Zinc finger proteins containing the Kruppel associated box (KRAB-ZFPs) constitute the largest individual family of transcriptional repressors encoded by the genomes of higher organisms. KRAB domain, positioned at the NH2 terminus of the KRAB-ZFPs, interacts with a scaffold protein, KAP-1, which is able to recruit various transcriptional factors causing repression of genes to which KRAB ZFPs bind. The relevance of such repression is reflected in the large number of the KRAB zinc finger protein genes in the human genome. However, in spite of their numerical abundance little is currently known about the gene targets and the physiological functions of KRAB- ZFPs. However, emerging evidence links the transcriptional repression mediated by the KRAB-ZFPs to cell proliferation, differentiation, apoptosis and cancer. Moreover, the fact that KRAB containing proteins are vertebrate-specific suggests that they have evolved recently, and that their key roles lie in some aspects of vertebrate development. In this review, we will briefly discuss some regulatory functions of the KRAB-ZFPs in different physiological and pathological states, thus contributing to better understand their biological roles.

We have identified a new mutation in the FBP (fructose 1,6-bisphosphate) aldolase A gene in a child with suspected haemolytic anaemia associated with myopathic symptoms at birth and with a subsequent diagnosis of arthrogryposis multiplex congenita and pituitary ectopia. Sequence analysis of the whole gene, also performed on the patient's full-length cDNA, revealed only a Gly³⁴⁶→Ser substitution in the heterozygous state. We expressed in a bacterial system the new aldolase A Gly³⁴⁶→Ser mutant, and the Glu²⁰⁶→Lys mutant identified by others, in a patient with an aldolase A deficit. Analysis of their functional profiles showed that the Gly³⁴⁶→Ser mutant had the same K_m as the wild-type enzyme, but a 4-fold lower k_cat. The Glu²⁰⁶→Lys mutant had a K_m approx. 2-fold higher than that of both the Gly³⁴⁶→Ser mutant and the wild-type enzyme, and a k_cat value 40% less than the wild-type. The Gly³⁴⁶→Ser and wild-type enzymes had the same T_m (melting temperature), which was approx. 6–7 °C higher than that of the Glu²⁰⁶→Lys enzyme. An extensive molecular graphic analysis of the mutated enzymes, using human and rabbit aldolase A crystallographic structures, suggests that the Glu²⁰⁶→Lys mutation destabilizes the aldolase A tetramer at the subunit interface, and highlights the fact that the glycine-to-serine substitution at position 346 limits the flexibility of the C-terminal region. These results also provide the first evidence that Gly³⁴⁶ is crucial for the correct conformation and function of aldolase A, because it governs the entry/release of the substrates into/from the enzyme cleft, and/or allows important C-terminal residues to approach the active site.

Two recombinant clones, pA2 and pA3, containing cDNA sequences for human aldolase B have been isolated from a full length human liver cDNA library. The larger one, pA3, has been subcloned in M13 phage and completely sequenced with the chain terminator method. The sequence covers 1,600 nucleotides including the whole coding region (1,050 nucleotides), 67 nucleotides from the 5' non-coding region and the whole 3' non-coding region, 440 nucleotides long, down to the poly-A tail. Comparison with rabbit aldolase A and with a partial sequence of rat aldolase B, shows a homology of about 76% for aldolase A and of about 94% for aldolase B, which indicates that the sequenced cDNA codes for the liver isoenzyme. This is the first complete sequence reported for human aldolase B. The pA3 clone strongly hybridizes to 18S mRNA from human adult liver as expected from the size of the isolated cDNA.

The phosphate carrier (PiC) catalyses the import of phosphate into mitochondria where it is needed for ATP synthesis. We have analysed the 5'-flanking region of the human PiC gene and found that it has a single transcriptional initiation site and lacks a TATA box. Through deletion analysis of the -1213/-25 nt region, we identified an activation domain (-223/-25) and an inhibition domain (-1017/-814). The most effective promoter activity in transfected HeLa cells corresponded to the region containing putative binding sites for Sp1 (-163/-142; where Sp1 stands for stimulating protein-1) and CREB (-138/-116; where CREB stands for cAMP-response-element-binding protein). These DNA sequences were active in gel-shift assays in the presence of HeLa cell nuclear extracts or recombinant Sp1 and CREB respectively. Forskolin increased PiC promoter activity via the CREB site. Both footprinting and transfection of deletion constructs of the inhibition region (-1017/-814) showed that PiC silencer activity extends over 25 nt (-943/-919), which specifically binds two proteins present in HeLa cell nuclear extracts. These transcription factors were purified by DNA affinity, analysed by MS and identified as p54(nrb)/NonO (nuclear RNA binding protein) and PSF (protein-associated splicing factor). The PiC silencer region cloned in front of the ferritin promoter conferred a strong inhibition to the heterologous promoter. These findings may provide insight into control of PiC gene expression in different cell types and under different growth conditions. To our knowledge, this is the first study to analyse the regulation of the PiC gene expression in any cell.