Multicellular eukaryotic organisms contain identical genetic information within each cell, stored
within the nucleus as chromatin fibers. The regulation of gene expression allows cell-type-specific
temporal and spatial gene activation or repression despite the identical DNA sequence that determines
differentiation and cell identity. Chromatin 'writer' enzymes can alter chromatin by installing
post-translational modifications (PTMs) on histone proteins, which are then recognized by 'reader'
proteins. PTMs are epigenetic marks that regulate a dynamic yet balanced gene expression. Polycomb
group (PcG) proteins form a regulatory system of 'readers' and 'writers', essential for
transcriptional repression during development and differentiation.
Polycomb repressive complexes 1 (PRC1), a key member of the PcG family, function as the primary
E3 ligase 'writers' of ubiquitination on histone H2A at lysine 119 (H2AK119ub). The H2AK119ub
modification recruits Polycomb repressive complexes 2 (PRC2) to install H3K27me1-3 and form
repressive chromatin domains. Thus, variant PRC1 (vPRC1) is the primary catalyst of PcG-initialized
gene repression, with its activity dependent on its multisubunit composition, among others, the
Polycomb group of ring finger (PCGF) subunit. vPRC1 complexes dynamically engage chromatin,
but how these transient interactions are coupled to ubiquitin writing is unknown.
Here, I present a single-molecule fluorescence approach to directly observe ubiquitination dynamics
by vPRC1 in real-time on defined chromatin. Initially, I dissected the recruitment dynamics of
PRC1 complexes on immobilized synthetic chromatin. Subsequently, I introduced vPRC1 with a
preloaded E2~Ub to initiate H2AK119 ubiquitination. The observed binding and enzymatic kinetics
were used to construct a mechanistic model of real-time chromatin 'reading' and 'writing'.
vPRC1 transiently recruited chromatin fibers with non-specific and specific binding modes characterized
by short and long residence times. The presence of E2 increased the specific
residence time and the association rate, while a pre-installed H2AK119ub rescued the dissociation
and association times observed on wild-type chromatin. vPRC1 complexes dynamically searched
chromatin until they bound nucleosomes in a catalytically competent state, followed by E2~Ub
recruitment and ubiquitin transfer. The ubiquitination rate increased over time on wild-type H2A
chromatin fibers, while the ubiquitination efficiency on H2AK119R/K120R was minimal. vPRC1
showed a small processivity with up to two ubiquitin being transferred per single binding event.
The reactive binding and catalytic times of isolated events were independent of vPRC1 subunit
composition, while PCGF1-containing enzymes showed faster global ubiquitination due to more
frequent catalytically competent states. The efficiency of E2~Ub recruitment and the formation
of the catalytically active state determined the global activity differences between PRC1 subtypes
containing either PCGF1 or PCGF4. The findings demonstrate that the key factor influencing vPRC1
ubiquitination activity is the dynamic assembly of a ternary complex between chromatin, vPRC1
and E2.
The methodology may be expanded to study the enzymatic kinetics of other E3 ligase enzymes
and deubiquitinases on chromatin or other substrates. In particular, single-molecule enzymology
can be applied to characterize drugs' effects on the binding and enzymatic kinetics of E3 ligas