing BMP4 signals.…”