Phosphorylation of HOX11/TLX1 on Threonine-247 during mitosis modulates expression of cyclin B1.
BACKGROUND
The HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11.


RESULTS
Cell cycle specific expression studies revealed a phosphorylated form of HOX11 detectable only in the mitotic fraction of cells after treatment with inhibitors to arrest cells at different stages of the cell cycle. Mutational analyses revealed phosphorylation on threonine-247 (Thr247), a conserved amino acid that defines the HOX11 gene family and is integral for the association with DNA binding elements. The effect of HOX11 phosphorylation on its ability to modulate expression of the downstream target, cyclin B1, was tested. A HOX11 mutant in which Thr247 was substituted with glutamic acid (HOX11 T247E), thereby mimicking a constitutively phosphorylated HOX11 isoform, was unable to bind the cyclin B1 promoter or enhance levels of the cyclin B1 protein. Expression of the wildtype HOX11 was associated with accelerated progression through the G2/M phase of the cell cycle, impaired synchronization in prometaphase and reduced apoptosis whereas expression of the HOX11 T247E mutant restored cell cycle kinetics, the spindle checkpoint and apoptosis.


CONCLUSIONS
Our results demonstrate that the transcriptional activity of HOX11 is regulated by phosphorylation of Thr247 in a cell cycle-specific manner and that this phosphorylation modulates the expression of the target gene, cyclin B1. Since it is likely that Thr247 phosphorylation regulates DNA binding activity to multiple HOX11 target sequences, it is conceivable that phosphorylation functions to regulate the expression of HOX11 target genes involved in the control of the mitotic spindle checkpoint.
Chen
Edwin
E
Institute of Medical Science, University of Toronto, Toronto, Ontario M5S1A8, Canada.
Huang
Xiaoyong
X
Zheng
Yanzhen
Y
Li
You-Jun
YJ
Chesney
Alden
A
Ben-David
Yaacov
Y
Yang
Eric
E
Hough
Margaret R
MR
D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D047369:Chromatin Immunoprecipitation; D056744:Cyclin B1 / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D013912:Threonine / Q000378:metabolism*
D056744:Cyclin B1; D018398:Homeodomain Proteins; C487378:Tlx1 protein, mouse; D013912:Threonine