Paolo V. Pedone

In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions.

Summary The maintenance of H3K9 and DNA methylation at imprinting control regions (ICRs) during early embryogenesis is key to the regulation of imprinted genes. Here, we reveal that ZFP57, its cofactor KAP1, and associated effectors bind selectively to the H3K9me3-bearing, DNA-methylated allele of ICRs in ES cells. KAP1 deletion induces a loss of heterochromatin marks at ICRs, whereas deleting ZFP57 or DNMTs leads to ICR DNA demethylation. Accordingly, we find that ZFP57 and KAP1 associated with DNMTs and hemimethylated DNA-binding NP95. Finally, we identify the methylated TGCCGC hexanucleotide as the motif that is recognized by ZFP57 in all ICRs and in several tens of additional loci, several of which are at least ZFP57-dependently methylated in ES cells. These results significantly advance our understanding of imprinting and suggest a general mechanism for the protection of specific loci against the wave of DNA demethylation that affects the mammalian genome during early embryogenesis.

Expression and parental imprinting of the H19 gene in human rhabdomyosarcoma.

The expression of Insulin-like Growth Factor 2 (IGF-2) and H19, two genes located on human chromosome 11p15 and provided with cell growth modulating activity, is regulated by parental imprinting, in that the activity of their alleles is dependent on the parental origin. Parental bias in the genetic alterations of chromosome 11p15 observed in several pediatric cancers suggests the involvement of imprinted genes in tumor development. We have previously reported that the number of functional IGF-2 alleles is frequently increased in rhabdomyosarcoma (RMS), as a consequence of either relaxation of imprinting (LOI) or gene duplication. Here we show that the expression of the H19 gene is significantly suppressed with respect to normal muscle tissue in 13 out of 15 rhabdomyosarcomas with embryonal histology (ERMS) and in three out of 11 rhabdomyosarcomas classified as alveolar subtype (ARMS). Since a growth-inhibitory activity has been found associated with the H19 gene, the extinction of its expression can contribute to RMS development. Parental imprinting of the H19 gene was found conserved in all informative RMSs, including those whose IGF-2 imprinting was relaxed, indicating that LOI is a gene-specific event. Seven ERMSs and one ARMS displaying low H19 RNA levels showed an underrepresentation of the expressed allele in their genotype. This result is consistent with the paternal imprinting of the H19 gene and with the preferential loss of the maternal 11p15 alleles in these neoplasms. Low H19 expression was also found in four out of eight RMSs retaining the heterozygosity at 11p15, but showing IGF-2 LOI. These findings suggest that the genetic and epigenetic alterations affecting chromosome 11p15 in a high number of RMSs cause deregulation of more than one imprinted gene, possibly affecting tumor growth, including the extinction of H19 expression and an increase in the number of active IGF-2 alleles.

Relaxation of insulin-like growth factor 2 imprinting and discordant methylation at KvDMR1 in two first cousins affected by Beckwith-Wiedemann and Klippel-Trenaunay-Weber syndromes.

Beckwith-Wiedeman syndrome (BWS) and Klippel-Trenaunay-Weber syndrome (KTWS) are different human disorders characterized, among other features, by tissue overgrowth. Deregulation of one or more imprinted genes located at chromosome 11p15.5, of which insulin-like growth factor 2 (IGF2) is the most likely candidate, is believed to cause BWS, whereas the etiology of KTWS is completely obscure. We report a case of BWS and a case of KTWS in a single family. The probands, sons of two sisters, showed relaxation of the maternal IGF2 imprinting, although they inherited different 11p15.5 alleles from their mothers and did not show any chromosome rearrangement. The patient with BWS also displayed hypomethylation at KvDMR1, a maternally methylated CpG island within an intron of the KvLQT1 gene. The unaffected brother of the BWS proband shared the same maternal and paternal 11p15.5 haplotype with his brother, but the KvDMR1 locus was normally methylated. Methylation of the H19 gene was normal in both the BWS and KTWS probands. Linkage between the insulin-like growth factor 2 receptor (IGF2R) gene and the tissue overgrowth was also excluded. These results raise the possibility that a defective modifier or regulatory gene unlinked to 11p15.5 caused a spectrum of epigenetic alterations in the germ line or early development of both cousins, ranging from the relaxation of IGF2 imprinting in the KTWS proband to disruption of both the imprinted expression of IGF2 and the imprinted methylation of KvDMR1 in the BWS proband. Analysis of these data also indicates that loss of IGF2 imprinting is not necessarily linked to alteration of methylation at the KvDMR1 or H19 loci and supports the notion that IGF2 overexpression is involved in the etiology of the tissue hypertrophy observed in different overgrowth disorders, including KTWS.

The H19 endodermal enhancer is required for Igf2 activation and tumor formation in experimental liver carcinogenesis.

The expression of the linked but reciprocally imprinted Igf2 and H19 genes is activated in adult liver in the course of tumor development. By in situ hybridization analysis we have shown that both the Igf2 and H19 RNAs are expressed in the majority of the neoplastic nodules, and that hepatocellular carcinomas are developed in an experimental model of liver carcinogenesis. H19 is also highly activated in smaller and less distinct hyperplastic regions. The few neoplastic areas showing Igf2 but no H19 RNA display loss of the maternally inherited allele at the Igf2/H19 locus. These data are compatible with the existence of a common activation mechanism of these two genes during liver carcinogenesis and with a stronger H19 induction in the pre-neoplastic lesions. By using mice carrying a deletion of the H19 endodermal enhancer, we show that this regulatory element is necessary for the activation of the Igf2 and H19 genes upon induction of liver carcinogenesis. Furthermore, multiple sites of the H19 endodermal enhancer region become hypersensitive to DNase I when the carcinogenesis process is induced. Lastly, liver tumors developed in mice paternally inheriting the H19 enhancer deletion are found to have marked growth delays, increased frequency of apoptotic nuclei, and lack of Igf2 mRNA expression, thus indicating that this regulatory element plays a major role in the progression of liver carcinogenesis, since it is required for the activation of the anti-apoptotic Igf2 gene.

Zinc to cadmium replacement in the A. thaliana SUPERMAN Cys2His2 zinc finger induces structural rearrangements of typical DNA base determinant positions

Among heavy metals, whose toxicity cause a steadily increasing of environmental pollution, cadmium is of special concern due to its relatively high mobility in soils and potential toxicity at low concentrations. Given their ubiquitous role, zinc fingers domains have been proposed as mediators for the toxic and carcinogenic effects exerted by xenobiotic metals. To verify the structural effects of zinc replacement by cadmium in zinc fingers, we have determined the high resolution structure of the single Cys₂ His₂ zinc finger of the Arabidopsis thaliana SUPERMAN protein (SUP37) complexed to the cadmium ion by means of UV-vis and NMR techniques. SUP37 is able to bind Cd(II), though with a dissociation constant higher than that measured for Zn(II). Cd-SUP37 retains the ββα fold but experiences a global structural rearrangement affecting both the relative orientation of the secondary structure elements and the position of side chains involved in DNA recognition: among them Ser17 side chain, which we show to be essential for DNA binding, experiences the largest displacement.

Relaxation of insulin-like growth factor-2 imprinting in rat cultured cells

The parental-specific expression of the insulin-like growth factor-2 (Igf-2) and H19 genes was studied in rat fibroblast cells derived from a 3 day-old first-generation hybrid animal obtained by crossing Fisher and Wistar strains (F x W cells). Results showed that the reciprocal imprinting of the Igf-2 and H19 genes was conserved in the rat tissues and in the derived F x W cells when cultured with frequent transfer. Igf-2 and H19 gene expression was coordinately up-regulated upon reaching confluence, but Igf-2 RNA levels were further increased in a time-dependent manner and the repressed state of the maternal Igf-2 allele was progressively relaxed in cultures held in the confluent state and in the presence of low serum for more than 3 days. The active expression and relaxed imprinting status of the Igf-2 gene persisted over cell generations when the growth-constraining conditions were released by trypsinization and dilution. On the contrary, the imprinting of the H19 gene appeared to be unaffected by changes in growth conditions and its expression was down-regulated when the confluent cells were passaged. Methylation of the H19 promoter and Igf-2 coding regions was increased in the F x W cells extensively held under confluence and in the derived post-confluent cultures. The heritable changes in the expression, and imprinting status of the Igf-2 and H19 genes observed in the F x W cells closely resembles events described in human embryonal cancers and cancer-predisposing syndromes. The occurrence of imprinting relaxation under strong growth-inhibitory conditions supports the hypothesis that it is an epigenetic change.

The prokaryotic zinc-finger: structure, function and comparison with the eukaryotic counterpart

Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. The Ros Cys2His2 ZF binds DNA in a peculiar mode and folds in a domain significantly larger than its eukaryotic counterpart consisting of 58 amino acids (the 9–66 region) arranged in a βββαα topology, and stabilized by a conserved, extensive, 15‐residue hydrophobic core. The prokaryotic ZF domain, then, shows some intriguing new features that make it interestingly different from its eukaryotic counterpart. This review will focus on the prokaryotic ZFs, summarizing and discussing differences and analogies with the eukaryotic domains and providing important insights into their structure/function relationships.

Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the ~30 amino acid eukaryotic zinc finger motif in which a β-sheet and an α-helix are clustered around a zinc ion by various combinations of four ligands. The prokaryotic zinc finger domain (found in the Ros protein from Agrobacterium tumefaciens) is different from the eukaryotic counterpart as it consists of 58 amino acids arranged in a βββαα topology stabilized by a 15-residue hydrophobic core. Also, this domain tetrahedrally coordinates zinc and unfolds in the absence of the metal ion. The characterization of proteins belonging to the Ros homologs family has however shown that the prokaryotic zinc finger domain can overcome the metal requirement to achieve the same fold and DNA-binding activity. In the present work, two zinc-lacking Ros homologs (Ml4 and Ml5 proteins) have been thoroughly characterized using bioinformatics, biochemical and NMR techniques. We show how in these proteins a network of hydrogen bonds and hydrophobic interactions surrogate the zinc coordination role in the achievement of the same functional fold.

NMR Structure and Dynamics of the Resuscitation Promoting Factor RpfC Catalytic Domain.

Mycobacterium tuberculosis latent infection is maintained for years with no clinical symptoms and no adverse effects for the host. The mechanism through which dormant M. tuberculosis resuscitates and enters the cell cycle leading to tuberculosis is attracting much interest. The RPF family of proteins has been found to be responsible for bacteria resuscitation and normal proliferation. This family of proteins in M. tuberculosis is composed by five homologues (named RpfA-E) and understanding their conformational, structural and functional peculiarities is crucial to the design of therapeutic strategies.Therefore, we report the structural and dynamics characterization of the catalytic domain of RpfC from M. tubercolosis by combining Nuclear Magnetic Resonance, Circular Dichroism and Molecular Dynamics data. We also show how the formation of a disulfide bridge, highly conserved among the homologues, is likely to modulate the shape of the RpfC hydrophobic catalytic cleft. This might result in a protein function regulation via a “conformational editing” through a disulfide bond formation.

The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain.

The possibility of choices of protein ligands and coordination geometries leads to diverse Zn(II) binding sites in zinc-proteins, allowing a range of important biological roles. The prokaryotic Cys2His2 zinc finger domain (originally found in the Ros protein from Agrobacterium tumefaciens) tetrahedrally coordinates zinc through two cysteine and two histidine residues and it does not adopt a correct fold in the absence of the metal ion. Ros is the first structurally characterized member of a family of bacterial proteins that presents several amino acid changes in the positions occupied in Ros by the zinc coordinating residues. In particular, the second position is very often occupied by an aspartic acid although the coordination of structural zinc by an aspartate in eukaryotic zinc fingers is very unusual. Here, by appropriately mutating the protein Ros, we characterize the aspartate role within the coordination sphere of this family of proteins demonstrating how the presence of this residue only slightly perturbs the functional structure of the prokaryotic zinc finger domain while it greatly influences its thermodynamic properties.