# High resolution chromatin structure of purified eukaryotic genes

> **NIH NIH F32** · JOHNS HOPKINS UNIVERSITY · 2020 · $65,310

## Abstract

PROJECT SUMMARY/ABSTRACT
 In eukaryotes, genomic DNA is condensed into chromatin by associating with histone proteins, and the
folding of chromatin organizes the genome within the cell nucleus and plays an essential role in regulating gene
transcription. Chromatin dysfunction and the resulting dysregulation of gene expression is a well-recognized
contributor to many human diseases, including cancers and neurological disorders. Despite decades of research
and its crucial role in the expression of genes, the native chromatin structure of coding and regulatory regions of
the genome remains poorly resolved. While conventional structural biology methods have yielded many high-
resolution structures of chromatin components, a general limitation of these studies is that the conditions used
to prepare samples in vitro do not faithfully recapitulate the native chromatin environment in vivo, due to complex
and variable composition of native chromatin. On the other hand, methods that have been geared towards
probing the native chromatin landscape in situ are well-suited to provide information of a broad scale (e.g.
genome wide) but are limited in resolution or dimensionality. I aim to bridge the gap between these traditional
approaches by analyzing the composition and structure of natively assembled, yet purified, chromatin of specific
yeast gene loci. The initial phase of this work will be aimed at optimizing the preparation of isolated chromosomal
fragments in order to maximize preservation of their native composition and structure, using a combination of
electron microscopy (EM) and mass spectrometry proteomics as quality control assays. Quantitative mass
spectrometry of isotope-labeled samples will also be used to perform “chromatin proteomics” by analyzing
changes in chromatin composition between the induced and repressed forms of the minichromosome genes.
Following optimization of purification conditions, negative stain and rotary shadowing EM will be used to generate
high-resolution two-dimensional maps of nucleosome positioning across populations of a given purified yeast
gene in its active or repressed state. Finally, utilizing state-of-the-art instrumentation available at Johns Hopkins
University, modern cryo-EM and cryo-electron tomography techniques will be used to analyze the three-
dimensional chromatin structure of the purified gene, including an assessment of the variability in chromatin
structure amongst populations of repressed or induced genes. Ultimately, this approach should provide an
overview of the three-dimensional “structure” of a gene in both repressed and activated states, as well as
“zoomed-in” views of specific gene fragments, showing for instance the interplay between histones and non-
histone proteins at the promoter region of genes. The results of this study will significantly advance our
understanding of the mechanisms behind transcriptional regulation via chromatin, and could provide insights into
mechanisms of transcripti...

## Key facts

- **NIH application ID:** 10014622
- **Project number:** 5F32GM133151-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Robert Kenneth Louder
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $65,310
- **Award type:** 5
- **Project period:** 2019-09-16 → 2022-09-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10014622

## Citation

> US National Institutes of Health, RePORTER application 10014622, High resolution chromatin structure of purified eukaryotic genes (5F32GM133151-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10014622. Licensed CC0.

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