Tracking how molecular machines propagate epigenetic information in time and space

ABSTRACT
Heterochromatin, a gene-repressive nuclear ultrastructure, is required for the normal patterning of the genome
into active and inactive regions, to preserve structural integrity and drive and maintain developmental fates.
While heterochromatin assembly is locally nucleated by DNA-sequences, the majority of the patterning process
requires it to spread along the chromatin template. A major form of heterochromatin involved in this patterning
is signaled by methylation (me) at Lysine 9 (K9) of histone 3 (H3). Major questions have remained unanswered
about heterochromatin spreading, which has limited our ability to effectively manipulate this process for
regenerative medicine or synthetic biology: 1. What are the biochemical mechanisms underlying it? 2. How can
heterochromatin spread over loci of vastly different chemical, structural and stability regimes? And 3. How is the
reaction tuned to expand or contract during development to stabilize cell fate switches? Over the last four years,
my laboratory has devised strategies to tackle these questions. We have developed single-cell sensors of
heterochromatin spreading that have enabled us to document the intrinsic behavior of the reaction in real-time
(Al-Sady et al, 2016; Greenstein et al, 2018), how euchromatic features sculpt the spreading reaction (Greenstein
et al 2019) and defined genes that enable this process in different chromatin environments (Greenstein & Ng et
al, 2020). Additionally, we have developed single molecule systems to study histone methylation on individual
chromatin strands and bulk biochemical methods to probe the function of the H3K9me “writer machines”. Over
the next five years, we will deploy these experimental systems to fully illuminate the heterochromatin spreading
process from three angles: 1. The writer machine: We will use single molecule and biochemical sequencing
approaches to unravel the mechanisms and molecular trajectories by which the enzyme complexes “write”
H3K9me along the chromatin template. 2. The substrate: Heterochromatin spreading occurs over radically
different chromatin landscapes and cannot fit a “one-size-fits-all” model. We will use single-cell heterochromatin
spreading sensors in fission yeast to examine how chromatin loci of different activity states or the same locus
with different histories impact the reaction. Further, we will define the genetic circuitry that enables and tunes
spreading in different chromatin environments. 3. The view form development: We focus on the developmentally
crucial H3K9 methylase G9a/GLP and will distinguish different hypotheses on how developmentally triggered,
G9a/GLP-dependent heterochromatin expansions and contractions are implemented in mammalian stem cells.
Further, since the relationship between H3K9 methylation by G9a/GLP and silencing is elusive, we will define
the steps that must occur for gene repression after H3K9 methylation. Together, this suite of projects connects
the intrinsic biochemical features of heterochromatin spreading, to the steady-state and developmentally
dynamic genome partitioning function of this unique ultrastructure, which underlies genome and cell fate stability.

Grant Number: 5R35GM141888-05
NIH Institute/Center: NIH
Principal Investigator: Bassem Al-Sady