July 1, 2008;
Transcription factor HNF1beta and novel partners affect nephrogenesis.
Heterozygous mutations of the tissue
-specific transcription factor hepatocyte
nuclear factor (HNF)1beta, cause maturity onset diabetes of the young (MODY5) and kidney
anomalies including agenesis, hypoplasia, dysplasia and cysts. Because of these renal anomalies, HNF1beta
is classified as a CAKUT (congenital anomalies of the kidney
and urinary tract) gene. We searched for human fetal kidney
proteins interacting with the N-terminal region of HNF1beta
using a bacterial two-hybrid system and identified five novel proteins along with the known partner DCoH
. The interactions were confirmed for four of these proteins by GST pull-down assays. Overexpression of two proteins, E4F1
, in Xenopus embryos interfered with pronephros
formation. Further, in situ hybridization showed overlapping expression of HNF1beta
in the developing pronephros
is present largely in the nucleus
where it colocalized with E4F1
. However, ZFP36L1
was located predominantly in the cytoplasm
. A nuclear function for ZFP36L1
was shown as it was able to reduce HNF1beta
transactivation in a luciferase reporter system. Our studies show novel proteins may cooperate with HNF1beta
in human metanephric development and propose that E4F1
are CAKUT genes. We searched for mutations in the open reading frame of the ZFP36L1
gene in 58 patients with renal anomalies but found none.
Disease Ontology terms:
maturity-onset diabetes of the young type 5
DIABETES MELLITUS, NONINSULIN-DEPENDENT; NIDDM
[+] show captions
Figure 2. Four candidates interact with HNF1bbb in GST pull-down assays. Full-length interaction partners with an N-terminal myc-tag were produced in a rabbit reticulocyte system (TNT, Promega) and incubated with the recombinant GST–HNF1bbb fusion protein bound to glutathione sepharose beads. After washing, proteins were recovered and detected by 9E10 mouse anti-myc antibody in Western blots. As controls, GST bound to sepharose or sepharose alone were used.
Figure 3. Influence of over-expressed HNF1β interaction partners on pronephros development in Xenopus larvae. (a) The size of pronephros on injected versus control side is given. The size was determined in lateral views by measuring the area through the widest part of the immunostained pronephros as described.17 Data were tested for Gaussian distribution and accordingly the Student's t-test was used to score significant differences. The P-value is given by comparing embryos injected with interaction partner plus GFP mRNA as marker with those injected with GFP mRNA only. N is the number of animals investigated. (b) E4F1-injected larvae are immunostained to detect pronephric tubule and duct using the antibodies 3G8 and 4A6 as described.17 The upper panel is a dorsal view and the middle panel gives lateral views (right and left) of the same larvae (animal 1). The bottom panel represents the lateral views from a distinct individual (animal 2). In all the lateral views, anterior is up and the injected side is marked by an arrow. (c) Lateral views of a TRIM26-injected larvae (animal 3) prepared as in (b). (d) ZFP36L1-injected larvae prepared as in (b) with a dorsal view (top panel, animal 4) and lateral views of three distinct larvae (animals 5–7). Bar=500 and 200 μm in the dorsal view and lateral views, respectively.
Figure 4. Expression of HNF1β, ZFP36L1, and E4F1 in Xenopus embryos. (a) Whole-mount in situ hybridization of HNF1β, E4F1, and ZFP36L1 in Xenopus neurula and tailbud embryos using antisense probes. HNF1β is predominantly expressed in the pronephros anlage in neurula stage (arrow) as well as in the anlage of the pronephric tubule (arrow) and duct (arrow head) of the tailbud stage. Arrows and arrow heads mark the pronephric tubule and duct anlage, respectively, observed in tailbud embryos probed with E4F1 or ZFP36L1. ZFP36L1 is the Xenopus ortholog C3H-2. (b) Whole-mount in situ hybridizations with the corresponding sense probes.
e4f1 (E4F transcription factor 1) gene expression in X.laevis embryo, NF stage 18, assayed via in situ hybridization, anterior right, dorsal up.
e4f1 ( E4F transcription factor 1 ) gene expression in X.laevis embryo, NF stage 27/28, assayed via in situ hybridization, anterior right, dorsal up.
zfp36l1 (ZFP36 ring finger protein-like 1) gene expression in X.laevis embryo, NF stage 18, assayed via in situ hybridization, anterior right, dorsal up.
zfp36l1 (ZFP36 ring finger protein-like 1) gene expression in X.laevis embryo, NF stage 27/28, assayed via in situ hybridization, anterior right, dorsal up.
Figure 5. Cellular localization of HNF1β and the interaction partners E4F1 or ZFP36L1 in HEK293 cells. HEK293 cells were transfected with 0.9 μg Rc/CMV-GFP-HNF1β and 0.9 μg pCS2+mt-E4F1 or 0.9 μg Rc/CMV-GFP-HNF1β and 0.3 μg pCS2+mt-ZFP36L1. HNF1β is visible by green fluorescence of the GFP tag, whereas the myc-tagged E4F1 or ZFP36L1 is marked by a Cy3-coupled secondary antibody. Right panels are overlays of confocal microscopy.
Figure 6. The effect of E4F1 and ZFP36L1 on the transactivation potential of HNF1β. HEK293 cells were transfected with the luciferase reporter construct driven by the osteopontin (OPN), Ksp-cadherin or the artificial promoter containing four HNF1-binding site linked to the tk promoter (HNF14-tk). The expression vector Rc/CMV-HNF1β and either (a) pCS2+mt-E4F1 or (b) pCS2+mt-ZFP36L1 were included in the transfection as indicated. The luciferase activity without expression vector was used as standard. The standard deviation from a least six measurements are given and significant activation (P-value <0.05) by the transcription factors alone are indicated by the corresponding P-value. The bracket in B indicates the significant downregulation by ZFP36L1 (P-value 1.9 × 10−5). The ACE231 and pkhd113 promoter were also affected by E4F1 and ZFP36L1 in the absence of HNF1β (data not shown