【For INTERNATIONAL COLLABORATIVE RESEARCH FY2023】

SUMMARY ofDATE: 10May2024

FY2023 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University
Research Title

Determining the protein structure of TBX1 variants by ultra-high magnetic
field NMR spectroscopy

ApplicantName

Noboru Hiroi

Affiliation

Department of Pharmacology, Department of Cellular and Integrative
Physiology, Department of Cell Systems and Anatomy UT Health San
Antonio

Present Title

Professor

Research Collaborator (Host PI)

Yohei Miyanoiri

Summary
Hemizygous deletion at human chromosome 22q11.2 is associated with elevated rates ofschizophrenia and diverse neurodevelopmental disorders, including ASD, intellectual disability, andattention-deficit hyperactivity disorder. Ultra-rare variants of some 22q11.2 CNV have been
identified in samples of ASD, schizophrenia, and neurodevelopmental disorders. In particular, a few
cases of ultra-rare TBX1 varaints (e.g., frameshift deletions) are associated with ASD andschizophrenia. Genetic mouse models for dose alterations of small segments and single genes within
22q11.2 CNV have pinpointed some segments and driver genes for distinct dimensional aspects ofmental illnesses. Dose alterations of a ~200kb segment, including Tbx1, impacts social andsensorimotor gating dimensions in mice. Constitutive heterozygosity of Tbx1 in a homogeneous
genetic background impairs social communication and interaction in mice. To further examine theprecise impact of Tbx1 variants, we have recently developed a mouse model of Tbx1 frameshiftdeletion. This variant is located in an exon that encodes a protein sequence needed for thetranslocation of this transcription factor into the cell nucleus. Given its functional relevance to many
aspects of neurodevelopmental disorders, it is imperative to achieve a better understanding of thestructural basis of TBX1 variants on the function of a transcription factor. However, the 3D structureof the TBX1 protein, as well as how this structure is altered by protein-modifying variants, remains
unknown. Additionally, details about the interacting factors of TBX1 are not yet understood.
In this collaborative research, we aim to elucidate the function of TBX1 by analyzing its 3D
structure and interaction modes using solution NMR methid. So far, we have successfully prepared astable isotope-labeled human TBX1 T-box domain and measured its NMR spectrum. The molecularweight of the T-box domain is approximately 20 kDa, but by preparing the 2H,15N-labeled T-box andutilizing a 950 MHz ultra-high-field NMR, we have successfully observed nearly all amide signals
on the 1H-15N TROSY HSQC spectrum. Additionally, the spectral pattern confirmed that the T-box
retains its 3D structure. Furthermore, titration experiments with the target DNA successfully
captured changes in chemical shifts and signal sensitivities of specific amide signals. This indicatesthat the T-box domain is involved in interactions with the target DNA.
*Deadline: May 10, 2024
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