Development of a low input chromatin profiling technique to examine the role of PRC2 proteins in development and leukaemia

Sondermann, Nathalie Allegra Faye

Development of a low input chromatin profiling technique to examine the role of PRC2 proteins in development and leukaemia

Document Type

Doctoral thesis
Published Version

Author

Sondermann, Nathalie Allegra Faye

Director

Sabatel, Céline

Publication Date

2024-03-21

Pages

191 p.

Embargo date

2026-03-21T01:00:00Z

Citation

Sondermann, N. Development of a low input chromatin profiling technique to examine the role of PRC2 proteins in development and leukaemia. Universitat Pompeu Fabra; 2024. handle: http://hdl.handle.net/10803/690634

This citation was generated automatically.

Doctoral program

Programa de Doctorat en Biomedicina

Abstract

Polycomb repressive complexes (PRC1/2) are key chromatin regulators in development. Their deregulation is implicated in various cancers including leukaemia. Meanwhile, MLL-rearrangements by chromosomal translocations and the resulting fusion proteins as MLL-AF9 are a known cause of leukaemia. Recent re-search show an interplay between PRC1/2 and MLL-AF9 during leukemogenesis. This raises the question of a potential role in embryonic development as well. However, conventional chromatin immunoprecipitation (ChIP) is not suited to ex-amine non-histone proteins in fewer than millions of cells and still has a high background signal. Recent alternatives as CUT&RUN and CUT&Tag show limitations as well, despite a promising, targeted approach. Here, an improved CUT&Tag protocol has been developed and validated for the examination of PRC1/2 in low cell numbers. Moreover, it has been used to examine MLL-AF9 activity during differentiation of mouse embryonic stem cells into embryoid bodies, and implications for PRC1/2 executing their silencing activity on key genes.
Los complejos represivos Polycomb (PCR1/2) son reguladores clave de la cromatina durante el desarrollo cuya desregulación está implicada en varios tipos de cáncer incluyendo leucemia. En MLL, algunas de las causas conocidas son los reordenamientos cromosómicos debido a translaciones y la generación de proteínas resultantes de fusión, como MLL-AF9. Estudios recientes han demostrado la relación entre PCR1/2 y MLL-AF9 durante leucemogénesis lo cual apunta al desarrollo embrionario como un factor importante. Sin embargo, la inmunoprecipitación de cromatina convencional (ChIP) no es adecuada para el estudio de proteínas asociadas a la cromatina en experimentos con pocas células pues arriesga excesivo ruido de fondo. A pesar de su prometedor enfoque y especificidad, recientes alternativas como CUT&RUN y CUT&Tag todavía muestran limitaciones. Aquí, hemos desarrollado un protocolo mejorado de CUT&Tag validado para el estudio de PRC1/2 en pocas células. Además, hemos examinado la actividad de MLL-AF9 durante la diferenciación de células madre embrionarias de ratón en cuerpos embrionarios junto con cambios asociados a PRC1/2 en el silenciamiento de genes.