ABSTRACT
Transcription factors require coactivators to communicate with the general transcription machinery and,
thereby, ensure that biological inputs are translated into specific gene-expression programs. The Mediator
complex is such a coactivator and acts as a ‘molecular bridge’ between transcription factor at enhancers and
RNA polymerase II (Pol II) at promoters. It is a large macromolecular complex further arranged in four modules
that confer high flexibility: the head, the middle, the tail and the kinase module. The member of the kinase
module Mediator 12 (MED12) has been found frequently mutated in both solid (endometrial, lung, cervical,
colon carcinomas) and blood (DLBCL, CLL, ALL, AML) cancers. However, the underlying mechanisms of
MED12 mutations and its role in disease initiation and progression remain elusive. We have recently focused
on the function of the kinase module and specifically MED12 in hematopoietic stem cell (HSC) differentiation
and transformation. We found that MED12 protein expression is controlled post-translationally by the ubiquitin
ligase FBXW7, a frequently mutated tumor suppressor. We also found that MED12 is an essential regulator of
HSC function, as in vivo deletion of MED12 compromises HSC survival and leads to mouse lethality. Together
with essential hematopoietic transcription factors, MED12 co-occupies HSC-specific enhancers. MED12
depletion destabilizes P300 binding thus leading to rapid enhancer “inactivation”, and loss of expression of key
HSC-specific genes. These data suggest that MED12 expression and function can be altered due to multiple
mechanisms, including somatic mutations targeting the gene itself or its regulators (FBXW7), and that this
aberrant function can lead to malignant transformation. This proposal aims to shed light on the molecular
mechanisms altered upon deregulation of a crucial regulator of enhancer activity, such as MED12. While it has
been suggested that MED12 mutations confer a “gain-of-function”, no mechanistic studies have been
performed up to date. To address this key question, we are studying chronic lymphocytic leukemia (CLL), the
most common adult leukemia in the western world. To dissect how disruption of Mediator function contributes
to this heterogeneous and complex disease, we use a combination of: a) transcriptional/epigenetic
characterization of human patient samples harboring MED12 mutations, b) CRISPR-modified and ES targeted
transgenic mouse models to investigate the ability of MED12 lesions to initiate and maintain disease, and, c) in
vitro transcriptome, epigenetic and 3D-chromosome topology in CRISPR-modified cell lines with MED12
mutations. Defining the mechanisms by which Mediator and enhancer regulation contribute to malignant
transformation will be beneficial for the development of novel therapies targeting blood malignancies and solid
tumors. The recent identification of small molecules targeting Mediator pharmacologically suggests that such
therapies are within reach.