# Functional Analysis of Epigenetic Complexes

> **NIH NIH R35** · MASSACHUSETTS GENERAL HOSPITAL · 2024 · $890,852

## Abstract

ABSTRACT
Research in our laboratory focuses on the mechanisms used by complexes required for transcriptional
‘memory’ during development. Development of complex large organisms requires that master
regulatory genes that specify a certain cell lineage be maintained in an ’on’ state throughout the lifetime
of an animal and that master regulatory genes that are inappropriate for expression in that lineage be
maintained in an ‘off’ state. This proposal is primarily focused on Polycomb-Group complexes,
especially Polycomb Repressive Complex 1 (PRC1), which are key to maintaining the repressed state.
The Polycomb-Group (PcG) of genes was initially identified in 1947 by Pam Lewis using Drosophila
genetic screens. It was subsequently found to be conserved in vertebrates and to be essential for
developmental patterning. In humans, paralogs of the fly PcG proteins are frequently found in families
of complexes. The PRC1 family of complexes contains over 100 members with overlapping but distinct
compositions. These are subdivided into two large sub-families: The non-canonical PRC1 family
ubiquitylates histone H2A on lysine 119, the canonical PRC1 family compacts chromatin locally, forms
long-range interactions in the genome, and is found in condensates called ‘Polycomb bodies’ both in
vitro and in vivo.
This proposal focuses on functions of the core protein components of canonical PRC1 (cPRC1). The
two defining members of the cPRC1 family are one of the CBX proteins (usually CBX2, 4, 7 or 8) and
either PHC1 or PHC2. These proteins are known to be involved in phase separation into condensates,
local compaction (CBX2,4,8) and long-range interactions (both PHC proteins.) cPRC1 complexes also
contain a PCGF paralog and either RING1A or RING1B.
We propose to study the function of canonical PRC1 paralogs using four approaches:
The first set of experiments examines the ability of CBX and PHC proteins to generate condensates
when mixed with nucleosomal arrays in a purified in vitro system. We will use TIRF microscopy in a
protocol that allows visualization and quantification of single nucleosomal arrays, single cPRC1
complexes or sub-complexes, and the condensates that form containing both arrays and PRC1.
Dynamics of the condensates will be examined in real time, precise stoichiometries will be measured,
and properties will be compared between cPRC1 complexes with different compositions. This will
determine the contributions of different CBX and PHC proteins to condensate behavior. The ability of
these condensates to interfere with mammalian SWI/SNF function will be measured.
The second set of experiments will use a chemically inducible dimerization system in tissue culture
cells to add various distinct regions of proteins known to promote phase separation to a mutant CBX2
protein that no longer generates condensates. These experiments will determine whether addition of an
condensate forming region of an unrelated protein can rescue PRC1 biological function. These ...

## Key facts

- **NIH application ID:** 10840182
- **Project number:** 2R35GM131743-06
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** ROBERT KINGSTON
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $890,852
- **Award type:** 2
- **Project period:** 2019-04-01 → 2029-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10840182

## Citation

> US National Institutes of Health, RePORTER application 10840182, Functional Analysis of Epigenetic Complexes (2R35GM131743-06). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10840182. Licensed CC0.

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