Publications by authors named "Ewelina Zasadzińska"

9 Publications

  • Page 1 of 1

Assuring Clonality on the Beacon Digital Cell Line Development Platform.

Biotechnol J 2020 Jan 27;15(1):e1900247. Epub 2019 Nov 27.

Drug Substance Technologies, Process Development, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA, 91320, USA.

During biomanufacturing cell lines development, the generation and screening for single-cell derived subclones using methods that enable assurance of clonal derivation can be resource- and time-intensive. High-throughput miniaturization, automation, and analytic strategies are often employed to reduce such bottlenecks. The Beacon platform from Berkeley Lights offers a strategy to eliminate these limitations through culturing, manipulating, and characterizing cells on custom nanofluidic chips via software-controlled operations. However, explicit demonstration of this technology to provide high assurance of a single cell progenitor has not been reported. Here, a methodology that utilizes the Beacon instrument to ensure high levels of clonality is described. It is demonstrated that the Beacon platform can efficiently generate production cell lines with a superior clonality data package, detailed tracking, and minimal resources. A stringent in-process quality control strategy is established to enable rapid verification of clonal origin, and the workflow is validated using representative Chinese hamster ovary-derived cell lines stably expressing either green or red fluorescence protein. Under these conditions, a >99% assurance of clonal origin is achieved, which is comparable to existing imaging-coupled fluorescence-activated cell sorting seeding methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/biot.201900247DOI Listing
January 2020

Inheritance of CENP-A Nucleosomes during DNA Replication Requires HJURP.

Dev Cell 2018 11 4;47(3):348-362.e7. Epub 2018 Oct 4.

Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA; Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

Centromeric chromatin defines the site of kinetochore formation and ensures faithful chromosome segregation. Centromeric identity is epigenetically specified by the incorporation of CENP-A nucleosomes. DNA replication presents a challenge for inheritance of centromeric identity because nucleosomes are removed to allow for replication fork progression. Despite this challenge, CENP-A nucleosomes are stably retained through S phase. We used BioID to identify proteins transiently associated with CENP-A during DNA replication. We found that during S phase, HJURP transiently associates with centromeres and binds to pre-existing CENP-A, suggesting a distinct role for HJURP in CENP-A retention. We demonstrate that HJURP is required for centromeric nucleosome inheritance during S phase. HJURP co-purifies with the MCM2-7 helicase complex and, together with the MCM2 subunit, binds CENP-A simultaneously. Therefore, pre-existing CENP-A nucleosomes require an S phase function of the HJURP chaperone and interaction with MCM2 to ensure faithful inheritance of centromere identity through DNA replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2018.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219920PMC
November 2018

Posttranslational mechanisms controlling centromere function and assembly.

Curr Opin Cell Biol 2018 06 2;52:126-135. Epub 2018 Apr 2.

Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

Accurate chromosome segregation is critical to ensure the faithful inheritance of the genome during cell division. Human chromosomes distinguish the location of the centromere from general chromatin by the selective assembly of CENP-A containing nucleosomes at the active centromere. The location of centromeres in most higher eukaryotes is determined epigenetically, independent of DNA sequence. CENP-A containing centromeric chromatin provides the foundation for assembly of the kinetochore that mediates chromosome attachment to the microtubule spindle and controls cell cycle progression in mitosis. Here we review recent work demonstrating the role of posttranslational modifications on centromere function and CENP-A inheritance via the direct modification of the CENP-A nucleosome and pre-nucleosomal complexes, the modification of the CENP-A deposition machinery and the modification of histones within existing centromeres.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ceb.2018.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004306PMC
June 2018

Orchestrating the Specific Assembly of Centromeric Nucleosomes.

Prog Mol Subcell Biol 2017;56:165-192

Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, 22908, USA.

Centromeres are chromosomal loci that are defined epigenetically in most eukaryotes by incorporation of a centromere-specific nucleosome in which the canonical histone H3 variant is replaced by Centromere Protein A (CENP-A). Therefore, the assembly and propagation of centromeric nucleosomes are critical for maintaining centromere identify and ensuring genomic stability. Centromeres direct chromosome segregation (during mitosis and meiosis) by recruiting the constitutive centromere-associated network of proteins throughout the cell cycle that in turn recruits the kinetochore during mitosis. Assembly of centromere-specific nucleosomes in humans requires the dedicated CENP-A chaperone HJURP, and the Mis18 complex to couple the deposition of new CENP-A to the site of the pre-existing centromere, which is essential for maintaining centromere identity. Human CENP-A deposition occurs specifically in early G1, into pre-existing chromatin, and several additional chromatin-associated complexes regulate CENP-A nucleosome deposition and stability. Here we review the current knowledge on how new CENP-A nucleosomes are assembled selectively at the existing centromere in different species and how this process is controlled to ensure stable epigenetic inheritance of the centromere.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-319-58592-5_7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004308PMC
May 2019

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.

J Clin Invest 2017 Jun 8;127(6):2365-2377. Epub 2017 May 8.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI88936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451240PMC
June 2017

Centromeres of a Different CAL-ibre.

Dev Cell 2016 Apr;37(2):105-6

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA; Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

Centromeres of higher eukaryotes are defined by the epigenetic inheritance of the centromere-specific CENP-A nucleosome. Reporting in Developmental Cell, Rosin and Mellone (2016) show that co-evolution of the CENP-A histone variant and its chaperone CAL1 accounts for species incompatibility between centromeric histones in Drosophila.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2016.04.006DOI Listing
April 2016

Licensing of Centromeric Chromatin Assembly through the Mis18α-Mis18β Heterotetramer.

Mol Cell 2016 Mar;61(5):774-787

Department of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, VA 22908, USA; Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA; Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, 320 E. Superior Street, Chicago, IL 60611, USA. Electronic address:

Centromeres are specialized chromatin domains specified by the centromere-specific CENP-A nucleosome. The stable inheritance of vertebrate centromeres is an epigenetic process requiring deposition of new CENP-A nucleosomes by HJURP. We show HJURP is recruited to centromeres through a direct interaction between the HJURP centromere targeting domain and the Mis18α-β C-terminal coiled-coil domains. We demonstrate Mis18α and Mis18β form a heterotetramer through their C-terminal coiled-coil domains. Mis18α-β heterotetramer formation is required for Mis18BP1 binding and centromere recognition. S. pombe contains a single Mis18 isoform that forms a homotetramer, showing tetrameric Mis18 is conserved from fission yeast to humans. HJURP binding disrupts the Mis18α-β heterotetramer and removes Mis18α from centromeres. We propose stable binding of Mis18 to centromeres in telophase licenses them for CENP-A deposition. Binding of HJURP deposits CENP-A at centromeres and facilitates the removal of Mis18, restricting CENP-A deposition to a single event per cell cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2016.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935545PMC
March 2016

Protein purification and crystallization artifacts: The tale usually not told.

Protein Sci 2016 Mar 26;25(3):720-33. Epub 2016 Jan 26.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.

The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.2861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815408PMC
March 2016

Dimerization of the CENP-A assembly factor HJURP is required for centromeric nucleosome deposition.

EMBO J 2013 Jul 14;32(15):2113-24. Epub 2013 Jun 14.

Department of Biotechnology and Food Sciences, Lodz University of Technology, Lodz, Poland.

The epigenetic mark of the centromere is thought to be a unique centromeric nucleosome that contains the histone H3 variant, centromere protein-A (CENP-A). The deposition of new centromeric nucleosomes requires the CENP-A-specific chromatin assembly factor HJURP (Holliday junction recognition protein). Crystallographic and biochemical data demonstrate that the Scm3-like domain of HJURP binds a single CENP-A-histone H4 heterodimer. However, several lines of evidence suggest that HJURP forms an octameric CENP-A nucleosome. How an octameric CENP-A nucleosome forms from individual CENP-A/histone H4 heterodimers is unknown. Here, we show that HJURP forms a homodimer through its C-terminal domain that includes the second HJURP_C domain. HJURP exists as a dimer in the soluble preassembly complex and at chromatin when new CENP-A is deposited. Dimerization of HJURP is essential for the deposition of new CENP-A nucleosomes. The recruitment of HJURP to centromeres occurs independent of dimerization and CENP-A binding. These data provide a mechanism whereby the CENP-A pre-nucleosomal complex achieves assembly of the octameric CENP-A nucleosome through the dimerization of the CENP-A chaperone HJURP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emboj.2013.142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730228PMC
July 2013