Alessia Buscaino - Academia.edu (original) (raw)

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Papers by Alessia Buscaino

Biochemical and Biophysical Research Communications, 2002

The modulator of the sea urchin a-H2A histone gene promoter is the only enhancer identified in th... more The modulator of the sea urchin a-H2A histone gene promoter is the only enhancer identified in the a-histone gene cluster. Binding of a single factor, denoted MBF-1, has previously detected in nuclear extracts from morula and gastrula embryos. Here, we describe the cloning of MBF-1 by screening a cDNA expression library with a tandem array of modulator binding sites. MBF-1 presents no similarity with other DNA binding proteins and contains nine Kr€ u uppel like Zn fingers. In vitro translated proteins and a factor from nuclear extracts interact with the modulator with identical specificity. In addition, MBF-1 expressed in human cells transactivates a reporter gene driven by an array of modulator sites. The DNA binding domain consists of the Zn fingers plus an adjacent basic region, while sequences in the N-terminal region mediates the transactivation function. MBF-1 is expressed in the unfertilized egg and in early and late developmental stages thus confirming that it is not a stage specific enhancer binding factor and that silencing of the a-H2A gene after hatching is not due to the lack of the transactivator. Ó 2002 Elsevier Science (USA). All rights reserved.

Proteomics, 2006

MS protein identification and quantitation are key proteomic techniques in biological research. B... more MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode – implemented on a quadrupole-TOF instrument – that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain.

Molecular Cell, 2006

Dosage compensation in Drosophila is dependent on MSL proteins and involves hypertranscription of... more Dosage compensation in Drosophila is dependent on MSL proteins and involves hypertranscription of the male X chromosome, which ensures equal X-linked gene expression in both sexes. Here, we report the purification of enzymatically active MSL complexes from Drosophila embryos, Schneider cells, and human HeLa cells. We find a stable association of the histone H4 lysine 16-specific acetyltransferase MOF with the RNA/protein containing MSL complex as well as with an evolutionary conserved complex. We show that the MSL complex interacts with several components of the nuclear pore, in particular Mtor/TPR and Nup153. Strikingly, knockdown of Mtor or Nup153 results in loss of the typical MSL X-chromosomal staining and dosage compensation in Drosophila male cells but not in female cells. These results reveal an unexpected physical and functional connection between nuclear pore components and chromatin regulation through MSL proteins, highlighting the role of nucleoporins in gene regulation in higher eukaryotes.

Molecular Cell, 2003

Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophil... more Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophila, equalization is achieved by hypertranscription of the male X chromosome. This process requires an RNA/protein containing dosage compensation complex (DCC). Here we use RNA interference of individual DCC components to define the order of complex assembly in Schneider cells. We show that interaction of MOF with MSL-3 leads to specific acetylation of MSL-3 at a single lysine residue adjacent to one of its chromodomains. We observe that localization of MSL-3 to the X chromosome is RNA dependent and acetylation sensitive. We find that the acetylation status of MSL-3 determines its interaction with roX2 RNA. Furthermore, we find that RPD3 interacts with MSL-3 and that MSL-3 can be deacetylated by the RPD3 complex. We propose that regulated acetylation of MSL-3 may provide a mechanistic explanation for spreading of the dosage compensation complex along the male X chromosome.

PLoS Genetics, 2012

Non-coding transcription can trigger histone post-translational modifications forming specialized... more Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin.

The EMBO Journal, 2013

Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feat... more Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular 'nucleation sites' by RNA interference (RNAi), ensuring the mitotic stability of centromere-bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6 HP1 are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6 HP1 operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin.

EMBO reports, 2010

RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptional... more RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptionally or post-transcriptionally. In fission yeast, RNAi is tightly coupled to template transcription and chromatin modifications that establish heterochromatin in cis. Exogenous double-stranded RNA (dsRNA) triggers seem to induce heterochromatin formation in trans only when certain silencing proteins are overexpressed. Here, we show that green fluorescent protein (GFP) hairpin dsRNA allows production of high levels of Argonaute-associated small interfering RNAs (siRNAs), which can induce heterochromatin formation at a remote locus. This silencing does not require any manipulation apart from hairpin expression. In cells expressing a ura4 þ -GFP fusion gene, production of GFP siRNAs causes the appearance of ura4 siRNAs from the target gene. Production of these secondary siRNAs depends on RNA-dependent RNA polymerase Rdp1 (RDRP Rdp1 ) function and other RNAi pathway components. This demonstrates that transitivity occurs in fission yeast and implies that RDRP Rdp1 can synthesize RNA from targeted RNA templates in vivo, generating siRNAs not homologous to the hairpin.

EMBO reports, 2006

In Drosophila, dosage compensation of X-linked genes is achieved by transcriptional upregulation ... more In Drosophila, dosage compensation of X-linked genes is achieved by transcriptional upregulation of the male X chromosome. Genetic and biochemical studies have demonstrated that malespecific lethal (MSL) proteins together with roX RNAs regulate this process. Here, we show that MSL-3 is essential for cell viability and that three domains in the protein have distinct roles in dosage compensation. The chromo-barrel domain (CBD) is not necessary for MSL targeting to the male X chromosome but is important for male viability and equalization of X-linked gene transcription. The polar region cooperates with the CBD in MSL-3 function, whereas the MRG domain is responsible for targeting the protein to the X chromosome. Our results demonstrate that MSL-3 localization to the male X chromosome and transcriptional upregulation of X-linked genes are two separable functions of the MSL-3 protein.

Current Opinion in Genetics & Development, 2010

Centromere assembly and propagation is governed by genetic and epigenetic mechanisms. A centromer... more Centromere assembly and propagation is governed by genetic and epigenetic mechanisms. A centromere-specific histone H3 variant, CENP-A is strongly favored as the epigenetic mark that specifies centromere identity. Despite the critical importance of centromere function, centromeric sequences are not conserved. This has prompted exploration of other genomic and chromatin features to gain an understanding of where CENP-A is deposited. In this review we highlight recent papers that advance our understanding of how the cell builds a centromere. We focus on what influences the choice of site for CENP-A deposition and therefore the site of centromere formation. We then briefly discuss how centromeres are propagated once the site of centromere assembly is chosen.