Functional and Biochemical Characterization of ZBTB11, a Novel Protein Critical for Myelopoiesis
Authors: Keightley Maria Cristina , Carradice Duncan P. , Lieschke Graham 
Published: October 2019

Abstract
Abstract 1309
In an unbiased approach for identifying novel factors important for myelopoiesis, a forward genetic screen in zebrafish was undertaken to recover mutants with defects in myeloid development.
One resultant mutant, marsanne (man), shows a decrease in the number of myeloperoxidase-expressing neutrophils and the causative mutation was mapped to the gene encoding zbtb11, the zebrafish ortholog of ZBTB11, resulting in a C116S amino acid substitution. Although the function and role of ZBTB11 in physiology is unknown, by sequence homology it belongs to the BTB-ZF family of transcription factors. This family is defined by a BTB (Broad complex, Tramtrack, Bric à brac) domain, required for protein-protein interaction, and several carboxy-terminal Krüppel-like C2H2 zinc fingers (ZF) implicated in DNA binding.
A series of Zbtb11 N- and C-terminal deletion and point mutation constructs were functionally tested by assaying their ability to restore the wild-type phenotype in man mutants. Mutation of conserved amino acids in the region containing the C116S mutation, which lies in an N-terminal extension unique to the Zbtb11 clade within the BTB-ZF family, renders the protein incapable of rescuing the man phenotype, implicating an important function for this highly conserved region. A series of deletion mutants indicate an in vivo requirement for the BTB domain but not for the ZF domain, the latter suggesting that if direct DNA binding occurs, it can occur through another region of Zbtb11, or else via tethering.
Transfection of ZF4 cells with wild-type and C116S mutant Zbtb11 expression constructs shows that Zbtb11 is a transcriptional repressor and that the mutation abrogates the ability of Zbtb11 to repress transcription of the metallothionein 2A reporter. A three-pronged approach is underway to determine the directly-interacting proteins and target pathways of ZBTB11: 1) CHIP-Seq - monoclonal antibodies have been designed and generated to recognize mouse and human ZBTB11 with two monoclonals currently being tested in immunoprecipitation applications, prior to use in CHIP-Seq; 2) co-immunoprecipitation of ZBTB11 followed by LC-MS/MS mass spectrometry; and 3) a yeast two-hybrid screen.
Mass spectrometry has revealed 187 proteins unique to lysates from ZBTB11-transfected cells versus control. Of these, 67 are known to localize to the nucleus, the likely site of ZBTB11 localization. Immunocytochemistry studies will be undertaken to confirm the cellular localization of wild-type and mutant ZBTB11. Candidate ZBTB11 interacting proteins are currently undergoing independent validation by co-immunoprecipitation/western blotting.
These studies furthering the characterization of ZBTB11 function and its role in hematopoietic development are all ongoing. Several BTB-ZF proteins are known to be important in hematologic malignancies, including PLZF and Bcl-6. Studying ZBTB11, a novel transcriptional repressor, may also uncover new pathways important for myelopoiesis and disease.

Disclosures:
No relevant conflicts of interest to declare.