# DNA Binding Properties of Zinc Finger Proteins

> **NIH NIH R01** · JACKSON LABORATORY · 2020 · $367,621

## Abstract

The human genome contains over 700 genes encoding proteins with zinc finger domains, more than half of
which contain eight or more fingers organized in a tandem fashion. Many of these genes function as
transcription factors, insulator binding proteins, or chromatin modifiers. Despite their importance, we still lack a
comprehensive knowledge on the rules that determine these proteins’ binding to DNA, and the existing
prediction programs do not perform satisfactorily. Recently, we have developed two new methods for isolation
and deep sequencing of zinc finger protein binding sites. The first, Affinity-seq directly determines the relative
affinity of tens of thousands of binding sites genome-wide with high binding specificity. It also provides the
opportunity for mutational analysis of binding site specificities using alternate sources of genomic DNA. The
second, Spec-sec, determines the changes in binding energy for thousands of variants of a preferred
sequence, and their sensitivity to DNA methylation. We propose to apply these methods for comprehensive
analysis of DNA binding sites of over twenty mouse and human natural protein variants of the recombination
regulator PRDM9, as well as over one hundred other human and mouse zinc finger proteins, which represent
different groups of long zinc finger array proteins, and whose binding sites has not been determined previously.
In Aim 1, we will determine the specificities of PRDM9 protein variants binding to DNA. Aim 1a will determine
how systematic changes in contact amino acids, numbers, and interactions between ZFs in PRDM9 protein
variants affect their DNA binding by Affinity-seq. Aim 1b will determine the quantitative specificity and
sensitivity to DNA methylation of each PRDM9 protein variant by Spec-seq. Aim 1c will use cell culture
approaches to determine how conserved features of ZF arrays and combinations of motifs in the same array
affect the biological activity of engineered PRDM9 protein variants. In Aim 2, we will determine whether DNA-
binding specificities of different laZFP groups co-evolve with their additional domains. Aim 2a will determine the
commonality or uniqueness of the rules governing binding to DNA of laZFPs belonging to BTB-, SCAN-, SET-,
and KRAB- containing groups, and those without additional domains, by Affinity-seq. Aim 2b will determine
their quantitative specificity and sensitivity to CpG methylation (mCpG) status by Spec-seq. In Aim 3, we will
develop new and improved computational algorithms for binding site modeling and motif prediction based on
laZFP sequences, including mCpG sensitivity. Aim 3a will develop enhanced specificity representations of
ZFPs that take full advantage of the Spec-seq data and don’t impose the positional independence inherent in
PWM models. Aim 3b will develop improved motif prediction models including methylation sensitivity.

## Key facts

- **NIH application ID:** 10006589
- **Project number:** 5R01GM125736-03
- **Recipient organization:** JACKSON LABORATORY
- **Principal Investigator:** Petko M Petkov
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $367,621
- **Award type:** 5
- **Project period:** 2018-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10006589

## Citation

> US National Institutes of Health, RePORTER application 10006589, DNA Binding Properties of Zinc Finger Proteins (5R01GM125736-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10006589. Licensed CC0.

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