We have searched the human genome for genes encoding new proteins that may be involved in three nuclear gene expression processes: transcription, pre-messenger RNA splicing and polyadenylation. A plethora of potential new factors are implicated by sequence in nuclear gene expression, revealing a substantial but selective increase in complexity compared with Drosophila melanogaster and Caenorhabditis elegans. Although the raw genomic information has limitations, its availability offers new experimental approaches for studying gene expression.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of ammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA framents distributed along a given genomic region. Terminal differentiation of HL-60 was achieved with retinoic acid and dimethylformamide, as described (17).Transfection. Plasmid pAWTSV (=9 kb), a kind gift of Cesare Vesco (Institute of Cell Biology, Rome), carries the whole simian virus 40 (SV40) genome inserted in the BamHI site of pAT153 (18). Six 10-cm tissue culture plates, containing about 106 COS-1 cells each, were transfected with 10 pg of pAWTSV by the calcium phosphate precipitation technique. After 10 hr of incubation in calcium phosphate solution, cells were extensively washed and fresh medium was added, containing 10 nCi of [14C]thymidine per ml. After 18 hr of incubation, BrdUrd (100 uM final concentration) and[3H]deoxycytidine (1 jAM final concentration) were added.After 1 min of incubation, cells were killed by addition of sodium azide and DNA was extracted as described below.Extrction and Purification of Newly Syntez DNA.Total DNA was extracted, denatured, and size-fractionated by sedimentation through neutral sucrose gradients as described (15).In the experiment involving transfection of plasmid pAW-TSV, DNA (700 /4 final volume) was fractionated on four 5-20%6 (wt/vol) linear sucrose gradients (5 ml each) for 210 min at 200C in a Beckman SW55Ti rotor at 55 krpm; 24 fractions of 200 j4 were collected.In the experiment with synchronized HL-60 cells, DNA (2 ml final volume) was fractionated on eight 5-30% sucrose Abbreviations: DHFR, dihydrofolate reductase; SV40, simian virus 40. tTo whom reprint requests should be addressed. 7119The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

THC2, an autosomal-dominant thrombocytopenia described so far in only two families, has been ascribed to mutations in MASTL or ACBD5. Here, we show that ANKRD26, another gene within the THC2 locus, and neither MASTL nor ACBD5, is mutated in eight unrelated families. ANKRD26 was also found to be mutated in the family previously reported to have an ACBD5 mutation. We identified six different ANKRD26 mutations, which were clustered in a highly conserved 19 bp sequence located in the 5' untranslated region. Mutations were not detected in 500 controls and are absent from the 1000 Genomes database. Available data from an animal model and Dr. Watson's genome give evidence against haploinsufficiency as the pathogenetic mechanism for ANKRD26-mediated thrombocytopenia. The luciferase reporter assay suggests that these 5' UTR mutations might enhance ANKRD26 expression. ANKRD26 is the ancestor of a family of primate-specific genes termed POTE, which have been recently identified as a family of proapoptotic proteins. Dysregulation of apoptosis might therefore be the pathogenetic mechanism, as demonstrated for another thrombocytopenia, THC4. Further investigation is needed to provide evidence supporting this hypothesis.

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3), leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc–induced neuroblastoma.

Human T-cell leukaemia virus type I (HTLV-I) Tax protein increases the DNA binding of many cellular transcription factors that contain a basic region-leucine zipper (bZIP) DNA-binding domain. bZIP domains comprise a leucine-rich dimerization motif and a basic region that mediates DNA contact. How Tax recognizes diverse bZIPs is not understood. Here we show that no specific sequence of the leucine zipper is required for a Tax response. In contrast, the basic region is essential for the Tax-mediated DNA-binding increase, which can be eliminated by single substitutions of several conserved amino acids. Surprisingly, Tax alters the relative affinity of a bZIP for different DNA binding sites. Thus, through recognition of the conserved basic region. Tax increases DNA binding and modifies DNA site selection. Tax provides a model for how a single auxiliary factor can regulate multiple sequence-specific DNA-binding proteins.