Biology

Dr. Ramesh Yelagandula
Institute of Molecular Biotechnology (IMBA)-Vienna Biocenter, Vienna, Austria

Abstract:

Heterochromatin is an essential feature of eukaryotic genomes with important
functions in gene regulation, chromosome segregation, genome stability and
development. At the molecular level, heterochromatin is characterized by specific
chromatin modifications including DNA methylation and histone H3 lysine 9
methylation (H3K9me3) which are critical for transcriptional silencing of transposons,
repetitive DNA elements and genes. H3K9me3 bookmarks nucleosomes for
interaction with Heterochromatin Proteins (HP1s) which in turn leads to targeting of
many additional heterochromatin regulators. Furthermore, HP1s themselves can
dimerize through the chromo-shadow domain supporting staple-like connections
between HP1-associated chromatin regions, which could ultimately generate a
compact, liquid-like nuclear compartment visualized as constitutive chromocenters.
Importantly, in proliferating cells, genome replication presents a major disruption of
heterochromatin integrity and transcriptional silencing. Hence epigenetic
mechanisms are required to ensure faithful restoration of repressive chromatin
structure. While many important regulators of heterochromatin have been identified,
relatively little is known about their contribution to epigenetic inheritance and the
underlying molecular mechanism of H3K9me3 propagation through genome
replication.
To address this question, I have updated a powerful reductionist assay previously
developed in my current lab to recapitulate inheritance of heterochromatin in mouse
embryonic stem cells (mESCs) cells using reversible HP1 tethering to chromatin via
the Tet-OFF system. Using this biosynthetic approach, I have found that heritable
H3K9me3 retention and reporter silencing is tightly linked to replication-dependent
maintenance of DNA methylation suggesting positive feedback loop between
H3K9me3 and DNA methylation in mammals similar to plants and fungi. To uncover
novel regulators involved in the feedback loop between DNA methylation and
H3K9me3, I have used our phenotypic reporter assay in combination with CRISPR
screening technology. In addition to known regulators (H3K9 methylases, Dnmt1 and
UHRF1), I have identified several novel hits without previous links to
heterochromatin maintenance. Currently, I am further characterizing the role of two
novel ZFPs in maintenance of heterochromatin. Interestingly, deletion of one of the
ZFPs leads to decrease in global DNA methylation, activation of transposable
elements and totipotent cell markers. These results suggest that identified ZFP plays
an important role in progression of early embryogenesis through assembly of
heterochromatin.