The 86K immediate early (IE) 2 protein of human cytomegalovirus trans-activates a number of homologous and heterologous promoters, including the cellular promoter for the 70K heat-shock protein (hsp70), and the human immunodeficiency virus long terminal repeat. We have previously shown that IE2 trans-activates these two promoters in a TATA-dependent manner, and that IE2 is able to form a direct contact with TATA-box binding protein (TBP) in vitro. We now show that IE2 binds to the basic repeat region of TBP. In addition IE2 can contact a second general transcription factor, TFIIB. We have mapped the TBP-and TFIIB-binding regions within IE2 and show that these regions overlap, and also lie within parts of the protein previously identified as being required for the trans-activation and autoregulation functions of IE2.
Human cytomegalovirus (HCMV) immediate-early (IE) proteins are known potent transregulators of viral and cellular gene expression upon HCMV infection. HCMV is known to activate a number of cellular genes intimately associated with the cell cycle and DNA replication by mechanisms involving the viral major IE 86-kDa protein (IE2). We have recently shown that IE2 mediates this activation in a TATA-dependent manner and interacts directly with the TATA-binding protein. However, a number of TATA-less cellular promoters, e.g., DNA polymerase ␣ and dihydrofolate reductase, are also activated by HCMV infection. Consequently, we have asked how HCMV mediates this activation. We show that, consistent with its known TATA dependency, IE2 does not activate the DNA polymerase ␣ promoter. In contrast, this promoter is strongly activated by the major IE 72-kDa protein (IE1). Whilst deletion of ATF or E2F sites within the DNA polymerase ␣ promoter had little effect on IE1-mediated activation, removal of the CCAAT box appeared to abolish high levels of activation by IE1. Consistent with this observation, we also find that IE1 interacts directly with the CCAAT box binding factor CTF1 in vitro and massively augments CTF1-mediated activation of the DNA polymerase ␣ promoter in transient transfection assays.
Homozygous truncating mutations in the helix-loop-helix transcription factor PTF1A are a rare cause of pancreatic and cerebellar agenesis. The correlation of Ptf1a dosage with pancreatic phenotype in a mouse model suggested the possibility of finding hypomorphic PTF1A mutations in patients with pancreatic agenesis or neonatal diabetes but no cerebellar phenotype. Genome wide SNP typing in two siblings with neonatal diabetes from a consanguineous pedigree revealed a large shared homozygous region (31 Mb) spanning PTF1A. Sanger sequencing of PTF1A identified a novel missense mutation, p.P191T. Testing of 259 additional patients using a targeted next generation sequencing assay for 23 neonatal diabetes genes detected one additional proband and an affected sibling with the same homozygous mutation. All 4 cases were diagnosed with diabetes at birth and are insulin treated. Two of the 4 had exocrine pancreatic insufficiency requiring replacement but none of the affected individuals have neurodevelopmental delay. Transient transfection assays of the mutant protein demonstrated a 75% reduction in transactivation activity. This study shows that the functional severity of a homozygous mutation impacts on the severity of clinical features found in patients.
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