| Abstract |
The MHCII genes of the major histocompatibily complex play an important
role in immune response. Their regulation implicates proteins that synergistically bind
to the promoter and constitute the MHCII enhanceosome (MCE). Although necessary,
MCE is not sufficient to drive transcription. The master coactivator CIITA is required
for efficient transcription initiation and elongation. This study approaches the
regulation of MHCII genes through the cell cycle and particular during mitosis. The
existence of gene bookmarking was examined, an important process for the cell in
order to maintain the gene expression profile to the next cell cycle.
Immunostaining experiments for components of MCE showed their
association on mitotic chromatin in a dynamic manner, although with variable kinetics
in different stages of the cell cycle. After the synchronization of a B lymphocyte
culture and chromatin immunoprecipitation assays in different cell cycle stages were
studied the kinetics of the recruitment of different regulatory factors on the DRA
promoter, a prototypical MHCII gene. The recruitment of the regulatory factors was
also identified in a better defined mitotic environment.
To compare the identified bookmarking mechanism in non-lymphoblastoid
cells, epithelial cells, modified to express MHCII genes constitutively, were used.
Chromatin immunoprecipitation assays were showed that mitotic occupancy of the
factors on the DRA promoter was diminished, and so does the accessibility, compared
to B lymphocytes. Furthermore transcription was abolished contrary to B lymphocyte
that it maintained in low levels and can be fully rescued even by providing CIITA
inside mitosis.
Also, another regulatory element of the DRA gene, LCR/XL4 was studied.
Results showed the mitotic occupancy of the NFY complex on this region and the
recruitment of a phosphatase, PP2A, through interaction with NFYA transcription
factor. Then, with PP2A knocking down was identified that PP2A bookmarks the
DRA gene, through chromatin decondensation, for transcription timing upon entry
into the G1 phase of the cell cycle. Finally, chromatin decondensation could rescue
the DRA expression in epithelial cells during mitosis.
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