Enhancer landscape of normal and leukemic progenitors and alterations elicited by inactivation of Polycomb Ring1A and Ring1B

• Autores: Natalia Giner Laguarda
• Directores de la Tesis: Miguel Ángel Vidal Caballero (dir. tes.), Florian Grebien (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2024
• Idioma: inglés
• Número de páginas: 180
• Títulos paralelos:
  • Repertorio de potenciadores transcripcionales en progenitores normales y leucémicos y alteraciones inducidas por la inactivación de genes Polycomb Ring1A y RING1B
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Leukemic transformation arises from disruptions in default differentiation pathways of hematopoietic progenitors. Transcriptional networks of transcription factors and chromatin modifiers (epigenetic regulators) play decisive roles in normal but also in its aberrant deviations. The Polycomb group of genes represents one of the essential epigenetic regulators with well documented roles in hematopoiesis. The Polycomb products act as multiprotein assemblies endowed with an ability to modify histones through two large collection of complexes. Of these, the type I of Polycomb Repressive Complexes (PRC1) monoubiquitylates histone H2A, in a modification traditionally associated to gene silencing. The ubiquitylation module contains either RING1A or RING1B, essential paralogs in all PRC1 complexes. RING1A and RING1B were identified in the lab and previous work showed antagonistic roles regulating expansion of normal hematopoetic progenitors and restriction of differentiated progeny proliferation. We also showed that Ring1B is haploinsufficient in the transformation to AML cell types, via MLL-fusion proteins. In the present work, populations of non-transformed and leukemic progenitors were generated where the functions of RING1A and RING1B could be abrogated to investigate their function, in the transformation process. The work emphasized the characterization of enhancers, through mapping of histone modifications and chromatin accessibility assays, in combination with transcriptomic analysis.

    As normal, non-transformed hematopoietic progenitors we used wild type and Ring1B−/−, Ring1Bf/f , Cre-ER primary progenitors expanded ex-vivo by ectopic expression of HOXB8, under non-differentiating conditions (B8 cells). As a leukemic variant, we used the MLLAF9 and N-RasG12D expressing RN2 cell line. Ring1A and Ring1B inactivation was achieved by tamoxifen treatment of B8 cells and by a step-wise CRISPR-Cas9-mediated inactivation of both paralogs in RN2 cells.

    Transcriptomic analysis of wild type and RING1-depleted cells showed, in addition to the expected repressing functions, a majority of transcriptionally active targets, being promoted or compatible with gene expression. In mutant cells, some aspects of normal and leukemic states were partially reverted, suggesting a function for RING1 paralogs in canalization of transcriptional programs involved in cell identity.

    Chromatin structure, of normal and leukemic types was assessed by DNA accessibility and mapping histone modifications pertinent to enhancer characterization (H3K4me1, H3K27ac, H3K27me3). We focused on distal (5 kb away from TSSs) enhancers: among thousands identified, a substantial proportion undergo redeployment with RING1 depletion. Prevalent among DNA motifs algorithmically predicted are transcription factors (TFs) of the ETS family (PU-1, FLI-1) and of the AP1 family. Motifs for RUNX-1 and CEBPα TFs were enriched too and actual binding, from published data, was confirmed at higher occupancy rates in active enhancers that in other enhancer classes (intermediate, poised, primed).

    RING1B occupancy was assessed in B8 cells where, besides promoters regions, binding occurs at a subset of active and poised enhancers. Computationally inferred superenhancers, relatively unaffected by RING1 depletion, were enriched in the set of RING1Bbound enhancers co-occupied by TFs (PU-1, FLI-1, RUNX-1, CEBPα).

    In a set of normal versus leukemic transformation, this study expands the conventionally accepted repressing functions of RING1 proteins, points at activities of these Polycomb products at enhancers and generated a viable cell line adapted without RING1 products