TGF- ß Signaling Cooperates with AT Motif-Binding Factor-1 for Repression of the α -Fetoprotein Promoter.

α-Fetoprotein (AFP) is known to be highly produced in fetal liver despite its barely detectable level in normal adult liver. On the other hand, hepatocellular carcinoma often shows high expression of AFP. Thus, AFP seems to be an oncogenic marker. In our present study, we investigated how TGF-ß signaling cooperates with AT motif-binding factor-1 (ATBF1) to inhibit AFP transcription. Indeed, the expression of AFP mRNA in HuH-7 cells was negatively regulated by TGF-ß signaling. To further understand how TGF-ß suppresses the transcription of the AFP gene, we analyzed the activity of the AFP promoter in the presence of TGF-ß. We found that the TGF-ß signaling and ATBF1 suppressed AFP transcription through two ATBF1 binding elements (AT-motifs). Using a heterologous reporter system, both AT-motifs were required for transcriptional repression upon TGF-ß stimulation. Furthermore, Smads were found to interact with ATBF1 at both its N-terminal and C-terminal regions. Since the N-terminal (ATBF1N) and C-terminal regions of ATBF1 (ATBF1C) lack the ability of DNA binding, both truncated mutants rescued the cooperative inhibitory action by the TGF-ß signaling and ATBF1 in a dose-dependent manner. Taken together, these findings indicate that TGF-ß signaling can act in concert with ATBF1 to suppress the activity of the AFP promoter through direct interaction of ATBF1 with Smads.

A homeodomain-zinc finger protein, ZFHX4, is expressed in neuronal differentiation manner and suppressed in muscle differentiation manner.

Human ZFHX4 has recently been shown to be a candidate gene for congenital bilateral isolated ptosis. Here, we report molecular cloning of the human ZFHX4 cDNA and genomic organization of this gene. Human ZFHX4 is about 180 kb long, containing 12 exons that encodes a 3599-amino acid protein carrying four homeodomains and 22 zinc fingers. The 11th exon is 3.2 kb in length and encodes all the four homeodomains together with four of the 22 zinc fingers. ZFHX4 is 90% homologous to mouse Zfhx4, 52% to human ATBF1A and 24% to Drosophila ZFH-2. ZFHX4 was mapped to human chromosome 8q13.3-q21.11 by fluorescence in situ hybridization using BAC clone RP11-48D4 as a probe. RT-PCR analysis showed that ZFHX4 transcripts were expressed in adult human brain, liver and muscle. This, together with the finding that Zfhx4 was expressed transiently in differentiating P19 embryonal carcinoma cells and C2C12 myoblasts, suggests that ZFHX4/Zfhx4 is involved in neural and muscle differentiation.

c-Myb inhibits myogenic differentiation through repression of MyoD.

c-Myb, known to play a central role in hematopoiesis, is also an important factor involved in myogenesis. Here, we found that the c-myb gene is expressed in proliferating C2C12 myoblasts and turned off in differentiating cells. Detailed analysis of c-myb RNAs revealed that the cell density is the essential factor determining c-myb expression. Both c-myb and its alternatively spliced form c-mybE9A RNAs are down-regulated in confluent cells. Constitutive expression of exogenous c-myb in C2C12 cells inhibits their terminal differentiation. It is shown that the c-Myb protein physically interacts with MyoD, the key regulator of myogenesis, and inhibits MyoD-dependent transcription. The interaction domains are the DNA binding domain of c-Myb and the bHLH motif of MyoD. Our data suggest that in proliferating cells c-Myb binds MyoD and inhibits its transcriptional activity until cell-cell contacts are established and c-myb expression is switched off. Thus, the c-Myb protein may be one of factors ensuring that proliferating myoblasts remain undifferentiated.

ATBF1-A protein, but not ATBF1-B, is preferentially expressed in developing rat brain.

The ATBF1 gene encodes transcription factors containing four homeodomains and multiple zinc finger motifs. However, the gene products have yet to be identified and the role remains unknown in vivo. In this study, we raised an antiserum for ATBF1 and found high levels of expression of ATBF1 in developing rat brain. Western and Northern blot analyses detected a 400 kDa protein and 12.5 kb mRNA in developing rat brain, respectively; both corresponding to ATBF1-A but not the B isoform. The protein was highly expressed in the midbrain and diencephalon and mRNA was highly expressed in the brainstem, mostly in embryo and neonatal brain. Immunohistochemistry identified postmitotic neurons in the brainstem as the major site of ATBF1 expression, and the expression levels varied depending on age of and location in the brain. Expression was transient and weak in the precursor cells at early neurogenesis. ATBF1 decreased postnatally, but remained in mature neurons, including those expressing DOPA decarboxylase (DDC). High levels of ATBF1 were expressed in precursor cells in accordance with neurogenesis and were continued to the mature neurons in specific areas such as the inferior colliculus. Expression was not significant from precursor cells to mature neurons in the cerebral cortex and hippocampus. ATBF1 and its Drosophila homolog, Zfh-2, are known to regulate cell differentiation and proliferation via the interaction with either of the basic helix-loop-helix transcription factors, c-myb, or the DDC gene. Together with these reported functions the expression features detected here suggest that ATBF1 may participate in the regulation of neuronal cell maturation or region-specific central nervous system differentiation.

Regulation of the alpha-fetoprotein gene by the isoforms of ATBF1 transcription factor in human hepatoma.

We investigated mechanisms regulating expression of alpha-fetoprotein (AFP) in 3 human hepatoma cell lines, HuH-7, HepG2, and huH-1, producing high, medium, and low levels of AFP, respectively. The silencer, a negative cis-acting element of the AFP gene, was highly activated in huH-1 and HepG2 to repress AFP enhancer activity by 91%, whereas only 26% repression was observed in HuH-7. To account for the difference in AFP production between HepG2 and huH-1, we investigated the roles of two isoforms of the AT motif-binding factor 1 (ATBF1) transcription factor, ATBF1-A and -B. Cotransfection assays showed that the ATBF1 isoforms regulated the AFP gene differently in HepG2 and huH-1. In huH-1 and HuH-7, both ATBF1 isoforms suppressed strongly enhancer activity and slightly promoter activity. In HepG2, on the other hand, ATBF1-A suppressed the enhancer and promoter activities, but surprisingly, ATBF1-B was found to stimulate enhancer activity while showing no effect on the promoter. Levels of ATBF1-A mRNA were similar in all 3 cell lines, whereas the expression ATBF1-B mRNA varied greatly, with the highest level seen in HepG2 followed by huH-1 and HuH-7. These results suggest that, in HepG2, ATBF1-B may have a dominant negative effect to relieve the transcriptional repression caused by its isoform. In support of this view, we found that the N-terminal region specific to the ATBF1-A molecule possessed transcriptional repressor activity. Thus, the use of the ATBF1 variants as well as the silencer may provide a unique mechanism that contributes to the determination of AFP levels in human hepatoma cell lines.