# Understanding CTCF Boundaries Controlling Hox Gene Expression

> **NIH NIH R01** · UNIVERSITY OF MIAMI SCHOOL OF MEDICINE · 2024 · $599,999

## Abstract

Project Summary
The formation of discrete chromatin-chromatin interactions in the genome during the
differentiation of mammalian cells leads to the specific insulation of repressed chromatin domains
from those actively engaged in transcription. This partitioning thwarts the inappropriate invasion
of actively transcribing regions into those that must stay repressed⏤this process is highly
regulated and diversified during development, giving rise to the specific gene expression profiles
reflective of and inherent to markedly distinct cell types. The key proteins recognized as fostering
chromatin-chromatin interacting structures are cohesin, CTCF, and the newly recognized MAZ.
Cohesin is believed to extrude a loop of chromatin such that the endpoints of this loop merge with
two chromatin-bound CTCF proteins, thereby anchoring cohesin. CTCF and MAZ interact
independently with cohesin and exert their function through binding to specific pairs of CTCF and
MAZ DNA elements, respectively. Notably, CTCF also interacts with RNA which leads to its
multimerization and CTCF mutant in any of its RNA binding domains [Zinc Finger 1 (ZF1), ZF10,
and RBRi] gives rise to disrupted chromatin loop formation. Of particular interest in this study is
the CTCF mutant in its ZF1 as it retains all other CTCF functions. The approach here exploits the
mammalian HoxA-D clusters as an ideal model for investigating the process of insulation as a
function of differentiation as they exhibit linear and temporal regulation during development. This
project proposes to expand upon previous successful investigations of Hox gene regulation, which
led to the discovery of MAZ as a DNA-site-specific insulation factor, our recent findings of another
possible “site-specific insulator”, PATZ1, the key role of CTCF/RNA interaction in fostering the
integrity of chromatin boundaries, as well as the construction of synthetic HoxA clusters that when
placed ectopically exhibit all the regulatory features inherent to the endogenous version. With this
foundation, investigations of the features requisite to chromatin boundary formation as a function
of differentiation will include: genetic manipulations in vivo in conjunction with molecular biological
approaches to define the roles of MAZ and PATZ1 in insulating a portion of the HoxA cluster, the
identification of other potential insulating proteins that function at the HoxA cluster posterior to
that of MAZ and PATZ1, and an investigation as to whether RNAs interacting with CTCF are
determinant to particular chromatin boundary formation. The results from these multi-pronged in
vivo and in vitro approaches will reveal the regulatory and mechanistic basis by which chromatin
boundaries are organized, thereby dictating appropriate gene expression.

## Key facts

- **NIH application ID:** 10801657
- **Project number:** 2R01NS100897-06A1
- **Recipient organization:** UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
- **Principal Investigator:** Esteban Orlando Mazzoni
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $599,999
- **Award type:** 2
- **Project period:** 2018-03-15 → 2029-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10801657

## Citation

> US National Institutes of Health, RePORTER application 10801657, Understanding CTCF Boundaries Controlling Hox Gene Expression (2R01NS100897-06A1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10801657. Licensed CC0.

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