# Synthetic HoxA to dissect transcriptional regulatory logic

> **NIH NIH R01** · NEW YORK UNIVERSITY · 2022 · $544,240

## Abstract

Project Summary
The “big picture” of this proposal is that using our newfound ability to build big synthetic DNAs (1 Mb scale) at
will and with great efficiency and precision, we propose a new paradigm for “systems genetics” of gene
regulation in the context of a unique master developmental regulator, the HoxA cluster. We use yeast to make
hundreds of mammalian gene loci variants rapidly, with high precision and cos-effective. Leveraging this
technology, we have already assembled and deliver 134kb and 170 kb long constructs containing the entire rat
HoxA cluster, as well as the mouse counterparts. We can deliver precision-engineered large HoxA constructs
to either an ectopic location at the Hprt1 locus, and working on delivering to the allelic location as well. Using
these powerful new tools, we describe how we can deliver the rat and mouse HoxA clusters to Hprt1 in mouse
ES cells. Using the heterologous rat HoxA clusters allows us to internally compare the Hox loci from both
species in the same cell using a range of chromatin “omics-based” readouts. The HoxA cluster is extremely
highly conserved at the DNA level; we will take advantage of the rat HoxA, since it is a rodent gene it is likely to
complement function in the mouse, but is densely carpeted with genomic variants (one per 10 bp on average
across HoxA). These experiments will be done in a data-rich system using in vitro differentiation of mES cells
to motor neurons. These experiments can be done in the context of already generated HoxA+/+, +/–, or –/– ES
cells. The aims include asking the question Do Hox clusters require their genomic context containing long-
distance regulatory elements for the establishment of chromatin boundaries and initial gene expression? (Aim
1), attempting functional complementation with our ectopic synthetic loci in HoA-/- knockout background (Aim
2) and asking Is it possible to establish novel regulatory domains within Hox clusters? (Aim 3).

## Key facts

- **NIH application ID:** 10470924
- **Project number:** 5R01GM138876-02
- **Recipient organization:** NEW YORK UNIVERSITY
- **Principal Investigator:** Esteban Orlando Mazzoni
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $544,240
- **Award type:** 5
- **Project period:** 2021-09-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10470924

## Citation

> US National Institutes of Health, RePORTER application 10470924, Synthetic HoxA to dissect transcriptional regulatory logic (5R01GM138876-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10470924. Licensed CC0.

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