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Papers by Daniele Fachinetti

Developmental cell, Jan 4, 2015

Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere p... more Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere position and function through a two-step mechanism relying on self-templating centromeric chromatin assembled with the histone H3 variant CENP-A, followed by CENP-A-dependent nucleation of kinetochore assembly. Nevertheless, natural human centromeres are positioned within specific megabase chromosomal regions containing α-satellite DNA repeats, which contain binding sites for the DNA sequence-specific binding protein CENP-B. We now demonstrate that CENP-B directly binds both CENP-A's amino-terminal tail and CENP-C, a key nucleator of kinetochore assembly. DNA sequence-dependent binding of CENP-B within α-satellite repeats is required to stabilize optimal centromeric levels of CENP-C. Chromosomes bearing centromeres without bound CENP-B, including the human Y chromosome, are shown to mis-segregate in cells at rates several-fold higher than chromosomes with CENP-B-containing centromeres...

Experimental Cell Research, 2012

The centromere is the fundamental unit for insuring chromosome inheritance. This complex region h... more The centromere is the fundamental unit for insuring chromosome inheritance. This complex region has a distinct type of chromatin in which histone H3 is replaced by a structurally different homologue identified in humans as CENP-A. In metazoans, specific DNA sequences are neither required nor sufficient for centromere identity. Rather, an epigenetic mark comprised of CENP-A containing chromatin is thought to be the major determinant of centromere identity. In this view, CENP-A deposition and chromatin assembly are fundamental processes for the maintenance of centromeric identity across mitotic and meiotic divisions. Several lines of evidence support CENP-A deposition in metazoans occurring at only one time in the cell cycle. Such cell cycle-dependent loading of CENP-A is found in divergent species from human to fission yeast, albeit with differences in the cell cycle point at which CENP-A is assembled. Cell cycle dependent CENP-A deposition requires multiple assembly factors for its deposition and maintenance. This review discusses the regulation of new CENP-A deposition and its relevance to centromere identity and inheritance.

Cell, 2009

Specialized topoisomerases solve the topological constraints arising when replication forks encou... more Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

Proceedings of the National Academy of Sciences, 2014

Molecular Cell, 2005

Via Adamello 16 thesis blocks or intra-S DNA damage (Foiani et al., 2000; 20141 Milano Rhind and ... more Via Adamello 16 thesis blocks or intra-S DNA damage (Foiani et al., 2000; 20141 Milano Rhind and Russell, 2000). In yeast, the replication check-Italy point is mediated by the Mec1 and Rad53 protein ki-2 Dipartimento di Scienze Biomolecolari nases (Zhou and Elledge, 2000). Active Mec1 and Rad53 e Biotecnologie modify the phosphorylation state of proteins implicated Università degli Studi di Milano in recombination and replication (Muzi-Falconi et al., Via Celoria 26 2003). In particular, the DNA polymerase ␣-primase 20133 Milano complex (pol-prim) and the single-strand DNA binding Italy protein RPA (Brush et al., 1996; Pellicioli et al., 1999) 3 Institute of Cell Biology are targeted by the checkpoint, probably to stabilize the ETH Hö nggerberg replisome-fork association when forks stall (Lucca et CH-8093 Zü rich al., 2004). Checkpoint-defective cells experiencing a hy-Switzerland droxyurea (HU)-induced replication block or intra-S DNA damage exhibit a variety of abnormal events: (i) the firing of dormant and pseudo origins (Santocanale and Diffley, Summary 1998; Shirahige et al., 1998; Sogo et al., 2002) that accelerates the completion of DNA synthesis in the presence The replication checkpoint coordinates the cell cycle of a damaged template (Tercero and Diffley, 2001) (it with DNA replication and recombination, preventing should be noted however that this phenotype contribgenome instability and cancer. The budding yeast utes only modestly to cell viability [Tercero et al., 2003]); Rad53 checkpoint kinase stabilizes stalled forks and (ii) the progressive dissociation of DNA polymerases replisome-fork complexes, thus preventing the accufrom stalled replication forks that affects replication remulation of ss-DNA regions and reversed forks at colsumption (Cobb et al., 2003; Lucca et al., 2004); (iii) and lapsed forks. We searched for factors involved in the the unscheduled formation of abnormal DNA structures processing of stalled forks in HU-treated rad53 cells. at replication forks that gives rise to recombination inter-Using the neutral-neutral two-dimensional electromediates and DNA breaks (Cha and Kleckner, 2002; phoresis technique (2D gel) and psoralen crosslinking Lopes et al., 2001; Sogo et al., 2002). In particular, checkcombined with electron microscopy (EM), we found point mutants accumulate single-stranded DNA molethat the Exo1 exonuclease is recruited to stalled forks cules (gapped and hemireplicated molecules) and fourand, in rad53 mutants, counteracts reversed fork acbranched structures (reversed forks) (Sogo et al., 2002). cumulation by generating ss-DNA intermediates. Hence, The gapped and hemireplicated molecules result from Exo1-mediated fork processing resembles the action lagging strand defects and, possibly, from nucleolytic of E. coli RecJ nuclease at damaged forks. Fork stabilevents (Lopes et al., 2001; Sogo et al., 2002). Reversed ity and replication restart are influenced by both DNA forks appear to result from the conversion of specialized polymerase-fork association and Exo1-mediated prosister chromatid junctions (SCJs) into four-branched cessing. We suggest that Exo1 counteracts fork restructures at collapsed forks (Lopes et al., 2003). The versal by resecting newly synthesized chains and X-shaped SCJ molecules form during origin firing under resolving the sister chromatid junctions that cause physiological conditions and branch migrate chasing regression of collapsed forks. replication forks (Lopes et al., 2003). The SCJs resemble hemicatenanes and likely contribute to the establish-Introduction ment of sister chromatid cohesion during S phase and assist sister-chromatid-mediated recombination and Cells coordinate chromosome replication with cell cycle replication bypass processes (Lopes et al., 2003). It has progression, repair, recombination, and sister chromabeen suggested that, in the absence of stable replisometid cohesion to prevent genome instability (Bell and fork complexes, the SCJs run off at stalled forks, Dutta, 2002; Muzi-Falconi et al., 2003; Nasmyth, 2001). allowing the ends of the daughter strands to pair to-A failure in the tuning of these pathways leads to chrogether, thus giving rise to reversed forks that can be mosome lesions, mutations, genome rearrangements, further processed by nucleolytic events (Lopes et al., and cancer. To avoid such problems, eukaryotic cells 2003). have developed the replication checkpoint (Muzi-Fal-It is still unclear whether the formation of gapped coni et al., 2003) that controls genome integrity by premolecules can influence the accumulation of reversed venting mitosis until replication has been completed forks or, vice versa, whether reversed fork formation is (Nyberg et al., 2002; Zhou and Elledge, 2000) and by a prerequisite for the accumulation of single-stranded regions at stalled forks. Further, it is unknown whether the pathological events at stalled forks in checkpoint-*Correspondence: marco.foiani@ifom-ieo-campus.it

Nature Cell Biology, 2013

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes ... more The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino-or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

Molecular Cell, 2010

Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. ... more Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes.

Genes & Development, 2012

Cell Cycle, 2003

Cells are continually challenged by genomic insults that originate from chemical and physical age... more Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.

Cell, 2009

Specialized topoisomerases solve the topological constraints arising when replication forks encou... more Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

Nature Structural & Molecular Biology, 2012

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Developmental cell, Jan 4, 2015

Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere p... more Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere position and function through a two-step mechanism relying on self-templating centromeric chromatin assembled with the histone H3 variant CENP-A, followed by CENP-A-dependent nucleation of kinetochore assembly. Nevertheless, natural human centromeres are positioned within specific megabase chromosomal regions containing α-satellite DNA repeats, which contain binding sites for the DNA sequence-specific binding protein CENP-B. We now demonstrate that CENP-B directly binds both CENP-A's amino-terminal tail and CENP-C, a key nucleator of kinetochore assembly. DNA sequence-dependent binding of CENP-B within α-satellite repeats is required to stabilize optimal centromeric levels of CENP-C. Chromosomes bearing centromeres without bound CENP-B, including the human Y chromosome, are shown to mis-segregate in cells at rates several-fold higher than chromosomes with CENP-B-containing centromeres...

Experimental Cell Research, 2012

The centromere is the fundamental unit for insuring chromosome inheritance. This complex region h... more The centromere is the fundamental unit for insuring chromosome inheritance. This complex region has a distinct type of chromatin in which histone H3 is replaced by a structurally different homologue identified in humans as CENP-A. In metazoans, specific DNA sequences are neither required nor sufficient for centromere identity. Rather, an epigenetic mark comprised of CENP-A containing chromatin is thought to be the major determinant of centromere identity. In this view, CENP-A deposition and chromatin assembly are fundamental processes for the maintenance of centromeric identity across mitotic and meiotic divisions. Several lines of evidence support CENP-A deposition in metazoans occurring at only one time in the cell cycle. Such cell cycle-dependent loading of CENP-A is found in divergent species from human to fission yeast, albeit with differences in the cell cycle point at which CENP-A is assembled. Cell cycle dependent CENP-A deposition requires multiple assembly factors for its deposition and maintenance. This review discusses the regulation of new CENP-A deposition and its relevance to centromere identity and inheritance.

Cell, 2009

Specialized topoisomerases solve the topological constraints arising when replication forks encou... more Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

Proceedings of the National Academy of Sciences, 2014

Molecular Cell, 2005

Via Adamello 16 thesis blocks or intra-S DNA damage (Foiani et al., 2000; 20141 Milano Rhind and ... more Via Adamello 16 thesis blocks or intra-S DNA damage (Foiani et al., 2000; 20141 Milano Rhind and Russell, 2000). In yeast, the replication check-Italy point is mediated by the Mec1 and Rad53 protein ki-2 Dipartimento di Scienze Biomolecolari nases (Zhou and Elledge, 2000). Active Mec1 and Rad53 e Biotecnologie modify the phosphorylation state of proteins implicated Università degli Studi di Milano in recombination and replication (Muzi-Falconi et al., Via Celoria 26 2003). In particular, the DNA polymerase ␣-primase 20133 Milano complex (pol-prim) and the single-strand DNA binding Italy protein RPA (Brush et al., 1996; Pellicioli et al., 1999) 3 Institute of Cell Biology are targeted by the checkpoint, probably to stabilize the ETH Hö nggerberg replisome-fork association when forks stall (Lucca et CH-8093 Zü rich al., 2004). Checkpoint-defective cells experiencing a hy-Switzerland droxyurea (HU)-induced replication block or intra-S DNA damage exhibit a variety of abnormal events: (i) the firing of dormant and pseudo origins (Santocanale and Diffley, Summary 1998; Shirahige et al., 1998; Sogo et al., 2002) that accelerates the completion of DNA synthesis in the presence The replication checkpoint coordinates the cell cycle of a damaged template (Tercero and Diffley, 2001) (it with DNA replication and recombination, preventing should be noted however that this phenotype contribgenome instability and cancer. The budding yeast utes only modestly to cell viability [Tercero et al., 2003]); Rad53 checkpoint kinase stabilizes stalled forks and (ii) the progressive dissociation of DNA polymerases replisome-fork complexes, thus preventing the accufrom stalled replication forks that affects replication remulation of ss-DNA regions and reversed forks at colsumption (Cobb et al., 2003; Lucca et al., 2004); (iii) and lapsed forks. We searched for factors involved in the the unscheduled formation of abnormal DNA structures processing of stalled forks in HU-treated rad53 cells. at replication forks that gives rise to recombination inter-Using the neutral-neutral two-dimensional electromediates and DNA breaks (Cha and Kleckner, 2002; phoresis technique (2D gel) and psoralen crosslinking Lopes et al., 2001; Sogo et al., 2002). In particular, checkcombined with electron microscopy (EM), we found point mutants accumulate single-stranded DNA molethat the Exo1 exonuclease is recruited to stalled forks cules (gapped and hemireplicated molecules) and fourand, in rad53 mutants, counteracts reversed fork acbranched structures (reversed forks) (Sogo et al., 2002). cumulation by generating ss-DNA intermediates. Hence, The gapped and hemireplicated molecules result from Exo1-mediated fork processing resembles the action lagging strand defects and, possibly, from nucleolytic of E. coli RecJ nuclease at damaged forks. Fork stabilevents (Lopes et al., 2001; Sogo et al., 2002). Reversed ity and replication restart are influenced by both DNA forks appear to result from the conversion of specialized polymerase-fork association and Exo1-mediated prosister chromatid junctions (SCJs) into four-branched cessing. We suggest that Exo1 counteracts fork restructures at collapsed forks (Lopes et al., 2003). The versal by resecting newly synthesized chains and X-shaped SCJ molecules form during origin firing under resolving the sister chromatid junctions that cause physiological conditions and branch migrate chasing regression of collapsed forks. replication forks (Lopes et al., 2003). The SCJs resemble hemicatenanes and likely contribute to the establish-Introduction ment of sister chromatid cohesion during S phase and assist sister-chromatid-mediated recombination and Cells coordinate chromosome replication with cell cycle replication bypass processes (Lopes et al., 2003). It has progression, repair, recombination, and sister chromabeen suggested that, in the absence of stable replisometid cohesion to prevent genome instability (Bell and fork complexes, the SCJs run off at stalled forks, Dutta, 2002; Muzi-Falconi et al., 2003; Nasmyth, 2001). allowing the ends of the daughter strands to pair to-A failure in the tuning of these pathways leads to chrogether, thus giving rise to reversed forks that can be mosome lesions, mutations, genome rearrangements, further processed by nucleolytic events (Lopes et al., and cancer. To avoid such problems, eukaryotic cells 2003). have developed the replication checkpoint (Muzi-Fal-It is still unclear whether the formation of gapped coni et al., 2003) that controls genome integrity by premolecules can influence the accumulation of reversed venting mitosis until replication has been completed forks or, vice versa, whether reversed fork formation is (Nyberg et al., 2002; Zhou and Elledge, 2000) and by a prerequisite for the accumulation of single-stranded regions at stalled forks. Further, it is unknown whether the pathological events at stalled forks in checkpoint-*Correspondence: marco.foiani@ifom-ieo-campus.it

Nature Cell Biology, 2013

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes ... more The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino-or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

Molecular Cell, 2010

Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. ... more Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes.

Genes & Development, 2012

Cell Cycle, 2003

Cells are continually challenged by genomic insults that originate from chemical and physical age... more Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.

Cell, 2009

Specialized topoisomerases solve the topological constraints arising when replication forks encou... more Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

Nature Structural & Molecular Biology, 2012

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