Identification and functional characterization of protein domains in the transcription factor TWIST
Saethre-Chotzen syndrome is an autosomal dominant inherited disorder with premature fusion of cranial sutures. It is caused by nucleotide sequence changes within or in proximity of the TWIST1 gene. This gene encodes for a bHLH transcription factor, which inhibits osteogenic differentiation by transc...
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|Saethre-Chotzen syndrome is an autosomal dominant inherited disorder with premature fusion of cranial sutures. It is caused by nucleotide sequence changes within or in proximity of the TWIST1 gene. This gene encodes for a bHLH transcription factor, which inhibits osteogenic differentiation by transcriptional control of various target genes. The aim of my work is to characterize functional domains in TWIST protein, and to determine the interacting partner for TWIST and its motifs particularly NSEEE and WR. The present study was thus undertaken to determine how TWIST1 gene mutations affect protein function. Evolutionary alignment of Twist proteins from different species, indicate TWIST contain 4 additional conserved regions such as NSEEE, NLS1, NLS2, and WR-domain besides the bHLH motif. The bHLH domain is thought to be responsible for heterodimerization with other bHLH proteins such as E12 protein or SEF2 protein. The functions of NSEEE, NLS1, NLS2, and conserved WR motifs are poorly understood at present.
First, I focused on functional characterization of the NLS1 and NLS2 domains as potential nuclear localization signals in TWIST., Specifically the effects of various NLS substitutions in TWIST on cellular localization was assayed by immunoflourescence assay. In particular, TWIST NLS1 altered at amino acid position K38R was found to be retained in the cytoplasm of transiently transfected U2-OS cells, suggesting that NLS1 is functional and essential for the nuclear transport of TWIST. Additionally, to understand the role of the TWIST NLS2 in nuclear localization, amino acid at positions 73, 76 and 77 were substituted in this motif. These results demonstrated that substitution at NLS2 position 76 does not play an essential role in the nuclear localization of TWIST, in contrast to the K73R and K77R that inhibit nuclear accumulation. Although K76R mutants cannot inhibit nuclear localization by itself, we demonstrate it plays a synergistic role with the NLS1 K38R mutation to further reduce nuclear localization. This synergistic effect is consistent with the observation that combined K38R (NLS1) and K76R (NLS2) mutants dramatically reduced nuclear localization, further suggesting that both NLS1 and NLS2 work together in regulating nuclear localization of TWIST protein.
TWIST belongs to class B bHLH proteins which are known to form stable heterodimers with members of class A bHLH transcription factors including gene products of E12 and E47, respectively. Accordingly, the subcellular localization of NLS1 and NLS2 in TWIST protein was investigated in U2-OS cells following co-transfection with E12. The cotransfections with heterodimerization partner E12 and NLS1-mutated TWIST led to a compensation of the mislocalization.
The second aim of my work was to identify the interacting proteins that could influence the functionality of TWIST using yeast-two-hybrid assay. I wanted to determine if the TWIST protein or its conserved motifs interact with other regulatory proteins to help to regulate the TWIST transcriptional activity. Using the entire coding sequence of TWIST1 gene, an interesting candidate was found belonging to the class A bHLH transcription factors that includes the SEF2 gene product. The direct interaction of SEF2 with TWIST was verified in a yeast mating assay and then confirmed in an in vivo NLS-rescue assay using U2-OS cells, showing that SEF2 forms a heterodimer with TWIST protein and co-localized into the nucleus.
Furthermore, two more highly conserved TWIST motifs NSEEE and WR were analyzed individually to find out their interacting proteins and their role in regulating the TWIST1 transcriptional activity. I found more than 1000 yeast clones for NSEEE motif and 120 clones for WR motif. The second screening of the yeast clones suggested some promising candidates protein as interacting partners with the NSEEE motif such as ETV5, SURF4, Spastin, Metalloproteinase 2, and ALR-like protein mRNA. By contrast I could not detect any interesting interacting partners with the WR domain. A possible explanation may be the requirement of bHLH to mediate WR interaction with other proteins.