AhR regulates cell differentiation through the control of transposon-containing pluripotency genes
Speaker: Pedro M. Fernández Salguero
Universidad de Extremadura [Badajoz, Spain]
Host: Pedro Lazo-Zbikowski
Salón de Actos del CICAhR regulates cell differentiation through the control of transposon-containing pluripotency genes
Antonio Morales-Hernández1, Francisco J. González-Rico1, Eva M. Rico-Leo1, Angel C. Román2, Nuria Moreno-Marín1, Eva M. Barrasa1, Cristina Vicente3, Lluís Montoliú3, Alejandra González4, Marcos Malumbres4, Laura Sánchez5, José L. García-Pérez5 and Pedro M. Fernández-Salguero1 (1) Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz; (2) Instituto Cajal, CSIC, Madrid; (3) Centro Nacional de Biotecnología (CNB), Madrid; (4) Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid; (5) Centro de Genómica e Investigación Oncológica (GENYO), Granada. The eukaryotic genome is organized into highly structured expression domains whose activation or repression largely depends on the sequence and conformation of the neighboring chromatin, which can allow or block gene expression from distal enhancers. Recent studies have proposed that such flanking chromatin regions in fact provide binding sites for transcription factors ultimately controlling insulator elements and chromatin boundaries. Widely spread transposable elements are likely to modulate genome organization and expression by providing such transcription factors binding sites. We have previously characterized a novel murine SINE-B1 retrotransposon (B1-X35S) with potent insulation/boundary activity that is conferred by its binding to the transcription factors dioxin receptor (AhR) and Slug (Snai2) (Roman et al., PNAS 2008; Genome Res 2011). In humans, three Alu elements heterologous to B1-X35S (XS-Alus) have been also identified that are present at the flanking upstream regions of the pluripotency-driver genes Oct4, Nanog, Shh, Sox2, Notch and Klf4. In undifferentiated embryonic carcinoma Ntera-2 cells, retinoic acid (RA)-induced differentiation repressed Oct4, Nanog and Shh expression in parallel with a major increase in AhR protein levels. Stable AhR knock-down restored a basal undifferentiated state and blocked RA-induced differentiation, rescuing Oct4, Nanog and Shh expression. Focusing on Oct4 and Nanog, the AhR- and RA-dependent repression of these genes was abolished by RNA Pol III inhibition, suggesting the involvement of non-coding RNAs in the regulatory process. RNAseq is underway to determine whether transcripts synthesized from the XS-Alu transposons flanking Oct4 and Nanog direct the AhR-mediated gene repression and cell differentiation. Such mechanism is consistent with the facts that transposition experiments revealed that Oct4 and Nanog XS-Alus are transcriptionally active while ChIP assays showed stronger AhR binding to such XS-Alus in differentiated vs undifferentiated cells. The role of XS-Alu elements in repressing Oct4 and Nanog was further evidenced by their intrinsic enhancer blocking activities, which are now under scrutiny for a potential boundary/chromatin barrier function. The relevance of AhR in maintaining a differentiated cell state was further supported by two sets of reprogramming experiments: (i) the Oct4-Klf4-Sox2-Myc cocktail was less efficient in inducing IPs cells from AhR+/+ than from AhR-/- MEFs; (ii) AhR knock-down enhanced whereas constitutive AhR activation blocked IPs reprogramming from MEFs derived from OKSM transgenic mice. These and other results from our laboratory highlight AhR as a regulator of cell differentiation by repressing pluripotency-associated genes harboring XS-Alu retrotransposons in their promoters.