Deconvolving Vertebrate Hox Binding Preferences and Activation Properties

Embryonic development culminates with an astonishing number of terminal differentiation cellular states. Transcription factors (TFs) bind to specific DNA sequences in the genome and are key regulators of gene transcription responsible for generating cell diversity. TFs are grouped in families with similar DNA binding preferences and sometimes similar activation or repression domains, yet they perform different functions during cell differentiation. Consequently, how different TFs of the same family bind and regulate gene expression is poorly understood. Hox genes code for homeodomain (HD) TFs, the second largest TF family in the mammalian genome. Vertebrate HOX TFs are divided into anterior (HOX1-5), central (HOX6-8), and posterior (HOX9-13) paralog groups. In vertebrates, Hox genes pattern various developing tissues. Notably, spinal cord neuronal diversity requires Hox gene activity along its rostro-caudal axis. The role of Hox genes in organizing the spinal cord presents an interesting conundrum. The limb-innervating expression program is controlled by central (HOX6 & HOX8) HOX TFs at the brachial level and posterior (HOX10) HOX TFs at the lumbar spinal cord. Thus, HOX TFs with different DNA sequence preferences induce a similar motor neuron fate. Meanwhile, the posterior HOXC9 induces thoracic fate. Thus, two posterior group genes, Hoxc9 and Hoxc10, induce different spinal cord fates. In agreement with their genomic cluster position, Hox13 paralogs are expressed late during development, distally, and in posterior regions. This application aims to understand how the mammalian posterior group Hox TFs diversify their function to pattern the spinal cord. Aim 1 will dwell more on the protein domain and specific HOX amino acids in Hoxc9 and Hoxc13 that mediate their ability to bind to inaccessible chromatin and DNA residence time. Aim 2 will take a complementary machine learning approach using ChIP-exo data coupled with chromatin features to distinguish individual pioneer Hox binding sites from non-pioneer sites. Aim 3 will investigate how the very variable N-terminus controls the transcriptional output. Project Number: 1R01NS149716-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Esteban Mazzoni (+2 co-PIs) | Institution: NEW YORK UNIVERSITY SCHOOL OF MEDICINE, NEW YORK, NY | Award Amount: $638,783 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 MGG-T (90)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11391795