# Structural and functional analysis of gene silencing

> **NIH NIH R01** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2020 · $364,425

## Abstract

Histones package genomic DNA in the eukaryotic nucleus into chromatin, whose structure controls all DNA-
based processes, including transcription, replication, recombination, and repair. A major mechanism regulating
chromatin structure is based on post-translational modifications of the histones, which are deposited by enzymes
or “writers” and recognized with high specificity and selectivity by “reader” domains of certain proteins to mediate
downstream functions. The interplay between writers and readers is essential for development and is perturbed
in many diseases, including cancer. A major focus in chromatin biology is thus to understand the detailed
mechanisms that control chromatin structure, which is greatly hampered by the relatively few high-resolution
structures of chromatin-bound proteins and protein complexes that deposit and recognize histone modifications.
Large segments of the eukaryotic genome are packaged into transcriptionally silent heterochromatin.
Heterochromatin is critical to maintain genome integrity and to prevent chromosomal defects. Interestingly,
heterochromatin formation and DNA replication are often coordinated through specific chromatin modifications.
For example, different methylation states of histone H4 at lysine 20 (H4K20) regulate distinct processes:
H4K20me3 is found in heterochromatin, whereas H4K20me2 is found at the origins of replication. Methylation of
H4K20 is catalyzed by two related histone methyltransferases, SUV4-20H1 and SUV4-20H2, but it is not known
how these enzymes are recruited to chromatin or how their catalytic activity is regulated. In addition,
heterochromatin formation and replication can be coordinated through readers, such as Orc1, a subunit of the
Origin Recognition Complex (ORC). Orc1 binds to chromatin using its bromo-adjacent homology (BAH) domain,
which interacts with histone modifications and heterochromatin proteins. The detailed mechanisms involved in
these processes are largely unknown. We will use structural and functional approaches to fill this critical
knowledge gap, with AIM 1 focusing on elucidating the mechanisms of SUV4-20H enzymes and AIM 2 focusing
on understanding the mechanisms of Orc1 BAH domains. We will use cryo-EM to determine structures of these
writers and readers bound to nucleosomes and complement the structures with functional experiments in vitro
and in vivo. The structural and functional studies of SUV4-20H writers and Orc1 BAH domain readers in complex
with nucleosomes will uncover general principles that underlie the deposition and recognition of histone
modifications. Our proposed comprehensive studies will provide crucial insights into the fundamental biological
processes of heterochromatin formation and replication and will provide invaluable insights into how deregulation
of these complexes and resulting aberrations in chromatin structure contribute to diseases.

## Key facts

- **NIH application ID:** 10122220
- **Project number:** 2R01GM115882-06
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** Karim Jean Armache
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $364,425
- **Award type:** 2
- **Project period:** 2015-07-17 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10122220

## Citation

> US National Institutes of Health, RePORTER application 10122220, Structural and functional analysis of gene silencing (2R01GM115882-06). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10122220. Licensed CC0.

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