Portrait of Dr Alessia Buscaino

Dr Alessia Buscaino

Senior Lecturer in Fungal Epigenetics
Director, Master by Research

About

Dr Alessia Buscaino graduated in Molecular Biology at the University of Palermo (Italy) in 2000.
She conducted her PhD research in the laboratory of Dr Asifa Akhtar at the European Molecular Biology Laboratory (EMBL-Germany) research institute. During her PhD, her interest in epigenetics and chromatin modifications flourished while investigating mechanisms of Dosage Compensation in Drosophila melanogaster.
In 2005, Alessia was awarded an EMBO long-term post-doctoral fellowship to conduct research in the laboratory of Professor Robin Allshire (WTCCB-Edinburgh). During her post-doc she investigated how heterochromatin assembles on large blocks of DNA repeats in the fission yeast Schizosaccharomyces pombe.
In 2013, she obtained an EMBO short-term Fellowship to investigate the chromatin status of Candida albicans repetitive DNA elements in Judith Berman's laboratory (TAU University- Tel-Aviv, Israel).
Alessia joined the University of Kent in 2013 as a Lecturer in Fungal Epigenetics. In 2016, Alessia was promoted to Senior Lecturer. Her group is part of the Kent Fungal Group

ORCID ID: 0000-0002-1704-3168

Research interests

Dr Buscaino's research group focuses in understanding how genome plasticity allows rapid and reversible adaptation to different environments. 
Research in the lab has two major themes:  

  1. Exploiting genome diversity in the yeast Scheffersomyces stipitis for improved bioethanol production The world population is growing rapidly and this rapid growth require high amount of energy. Lignocellulosic biomass is generated in large concentration as waste following agricultural, and forestry processing operations and could support bio-ethanol production at a level that would allow to reduce petroleum consumption by 30%. Lignocellulose is composed of different type of sugars: pentose sugars (such as xylose) and hexose sugars (such as glucose). The yeast Saccharomyces cerevisiae is usually the organism of choice for industrial production of ethanol. However, S. cerevisiae is not an ideal choice for the production of second-generation ethanol because it cannot ferment pentose sugars (such as xylose). Contrary to S. cerevisiae, the yeast Scheffersomyces stipitis, isolated from the gut of wood-eating beetles, can ferment xylose in addition to glucose. Despite its great potential, the use of S. stipitis for bioethanol production still faces many challenges. For example, S. stipitis fermentes xylose less efficiently than glucose and its growth is inhibited at high ethanol concentration. In addition, the plant cell-wall material of lignocellulosic biomass is difficult to deconstruct into fermentable sugars. The chemical pretreatment required to extract glucose and xylose generates by-products that inhibit S. stipitis growth and fermentation. Different S. stipitis isolates vary in their ability to ferment ethanol but the genetic basis underlying this improved ethanol production is largely unknown. Therefore, very little is known of S. stipitis genomic diversity and its contribution to ethanol production. The Buscaino lab aims to exploit S. stipitis genome diversity to improve bioethanol production
     
  2. Understanding epigenetic mechanisms of stress induced genome plasticity in the human fungal pathogen Candida albicans fungi cause more human deaths than either Malaria or Tuberculosis. Candida albicans is the most common human fungal pathogen causing life-threatening infections. The incidence of C. albicans strains resistant to anti-fungal drugs is increasing every year. C. albicans is a successful pathogen because it adapts to parts of our body by breaking and shuffling its DNA. The DNA is packaged into 'chromatin', a structure that allows the DNA to be wrapped up so that it fits inside cells. Different DNA regions are packaged into distinct chromatin types: some can prevent DNA shuffling, others promote DNA shuffling. The Buscaino lab aims to understand why DNA breaks occur in C. albicans, whether chromatin changes during infection and how chromatin controls DNA breaks. This research will help our understanding of C. albicans DNA shuffling and will help the design of strategies to block it, stopping infections and drug resistance.

Teaching

Alessia is the Module convenor of: 

  • Microbial Physiology and Genetics I - BI548   
  • Fungi as Human Pathogens - BI854 

Supervision

MSc-R projects available for 2019/20

  1. Improving second-generation bioethanol production by manipulating the genome of the non-conventional yeast Scheffersomyces stipitis
    Bench Fees: £1000 
  2. Centromere structure and Function in the bioethanol producing yeast Scheffersomyces stipitis.
    Bench Fees: £1000 
  3. Genome diversity and epigenetics in the most common human fungal pathogen Candida albicans 
    Bench Fees: £1000 
  4. Understanding mechanisms of RNA interference in the human fungal pathogen Candida albicans 
    Bench Fees: £1000 
  5. Drug resistance in fungal pathogens: the role of genome instability
    Bench Fees: £1000

Post-Doctoral Researcher, Ph.D. student and Research Master applications from UK, EU, US & Overseas are always considered. Various funding sources can be explored. 
Post-Doc fellowships: EMBO, FEBS, HFSP, Newton, Marie Curie, Wellcome Trust. Please send your CV and summary of your research interests to: a.buscaino@kent.ac.uk

Professional

Alessia is a Core Member of the BBSRC Committee C. 

Publications

Article

  • Freire-Beneitez, V., Price, J. and Buscaino, A. (2016). The chromatin of Candida albicans pericentromeric repeats bears features of both euchromatin and heterochromatin. Frontiers in Microbiology [Online]. Available at: http://dx.doi.org/10.3389/fmicb.2016.00759.
    Centromeres, sites of kinetochore assembly, are important for chromosome stability and integrity. Most eukaryotes have regional centromeres epigenetically specified by the presence of the histone H3 variant CENP-A. CENP-A chromatin is often surrounded by pericentromeric regions packaged into transcriptionally silent heterochromatin. Candida albicans, the most common human fungal pathogen, possesses small regional centromeres assembled into CENP-A chromatin. The chromatin state of C. albicans pericentromeric regions is unknown. Here, for the first time, we address this question. We find that C. albicans pericentromeres are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Pericentromeric chromatin is associated with nucleosomes that are highly acetylated, as found in euchromatic regions of the genome, and hypomethylated on H3K4, as found in heterochromatin. This intermediate chromatin state is inhibitory to transcription and partially represses expression of proximal genes and inserted marker genes. Our analysis identifies a new chromatin state associated with pericentromeric regions.
  • Freire-Beneitez, V. et al. (2016). Sir2 regulates stability of repetitive domains differentially in the human fungal pathogen Candida albicans. Nucleic Acids Research [Online]. Available at: http://dx.doi.org/10.1093/nar/gkw594.
    DNA repeats, found at the ribosomal DNA locus, telomeres and subtelomeric regions, are unstable sites of eukaryotic genomes. A fine balance between genetic variability and genomic stability tunes plasticity of these chromosomal regions. This tuning mechanism is particularly important for organisms such as microbial pathogens that utilise genome plasticity as a strategy for adaptation. For the first time, we analyse mechanisms promoting genome stability at the rDNA locus and subtelomeric regions in the most common human fungal pathogen: Candida albicans In this organism, the histone deacetylase Sir2, the master regulator of heterochromatin, has acquired novel functions in regulating genome stability. Contrary to any other systems analysed, C. albicans Sir2 is largely dispensable for repressing recombination at the rDNA locus. We demonstrate that recombination at subtelomeric regions is controlled by a novel DNA element, the TLO Recombination Element, TRE, and by Sir2. While the TRE element promotes high levels of recombination, Sir2 represses this recombination rate. Finally, we demonstrate that, in C. albicans, mechanisms regulating genome stability are plastic as different environmental stress conditions lead to general genome instability and mask the Sir2-mediated recombination control at subtelomeres. Our data highlight how mechanisms regulating genome stability are rewired in C. albicans
  • Freire-Benéitez, V. et al. (2016). Candida albicans repetitive elements display epigenetic diversity and plasticity. Scientific Reports [Online] 6:1-12. Available at: http://dx.doi.org/10.1038/srep22989.
    Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30 °C, while robust heterochromatin is assembled over these regions at 39 °C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation.
  • White, S. et al. (2014). The RFTS Domain of Raf2 Is Required for Cul4 Interaction and Heterochromatin Integrity in Fission Yeast. PLoS ONE [Online] 9. Available at: http://dx.doi.org/10.1371/journal.pone.0104161.
    Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 (equivalent to metazoan Suv3-9), a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-ACnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres.
  • Buscaino, A. et al. (2013). Distinct roles for Sir2 and RNAi in centromeric heterochromatin nucleation, spreading and maintenance. Embo Journal [Online] 32:1250-1264. Available at: http://dx.doi.org/10.1038/emboj.2013.72.
    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.
  • Buscaino, A. et al. (2012). Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification. PLoS Genetics [Online] 8:e1002499. Available at: http://dx.doi.org/10.1371/journal.pgen.1002499.
    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.
  • Simmer, F. et al. (2010). Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast. EMBO Reports [Online] 11:112-118. Available at: http://dx.doi.org/10.1038/embor.2009.273.
    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(+)-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.
  • Buscaino, A., Allshire, R. and Pidoux, A. (2010). Building centromeres: home sweet home or a nomadic existence? Current Opinion in Genetics and Development [Online] 20:118-126. Available at: http://dx.doi.org/10.1016/j.gde.2010.01.006.
    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.
  • Buscaino, A., Legube, G. and Akhtar, A. (2006). X-chromosome targeting and dosage compensation are mediated by distinct domains in MSL-3. EMBO Reports [Online] 7:531-538. Available at: http://dx.doi.org/10.1038/sj.embor.7400658.
    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 male-specific 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.
  • Almeida, R., Buscaino, A. and Allshire, R. (2006). Molecular biology: silencing unlimited. Current Biology [Online] 16:R635-R638. Available at: http://dx.doi.org/10.1016/j.cub.2006.07.033.
    Heterochromatin domains are essential for normal chromosome functions. The Eri1 ribonuclease is a negative regulator of the RNA interference machinery; recent studies have shown that, in fission yeast lacking Eri1, heterochromatin formation is more promiscuous.
  • Mendjan, S. et al. (2006). Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila. Molecular Cell [Online] 21:811-823. Available at: http://dx.doi.org/10.1016/j.molcel.2006.02.007.
    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.
  • Niggeweg, R. et al. (2006). A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separation. Proteomics [Online] 6:41-53. Available at: http://dx.doi.org/10.1002/pmic.200501332.
    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.
  • Nielsen, P. et al. (2005). Structure of the chromo barrel domain from the MOF acetyltransferase. Journal of Biological Chemistry [Online] 280:32326-32331. Available at: http://dx.doi.org/10.1074/jbc.M501347200.
    We report here the structure of the putative chromo domain from MOF, a member of the MYST family of histone acetyltransferases that acetylates histone H4 at Lys-16 and is part of the dosage compensation complex in Drosophila. We found that the structure of this domain is a beta-barrel that is distinct from the alpha + beta fold of the canonical chromo domain. Despite the differences, there are similarities that support an evolutionary relationship between the two domains, and we propose the name "chromo barrel." The chromo barrel domains may be divided into two groups, MSL3-like and MOF-like, on the basis of whether a group of conserved aromatic residues is present or not. The structure suggests that, although the MOF-like domains may have a role in RNA binding, the MSL3-like domains could instead bind methylated residues. The MOF chromo barrel shares a common fold with other chromatin-associated modules, including the MBT-like repeat, Tudor, and PWWP domains. This structural similarity suggests a probable evolutionary pathway from these other modules to the canonical chromo domains (or vice versa) with the chromo barrel domain representing an intermediate structure.
  • Buscaino, A. et al. (2003). MOF-regulated acetylation of MSL-3 in the Drosophila dosage compensation complex. Molecular Cell [Online] 11:1265-1277. Available at: http://dx.doi.org/10.1016/S1097-2765(03)00140-0.
    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.
  • Alessandro, C. et al. (2002). Identification of the enhancer binding protein MBF-1 of the sea urchin modulator alpha-H2A histone gene. Biochemical and Biophysical Research Communications [Online] 295:519-525. Available at: http://dx.doi.org/10.1016/S0006-291X(02)00708-8.
    The modulator of the sea urchin alpha-H2A histone gene promoter is the only enhancer identified in the alpha-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 Krüppel 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 alpha-H2A gene after hatching is not due to the lack of the transactivator.