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An aberration in the CTCF folding of the Hox 6 gene, results in extra ribs in this mouse.

DNA-binding protein critical to normal embryo

Scientists have found that CTCF, a DNA-binding protein, is essential in the body plan of a developing embryo.

Scientists at NYU Langone Medical Center and New York University, studied mouse brain cells and how they manage animal movements. Their research led to important details on how Hox genes keep cells in position — as well as in the right order front to back.

The work was published in Science, February 27, 2015.

Hox genes are arranged in organized clusters on an animal genome, but only a subset of Hox genes are active at any given time in a cell.

Researchers observed that a precise "memory" from mother cell to daughter cell of active and inactive Hox genes is fundamental to a normal body plan. If a failure occurs in that "memory" system, a body part could be produced in the wrong anatomical position.

"Previous research has shown that CTCF acts as a key insulating barrier preventing mistakes in cells as they multiply and differentiate," says Varun Narendra, the study's lead author, and a fifth-year graduate PhD student in developmental biology at NYU Langone and the Howard Hughes Medical Institute. "Now we have shown that correct positioning also depends on CTCF."

"The findings provide new insight into how cells faithfully transmit organizational information as embryos develop. When cell development goes awry, abnormal development and diseases such as cancer can occur. Information from this study could help in therapies to address developmental missteps in Hox genes and their regulators."

Danny Reinberg PhD, professor of biochemistry and molecular pharmacology at NYU Langone, Howard Hughes Medical Institute investigator and senior study investigator.

CTCF is a DNA-binding protein, which marks "insulator" regions on the DNA of animals. These regions act as boundaries affecting how cells package DNA. In specific regions, CTCF binding ensures segments of the genome are packaged to become active, while not interfering with neighboring segments that should not become active in any daughter cells generated. Using mouse embryo stem cells that generate motor neurons, scientists identified how CTCF isolates Hox genes in this way.

"We found that the purpose of CTCF is to divide the Hox cluster into segments, allowing a cluster to fold into strict domains that are either active or inactive on either side of CTCF."

Varun Narendra, lead author, graduate PhD student.

To demonstrate that CTCF binding is needed for correct Hox gene activation, researchers removed sites on the genome where CTCF would normally bind, and discovered that without CTCF binding, the Hox cluster would not fold properly. As a result, motor neurons activated the wrong set of Hox genes.

"By altering the folding pattern of the Hox cluster, we altered the motor neurons' understanding of their anatomical position. In doing so, we also altered their ability to send nerve signals to the appropriate muscle targets," said Esteban Mazzoni, PhD, a study co-investigator and assistant professor of biology and New York University.

Because the precise activation of Hox genes is essential for a cell's fate, Mazzoni says: "the research should prove extremely useful in developing novel embryonic stem cell-based therapies."

Abstract
Polycomb and Trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities.

Other contributors to the study, all from NYU Langone's Department of Biochemistry and Molecular Pharmacology, are Pedro P. Rocha, PhD; Disi An; Ramya Raviram, and Jane A. Skok, PhD.

Funding support for this study was provided by The Howard Hughes Medical Institute, National Institute of Health grants (R37-37120, RM-64844, T32 GM08652, GM112192, and R01 HD079682) and the P.A.L.S. Foundation.

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