Functional analysis of the zinc finger and activation domains of Glis3 and mutant Glis3(NDH1).
The Krüppel-like zinc finger protein Gli-similar 3 (Glis3) plays a critical role in pancreatic development and has been implicated in a syndrome with neonatal diabetes and hypothyroidism (NDH). In this study, we examine three steps critical in the mechanism of the transcriptional regulation by Glis3: its translocation to the nucleus, DNA binding and transcriptional activity. We demonstrate that the putative bipartite nuclear localization signal is not required, but the tetrahedral configuration of the fourth zinc finger is essential for the nuclear localization of Glis3. We identify (G/C)TGGGGGGT(A/C) as the consensus sequence of the optimal, high-affinity Glis3 DNA-binding site (Glis-BS). All five zinc finger motifs are critical for efficient binding of Glis3 to Glis-BS. We show that Glis3 functions as a potent inducer of (Glis-BS)-dependent transcription and contains a transactivation function at its C-terminus. A mutation in Glis3 observed in NDH1 patients results in a frameshift mutation and a C-terminal truncated Glis3. We demonstrate that this truncation does not effect the nuclear localization but results in the loss of Glis3 transactivating activity. The loss in Glis3 transactivating function may be responsible for the abnormalities observed in NDH1.
Beak
Ju Youn
JY
Cell Biology Section, LRB, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Kang
Hong Soon
HS
Kim
Yong-Sik
YS
Jetten
Anton M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D003409:Congenital Hypothyroidism / Q000235:genetics*; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins; D003920:Diabetes Mellitus / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D015513:Oncogene Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013577:Syndrome; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D000071676:Zinc Finger Protein GLI1; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C478000:Glis3 protein, mouse; D015513:Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D000071676:Zinc Finger Protein GLI1; D004247:DNA