NODAL/Activin signalling to chromatin: mechanisms of SMAD2-regulated transcription

NODAL/Activin signalling regulates key processes during embryonic development via SMAD2. How SMAD2 activates programmes of gene expression that are modulated over time, however, is not known. In this thesis, using the P19 embryonic teratoma cell line as a model system, I delineate the sequence of events that occur from SMAD2 binding to transcriptional activation, and the underlying mechanisms. I show that NODAL/Activin signalling induces dramatic changes in the chromatin landscape, and orchestrates a dynamic transcriptional network regulated by SMAD2, which acts via multiple mechanisms. By combining different genome-wide approaches, I have discovered two modes of SMAD2 binding. SMAD2 can bind pre-acetylated nucleosome-depleted sites, where it promotes a further increase in H3K9ac/H3K27ac. However, SMAD2 also binds to unacetylated, closed chromatin, independently of pioneer factors, where it induces nucleosome displacement and H3 acetylation. For a subset of genes, this requires cooperation with the remodeller SMARCA4 and the transcription factor FOXH1. I demonstrate that SMAD2 regulates RNA Polymerase II via de novo recruitment to target promoters, and that long term modulation of the transcriptional responses requires continued NODAL/Activin signalling. Moreover, SMAD2 binding does not necessarily equate with transcriptional kinetics, and my data suggest that SMAD2 recruits multiple co-factors during sustained signaling to shape the downstream transcriptional programme. I have used ATAC-seq to identify specific transcription factor footprints at SMAD2 binding sites, and future work will aim to unveil and characterise the network of transcription factors that collaborate with SMAD2 and enable cells to correctly interpret NODAL/Activin signaling over time

SMYD1 and G6PD modulation are critical events for miR-206-mediated differentiation of rhabdomyosarcoma

Rhadomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS cells resemble fetal myoblasts but are unable to complete myogenic differentiation. In previous work we showed that miR-206, which is low in RMS, when induced in RMS cells promotes the resumption of differentiation by modulating more than 700 genes. To better define the pathways involved in the conversion of RMS cells into their differentiated counterpart, we focused on 2 miR-206 effectors emerged from the microarray analysis, SMYD1 and G6PD. SMYD1, one of the most highly upregulated genes, is a H3K4 histone methyltransferase. Here we show that SMYD1 silencing does not interfere with the proliferative block or with the loss anchorage independence imposed by miR-206, but severely impairs differentiation of ERMS, ARMS, and myogenic cells. Thus SMYD1 is essential for the activation of muscle genes. Conversely, among the downregulated genes, we found G6PD, the enzyme catalyzing the rate-limiting step of the pentose phosphate shunt. In this work, we confirmed that G6PD is a direct target of miR-206. Moreover, we showed that G6PD silencing in ERMS cells impairs proliferation and soft agar growth. However, G6PD overexpression does not interfere with the pro-differentiating effect of miR-206, suggesting that G6PD downmodulation contributes to - but is not an absolute requirement for - the tumor suppressive potential of miR-206. Targeting cancer metabolism may enhance differentiation. However, therapeutic inhibition of G6PD is encumbered by side effects. As an alternative, we used DCA in combination with miR-206 to increase the flux of pyruvate into the mitochondrion by reactivating PDH. DCA enhanced the inhibition of RMS cell growth induced by miR-206, and sustained it upon miR-206 de-induction. Altogether these results link miR-206 to epigenetic and metabolic reprogramming, and suggest that it may be worth combining differentiation-inducing with metabolism-directed approaches

Proceedings Of The 23Rd Paediatric Rheumatology European Society Congress: Part Two

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