# N-terminal phosphorylation as a potentially tunable dial to modify the properties of HP1alpha-mediated heterochromatin

> **NIH NIH F32** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2021 · $34,281

## Abstract

Project Summary / Abstract
Appropriate gene expression underlies every aspect of cellular biology from viral replication to multicellular
organismal development to cancer. A key aspect of appropriate gene expression is the division of the genome
into heterochromatin, a more tightly compacted and transcriptionally silent portion of the genome, and
euchromatin, a more open, transcriptionally active portion. Euchromatic regions are thought to be defined by
the constant activity of sequence-specific transcription, but the interactions that define and maintain
heterochromatin are still an outstanding question in the field. Heterochromatin Protein 1α (HP1α) is a major
structural component of constitutive heterochromatin and is thought to mediate chromatin compaction. Recent
studies show that recombinantly purified HP1α undergoes liquid-liquid demixing on the addition of DNA to form
distinct protein-rich and protein-poor phases. Similarly, N-terminally phosphorylated HP1α (nPhos HP1α)
spontaneously demixes even in the absence of DNA. This phase separating ability has suggested new
potential mechanisms for heterochromatin function. In particular, the differential chromatin affinities and phase
separating abilities of nPhos and unmodified HP1α suggest that N-terminal phosphorylation may allow the cell
to tune its heterochromatin compartment. However, more work on nPhos HP1α biochemistry is required prior
to make targeted models of in vivo function.
The goal of this proposal is to determine the properties of nPhos HP1α available to the cell for the protection
and sequestration of heterochromatin. I will measure the strength and kinds of interaction between nPhos and
unmodified HP1α, and whether the two species form miscible phases in vitro using light and fluorescence
microscopy. I will also measure the viscoelasticity of and the diffusion of molecules within these phases
through correlated fluorescence fluctuations and micro-rheology to determine whether solvated molecules can
freely exchange between nPhos / HP1α phases. I will then correlate the physical material properties of pure
and mixed HP1α phases with measurements of chemical environment and heterochromatin-associated
enzymatic activity within HP1α droplets using established enzymatic assays as well as fluorescent pH, salt,
redox, and hydrophobicity probes. Lastly, I will determine the mesoscale structure of nPhos and HP1α droplets
containing heterochromatin-associated ligands using Soft X-ray Tomography (SXT). By generating HP1α
droplets in various heterochromatin contexts (chromatin, RNAi, known protein binding partners) I will be able to
determine how HP1α droplet structure is regulated, and how it relates to the corresponding material and
chemical properties. These studies will provide a comprehensive characterization of nPhos HP1α behavior,
and permit generation of targeted models for nPhos HP1α function for in vivo testing.

## Key facts

- **NIH application ID:** 10460025
- **Project number:** 3F32GM134567-02S1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Emily Wong
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $34,281
- **Award type:** 3
- **Project period:** 2019-09-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10460025

## Citation

> US National Institutes of Health, RePORTER application 10460025, N-terminal phosphorylation as a potentially tunable dial to modify the properties of HP1alpha-mediated heterochromatin (3F32GM134567-02S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10460025. Licensed CC0.

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