Emanuela Micheli

Chromatin Structure in Telomere Dynamics

The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation, and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to deprotected state and their role in telomere functions.

Selective G-quadruplex ligands: The significant role of side chain charge density in a series of perylene derivatives

The human telomeric G-quadruplex structure is a promising target for the design of cancer drugs. The selectivity of G-quadruplex ligands with respect to duplex genomic DNA is of especial importance. The high selectivity of polyamine conjugated perylene derivatives appears to be regulated by side-chain charge density, as indicated by data from a FRET melting assay and induced CD spectroscopy.

TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection.

AKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication.

Self-organization of G-quadruplex structures in the hTERT core promoter stabilized by polyaminic side chain perylene derivatives

hTERT core promoter regulates telomerase transcription in human cells, thus its structural features are of large interest. We have found that the G-rich hTERT core promoter region, corresponding to the major DNase I hypersensitive site in chromatin organization, contains nine putative G-quadruplex forming sequences (PQS) and is unfavorable for nucleosome formation. Here we show that four PQS are effectively able to form stable parallel intramolecular G-quadruplexes, using PAGE and CD spectroscopy analysis. The PQS-region, as a whole, appears to be organized in three self-interacting G-quadruplexes, probably giving rise to a helicoidal superstructure, as shown by CD and polymerase stop assay. POL-HPDI drugs, that we previously found useful in selectively stabilizing telomeric G-quadruplex, are able to stabilize both the single intramolecular G-quadruplex and the PQS-region superstructure. The features of their induced CD spectra suggest that POL-HPDIs bind to single G-quadruplexes and to whole PQS-region superstructure, mainly by end-stacking interactions.

The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response

Abstract Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin.

Superstructural self-assembly of the G-quadruplex structure formed by the homopurine strand in a DNA tract of human telomerase gene promoter

Abstract AFM imaging and physico-chemical studies provide evidence of the ability of the G-quadruplex structure, formed by a homopurine DNA sequence present in the human telomerase gene promoter, to self-assemble, giving rise to periodical linear superstructures. The reported study suggests the possibility of G-rich DNA sequences to form a new type of G-wire stabilized by end-to-end stacking between terminal G-quartets and characterized by loops of adenine extruded by parallel G-quadruplex repetitive elements.

WDR79/TCAB1 plays a conserved role in the control of locomotion and ameliorates phenotypic defects in SMA models

SMN (Survival Motor Neuron) deficiency is the predominant cause of spinal muscular atrophy (SMA), a severe neurodegenerative disorder that can lead to progressive paralysis and death. Although SMN is required in every cell for proper RNA metabolism, the reason why its loss is especially critical in the motor system is still unclear. SMA genetic models have been employed to identify several modifiers that can ameliorate the deficits induced by SMN depletion. Here we focus on WDR79/TCAB1, a protein important for the biogenesis of several RNA species that has been shown to physically interact with SMN in human cells. We show that WDR79 depletion results in locomotion defects in both Drosophila and Caenorhabditis elegans similar to those elicited by SMN depletion. Consistent with this observation, we find that SMN overexpression rescues the WDR79 loss-of-function phenotype in flies. Most importantly, we also found that WDR79 overexpression ameliorates the locomotion defects induced by SMN depletion in both flies and worms. Our results collectively suggest that WDR79 and SMN play evolutionarily conserved cooperative functions in the nervous system and suggest that WDR79/TCAB1 may have the potential to modify SMA pathogenesis.

Natural Aromatic Compounds as Scaffolds to Develop Selective G-Quadruplex Ligands: From Previously Reported Berberine Derivatives to New Palmatine Analogues

In this paper, the selective interactions of synthetic derivatives of two natural compounds, berberine and palmatine, with DNA G-quadruplex structures were reported. In particular, the previous works on this subject concerning berberine were further presented and discussed, whereas the results concerning palmatine are presented here for the first time. In detail, these palmatine derivatives were developed by inserting seven different small peptide basic chains, giving several new compounds that have never been reported before. The preliminary studies of the interactions of these compounds with various G-quadruplex-forming sequences were carried out by means of various structural and biochemical techniques, which showed that the presence of suitable side chains is very useful for improving the interaction of the ligands with G-quadruplex structures. Thus, these new palmatine derivatives might act as potential anticancer drugs.

Telomere Maintenance in the Dynamic Nuclear Architecture

Abstract Telomeres, the protective structures at the end of eukaryotic chromosomes, play a pivotal role in several regulatory pathways that determine the cell fate. Human telomeres consist of thousands of TTAGGG repeats organized in a peculiar compact chromatin and bound by the six-protein complex, shelterin. In germinal and embryonic stem cells, telomere length is maintained by the activity of telomerase that adds TTAGGG repeats at the 3′ ends of chromosomes. In contrast, telomerase is inactive in somatic cells, and consequently telomeres shorten at each replication cycle till they reach a critical length that triggers a DNA damage response pathway leading to cell growth arrest, a state known as replicative senescence. In this chapter, we review what is known about telomere structure and telomeric chromatin organization. We will discuss the dynamic changes of telomeres and the epigenetic and structural modifications linked to telomere shortening and the entry in replicative senescence.