Moshe Yaniv

Hepatocyte Nuclear Factor 1 Inactivation Results in Hepatic Dysfunction, Phenylketonuria, and Renal Fanconi Syndrome

HNF1 is a transcriptional activator of many hepatic genes including albumin, alpha1-antitrypsin, and alpha- and beta-fibrinogen. It is related to the homeobox gene family and is predominantly expressed in liver and kidney. Mice lacking HNF1 fail to thrive and die around weaning after a progressive wasting syndrome with a marked liver enlargement. The transcription rate of genes like albumin and alpha1-antitrypsin is reduced, while the gene coding for phenylalanine hydroxylase is totally silent, giving rise to phenylketonuria. Mutant mice also suffer from severe Fanconi syndrome caused by renal proximal tubular dysfunction. The resulting massive urinary glucose loss leads to energy and water wasting. HNF1-deficient mice may provide a model for human renal Fanconi syndrome.

Defective planar cell polarity in polycystic kidney disease

Morphogenesis involves coordinated proliferation, differentiation and spatial distribution of cells. We show that lengthening of renal tubules is associated with mitotic orientation of cells along the tubule axis, demonstrating intrinsic planar cell polarization, and we demonstrate that mitotic orientations are significantly distorted in rodent polycystic kidney models. These results suggest that oriented cell division dictates the maintenance of constant tubule diameter during tubular lengthening and that defects in this process trigger renal tubular enlargement and cyst formation.

ALTERED CONTROL OF CELLULAR PROLIFERATION IN THE ABSENCE OF MAMMALIAN BRAHMA (SNF2ALPHA )

The mammalian SWI–SNF complex is an evolutionarily conserved, multi‐subunit machine, involved in chromatin remodelling during transcriptional activation. Within this complex, the BRM (SNF2α) and BRG1 (SNF2β) proteins are mutually exclusive subunits that are believed to affect nucleosomal structures using the energy of ATP hydrolysis. In order to characterize possible differences in the function of BRM and BRG1, and to gain further insights into the role of BRM‐containing SWI–SNF complexes, the mouse BRM gene was inactivated by homologous recombination. BRM−/− mice develop normally, suggesting that an observed up‐regulation of the BRG1 protein can functionally replace BRM in the SWI–SNF complexes of mutant cells. Nonetheless, adult mutant mice were ∼15% heavier than control littermates. This may be caused by increased cell proliferation, as demonstrated by a higher mitotic index detected in mutant livers. This is supported further by the observation that mutant embryonic fibroblasts were significantly deficient in their ability to arrest in the G0/G1 phase of the cell cycle in response to cell confluency or DNA damage. These studies suggest that BRM participates in the regulation of cell proliferation in adult mice.

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function.

vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1.

vHNF1 and HNF1 are two nuclear proteins that bind to an essential element in the promoter proximal sequences of albumin and of many other liver‐specific genes. HNF1 predominates in hepatocytes but is absent in dedifferentiated hepatoma cells. These cells contain vHNF1 but fail to express most of the liver traits. In the present work we have isolated cDNA clones for vHNF1 and found that it is a homeoprotein homologous to HNF1 in regions important for DNA binding. Unexpectedly, vHNF1 transactivated the albumin promoter in transfection experiments. Like the HNF1 mRNA, the vHNF1 message was found in kidney, liver and intestine although in different proportions. The fact that vHNF1 and HNF1 readily form heterodimers in vitro and the biochemical characterization of vHNF1/HNF1 heterodimers in nuclear extracts of kidney, liver and several cell lines, strongly argue that such heterodimers exist in vivo. Our results raise the possibility that heterodimerization between homeoproteins could be a common phenomenon in higher eukaryotes, which may have implications in the regulatory network sustained between these factors.

The hbrm and BRG-1 proteins, components of the human SNF/SWI complex, are phosphorylated and excluded from the condensed chromosomes during mitosis.

In yeast, the SNF/SWI complex is believed to regulate transcription by locally altering the chromatin structure. At the present time, three human homologues of yeast SNF/SWI proteins have been characterized: hbrm and BRG‐1, homologues of SNF2/SWI2, and hSNF5, a homologue of SNF5. We show here that, during mitosis, hbrm and BRG‐1 are phosphorylated and excluded from the condensed chromosomes. In this phase of the cell cycle, the level of hbrm protein is also strongly reduced, whereas the level of BRG‐1 remains constant. The mitotic phosphorylation of hbrm and BRG‐1 is found not to disrupt the association of these proteins with hSNF5 but correlates with a decreased affinity for the nuclear structure in early M phase. We suggest that chromosomal exclusion of the human SNF/SWI complex at the G2‐M transition could be part of the mechanism leading to transcriptional arrest during mitosis.

SWI/SNF chromatin remodeling and cancer.

The SWI/SNF complex contributes to the regulation of gene expression by altering the chromatin structure. Depending on the context, it can be involved in either transcriptional activation or repression. Growing genetic and molecular evidence indicate that subunits of the SWI/SNF complex act as tumor suppressors in human and mice. Results from biochemical and transfection studies suggest also that SWI/SNF participates either in the inhibition or activation of several oncogenes and tumor suppressor genes and/or control their transcriptional activity. These activities provide molecular insight into the mechanism underlying SWI/SNF function in tumor suppression.

Stress-activated protein kinases are negatively regulated by cell density.

Stimulation by UV irradiation, TNFα, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N‐terminal domain of c‐Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c‐Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c‐Jun N‐terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G‐proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c‐Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress‐dependent signal cascade by confluency will restrict c‐Jun N‐terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.

Anatomy of a Homeoprotein Revealed by the Analysis of Human MODY3 Mutations

Hepatocyte nuclear factor 1α (HNF1α) is an atypical dimeric homeodomain-containing protein that is expressed in liver, intestine, stomach, kidney, and pancreas. Mutations in the HNF1α gene are associated with an autosomal dominant form of non-insulin-dependent diabetes mellitus called maturity-onset diabetes of the young (MODY3). More than 80 different mutations have been identified so far, many of which involve highly conserved amino acid residues among vertebrate HNF1α. In the present work, we investigated the molecular mechanisms by which MODY3 mutations could affect HNF1α function. For this purpose, we analyzed the properties of 10 mutants resulting in amino acid substitutions or protein truncation. Some mutants have a reduced protein stability, whereas others are either defective in the DNA binding or impaired in their intrinsic trans-activation potential. Three mutants, characterized by a complete loss of trans-activation, behave as dominant negatives when transfected with the wild-type protein. These data define a clear causative relationship between MODY3 mutations and functional defects in HNF1α trans-activation. In addition, our analysis sheds new light on the structure of a homeoprotein playing a key role in pancreatic β cell function.

The papillomavirus E2 protein: a factor with many talents

The products of the papillomavirus E2 open reading frame play a key role in the regulation of the viral cycle. E2 proteins can activate or repress viral promoters by several distinct mechanisms and viral DNA replication requires the expression of the full-length E2 protein together with the product of the E1 open reading frame. This is an interesting example of how a single eukaryotic DNA-binding protein has evolved to perform several different functions and it provides a valuable model system for studying the regulation of eukaryotic transcription and DNA replication.