Chromosome Cohesion ; DNA double strand break Damage ; DNA Replication dynamics; genomic interactions

José A. M. da Costa Nunes, MSc, DPhil (Oxon)

MSc (University of Lisbon, Portugal), DPhil (PhD - University of Oxford, UK)

email: j.dacostanunes@wolfson.oxon.org

ORCID: 0000-0002-9401-9954

https://www.researchgate.net/profile/Jose_Da_Costa-Nunes

KEYWORDS:

DNA replication

Chromosome cohesion and chromosome segregation

DNA double strand break (dsb) damage repair

Genome integrity

TADs, cohesin, CTCF

CRISPR

Cytology ; RNA in situ hybridisation ; DNA FISH ; DIC and immunofluorescence microscopy ; immunohistochemistry; Metaphase and Meiotic chromosome spreads ; karyotyping ; etc.

Molecular Biology ; PCR ; RT-CPR ; DNA ; RNA ; cloning ; sequencing ; southern ; northern ; FACS ( 2D and 3D ) ; Click-iT ; pull-down (magnetic beads); etc.

Next Generation Sequencing ; DNA libraries construction

Genetics (Forward and Reverse genetic screens) ; EMS mutagenesis ( and mutant screens ) ; etc.

Plant fertility / reproduction (meiosis ; gametogenesis developmental staging - male and female); pollen development cytological markers ; etc.

Arabidopsis ; cell culture ; calli ; plant transformation

human cell culture, mouse embryonic stem cells culture, E.coli, Agrobacterium tumefaciens, S pombe, Arabidopsis, tobacco

My work on the dynamics of DNA replication reports the progression of DNA replication during early S phase, with high resolution data. This data was yielded from different libraries made from newly replicated DNA harvested from cell cycle synchronised human cancer cells during the early stages of S phase. This work involved the yielding of Next Generation Sequencing data, but also involved the use of several other techniques such as FACS (2D and 3D), cytology (FISH and IF).

I am also interested in the biological mechanisms that are required for the maintenance of genome integrity in meiotic and somatic cells, and practical implications and applications of these phenomena (their effect on genetic recombination, genome integrity, ploidy, etc.).

Disruption of genome integrity (caused by the failed and/or flawed repair of the DNA dsb, or caused by chromosome mis-segregation) in meiotic cells can lead to sterility, while in somatic cells it can ultimately lead to the death of the organism (aneuploidy, cancer). Needless it is to say that research in plants, concerning mutations affecting chromosome segregation and recombination, and gametes and embryo formation (ultimately plant fertility) are of paramount importance for global food-production.

This work involved the characterisation of the cohesins of Arabidopsis (AtRAD21.1, AtRAD21.2 and AtRAD21.3), and also a contribution to the characterisation of the expression of their meiotic homologue DIF1 (AtREC8), as well as EMS mutants affecting meiotic chromosome segregation.

Work on mutants affecting gametogenesis in Arabidopsis, was also carried out.

(some) of the previous work

(in Arabidopsis: chromosome biology ; genome integrity ; DNA repair ; fertility)

AtRAD21 kleisins and DNA dsb repair

RAD21 kleisins/cohesins play several critical roles in chromosome biology; DNA double strand break (dsb) repair and chromosome cohesion are two of these functions. Therefore RAD21 plays a key role in genomic integrity. The RAD21 kleisin proteins are found (and functionally conserved) in different organisms (from yeast to humans) including Arabidopsis. Two of the Arabidopsis RAD21 homologues (AtRAD21.1 and AtRAD21.3) have been shown to be required for DNA dsb repair (da Costa-Nunes et al., 2006, 2014; Kozak et al., 2009). atrad21.1 and atrad21.3 mutants have a higher basal level of DNA dsb and their DNA dsb repair kinetics is affected. These mutations also contribute to the DNA dsb damage hyper-sensitivity phenotype (particularly in atrad21.1), a phenotype that is shared with other DNA repair mutants – see DNA damage sensitivity assay figure. Furthermore, the AtRAD21.1 gene expression levels enhance considerably and abruptly soon after exposure to DNA dsb damage agents – see fold variation in gene expression graphics – suggesting that AtRAD21.1 plays a role in an emergency response to DNA damage (da Costa-Nunes et al., 2006, 2014).

Genome integrity maintenance during meiosis

The deleterious DNA dsb breaks can be induced by abiotic factors

(such as exposure to chemicals, ionising radiation - gamma rays, UV rays, X-rays), and are also formed during meiosis (programmed DNA dsb formation) prior to meiotic recombination. To identify and characterise nove genes involved in the maintenance of genome stability during plant meiosis, I have generated an EMS meiotic mutant population and have used forward genetics to screen and study DNA repair and chromosome cohesion mutants exhibiting abnormal meiotic divisions (da Costa Nunes et al., 2009). Some of the identified mutants display disrupted genome integrity; their nuclei exhibit stretched, broken and mis-segregated chromosomes – see meiotic mutants four figures plate. Other mutants exhibited solely mis-segregation of the twenty intact chromosomes (sister chromatids) – see meiotic mis-segregation two figures plate.

2008 - 2011 - Research project

(funded by the Portuguese Research Funding Agency - FCT)

PTDC/BIA-BCM/64192/2006 - Chromosome Cohesion and DNA repair in Arabidopsis Funding: €52.000

(one of the 19% top proposals selected for funding, out of a total of 163 proposals - on Molecular and Cellular Biology - external funding)

Principal Investigator of the Project - José A. M. da Costa Nunes

Graduate Education and some relevant traineeships

1994-1998 Undergraduate degree (Agronomy Engineering; Plant breeding) (a 5 years degree),

Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Portugal

1994-1998 MSc degree (Plant Biotechnology),

Faculdade de Ciências, Universidade de Lisboa, Portugal

1996 Traineeship at I.G.E.R.

Institute for Grassland and Environmental Research, Aberystwyth, Wales, UK

1997-2001 DPhil (Oxon) (PhD degree),

Department of Plant Sciences, University of Oxford, UK

Supervisor: Professor Hugh Dickinson

1999 Course: "Arabidopsis Molecular Genetics" Cold Spring Harbour Course

Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y., USA

(a 21 days course)

Relevant Post-Doc Research training (Plant Sciences) and some other relevant courses and workshops attended :

2001 Short-term Post-Doctoral researcher

Department of Plant Sciences, University of Oxford, England, UK

Professor Hugh Dickinson's laboratory

2002 - 2005 junior Post-Doctoral researcher

Pflanzenbiologie Institut, Zürich Universität, Switzerland

Professor Ueli Grossniklaus' laboratory

2003 Workshop: "Teaching and Learning in Lab Courses", University of Zürich, Switzerland

2016 - 2021 Post-Doctoral researcher

SLS/GRE, University of Dundee, Scotland, UK

Professor Julian Blow's laboratory

2017 Course: "Next Generation Sequencing Bioinformatics Advanced Course"

Wellcome Genome Campus, Hixton (Cambridgeshire, UK)

(a 6 days course)

2018 Workshop "Active Learning Methodologies", Centre for Executive Education, Glasgow Caledonian University, UK

2022 - present Post-Doctoral researcher

I2BC, Universite' Paris-Saclay, France

Dr. Dann Noordermeer's laboratory

Peer reviewed Publications: Research Article(s)

da Costa-Nunes JA, Gierlinski M, Sasaki T, Haagensen EJ, Gilbert DM, Blow JJ (2023) The location and development of Replicon Cluster Domains in early replicating DNA. Wellcome Open Research.

DOI: 10.12688/wellcomeopenres.18742.1

da Costa-Nunes JA, Gierlinski M, Sasaki T, Haagensen EJ, Gilbert DM, Blow JJ (2023) The location and development of Replicon Cluster Domains in early replicating DNA. GitHub.

https://github.com/bartongroup/MG_EarlyReplication

da Costa-Nunes JA, Gierlinski M , Sasaki T, Haagensen EJ, Gilbert DM, Blow JJ (2023) The location and development of Replicon Cluster Domains in early replicating DNA Supp Figures S1-S5. Biostudies.

https://www.ebi.ac.uk/biostudies/studies/S-BSST966

da Costa-Nunes JA*, Capitão C, Kozak J, Costa-Nunes P, Ducasa GM, Pontes O, Angelis KJ (2014) The AtRAD21.1 and AtRAD21.3 Arabidopsis cohesins play a synergistic role in somatic DNA double strand break damage repair. BMC Plant Biology 14:353

http://www.biomedcentral.com/content/pdf/s12870-014-0353-9.pdf

https://doi.org/10.1186/s12870-014-0353-9

* as corresponding author and research coordinator

da Costa-Nunes JA* (2013) A novel Arabidopsis marker line that strongly labels uninucleate microspores and the subsequent male gametophyte development stages. SpringerPlus 2:237

http://dx.doi.org/10.1186/2193-1801-2-237

* as corresponding author and research coordinator

da Costa-Nunes JA*, Viegas W (2009) An approach to screen and identify novel meiotic mutants in an (Arabidopsis) EMS mutant population. In Shu QY (ed) Induced Plant Mutations in the Genomics Era. Food and Agriculture Organization of the United Nations, Rome, 2009, pages 126-128

http://www.fao.org/docrep/012/i0956e/i0956e04.pdf

* as corresponding author and research coordinator

Kozak J, West CE, White, C, da Costa-Nunes, JA, Angelis KJ (2009) Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance. DNA Repair 8: 413-419

http://dx.doi.org/10.1016/j.dnarep.2008.11.012

http://www.sciencedirect.com/science/article/pii/S1568786408003972

da Costa-Nunes JA, Bhatt AM, O'Shea S, West CE, Bray CM, Grossniklaus U, Dickinson HG (2006) Characterization of the three Arabidopsis thaliana RAD21 cohesins reveals differential responses to ionizing radiation. Journal of Experimental Botany 57: 971-983

http://dx.doi.org/10.1093/jxb/erj083

http://jxb.oxfordjournals.org/cgi/reprint/57/4/971

Page DR, Köhler C, da Costa-Nunes JA, Baroux C, Moore JM, Grossniklaus U (2004) Intrachromosomal excision of a hybrid Ds element induces large genomic deletions in Arabidopsis. Proc. Natl. Acad. Sci. USA. 101: 2969-2974

http://dx.doi.org/10.1073/pnas.0400089101

http://www.pnas.org/content/101/9/2969.full.pdf+html

Peer reviewed Publications: Review Article(s)

da Costa-Nunes JA, Noordermeer D (2023) TADs: Dynamic structures to create stable regulatory functions. Curr Opin Struct Biol. 81: 102622.

http://dx.doi/10.1016/j.sbi.2023.102622

da Costa-Nunes JA, Grossniklaus U (2003) Unveiling the gene-expression profile of pollen. Genome Biology 5: 205.1-205.3.

http://dx.doi.org/10.1186/gb-2003-5-1-205

http://genomebiology.com/content/pdf/gb-2003-5-1-205.pdf

Seminars and contribution to oral communications in International Conferences:

J. Costa-Nunes et al. (2020) "DNA replication dynamics during early S phase; a high resolution mapping of DNA replication in human cells" (University of Dundee, 28th July 2020).

J. Costa-Nunes et al. (2018) "The replication timing and how it responds to replicative stress" (University of Dundee, 24th July 2018).

A.M. Bhatt, J. Costa-Nunes, Y. Li & H.G. Dickinson (2001) "Functional analysis of Arabidopsis cohesins" Journal of Experimental Botany 52 (March Supplement): 102 (5th European Meiosis Meeting - 2nd - 6th April)

A.M. Bhatt, J. Costa-Nunes, Y. Li, F. Osman, H.G. Dickinson, C. Lister, C. Dean, G. Jones, P. Fransz (1999) "Functional analysis of Arabidopsis cohesins - DIF1 and AtRAD21" (4th European Meiosis Meeting, 18th - 22nd September 1999)

A.M. Bhatt, J. Costa-Nunes, F. Osman, C. Lister, P. Fransz, G.J.H. Jones, J. Doonan, C. Dean & H.G. Dickinson (1999) "Meiotic roles of the Arabidopsis cohesin DIF1" (XVI International Botanical Congress, St. Louis, Missouri, U.S.A.)

... some Meeting abstracts and posters in International and UK Conferences

J.A. da Costa-Nunes, M. Gierliński, T. Sasaki, E. Haagensen, J. Abbott, D.M. Gilbert, J.J.Blow (2019) “High resolution mapping, via NGS, of DNA replication domains at the early stages of S phase”. (Gordon Conference, “Genome Architecture in Cell Fate and Disease: The Eukaryotic Genome in 4 Dimensions: Integrative Approaches to Bridging Genotype and Phenotype”, The Hong Kong University of Science and Technology, Hong Kong, China, 4th- 9th August 2019)

J.A. da Costa-Nunes, M. Gierliński, T. Sasaki, E. Haagensen, J. Abbott, D.M. Gilbert, J.J.Blow (2019) “High resolution mapping, via NGS, of DNA replication domains at the early stages of S phase”. (2nd Scottish DNA Replication Meeting, University of St. Andrews, St. Andrews, UK, 2019, 8th May 2019)

J.A. da Costa-Nunes; M. Gierliński; T. Sasaki; E. Haagensen; J. Abbott; D.M. Gilbert; J.J.Blow (2018) “NGS based analysis of DNA replication stress induced modification of DNA replication dynamics”. (GRE Symposium, University of Dundee, Dundee, UK, 2017, 26th November 2018

J.A. da Costa-Nunes; M. Gierliński; T. Sasaki; E. Haagensen; J. Abbott; D.M. Gilbert; J.J.Blow (2018) “NGS based analysis of DNA replication stress induced modification of DNA replication dynamics”. (EMBO Workshop: DNA replication, chromosome segregation and fate decisions. Kyllini, Greece, 2018, 17th - 21st September 2018)

J.A. da Costa-Nunes; M. Gierliński; T. Sasaki; E. Haagensen; J. Abbott; D.M. Gilbert; J.J.Blow (2018) “Mapping DNA replication foci”. (1st Scottish DNA Replication Meeting, University of St. Andrews, St. Andrews, UK, 2018, 30th May 2018)

J.A. da Costa-Nunes; E. Haagensen, T. Sasaki, D.M. Gilbert, J.J.Blow (2017) “High Resolution Mapping of DNA replication domains dynamics in Early S phase”. (GRE Symposium, University of Dundee, Dundee, UK, 2017, 10th November 2017)

J. A. da Costa Nunes; R.B. Ferreira & W. Viegas (2009) "Early microspore development stage histological markers for sterile and semi-sterile mutant Arabidopsis screens", - 9th IPMB Congress - Conference Program. page 109 (IPMB - St. Louis, MO, USA, 25th - 30th October 2009)

J. A. da Costa Nunes & W. Viegas (2008) "An approach to screen and identify novel meiotic mutants in an EMS mutant population" IAEA-CN-167-321P - Food and Agriculture Organisation/International Atomic Energy Agency - International Symposium on Induced Mutations in Plants. – Book of Abstracts. page 36 (ISIMP – Vienna, Austria, 12th - 15th August 2008)

J.A. da Costa-Nunes; A.M. Bhatt, S.O’Shea, C.E. West, C.M. Bray, U. Grossniklaus & H.G. Dickinson (2006) "The AtRAD21 genes and their response to DNA damaging ionising radiation" POS-THU-590 - ISPMB, 8th International Congress of Plant Molecular Biology - Book of Abstracts. page 224 (ISPMB - Adelaide, Australia, 20th - 26th August 2006)

D.R. Page, C. Köhler, J. Costa-Nunes , C. Baroux, J.M. Moore, & U. Grossniklaus (2003) " Excision of a hybrid Ds element induces large genomic deletions in Arabidopsis" (Plant Genetics 2003; October 22th-26th, 2003; Snowbird Resort & Conference Center, Snowbird, Utah)

A.M. Bhatt, J. Costa-Nunes , A. Lagodienko & H.G. Dickinson (2002) "Functional analysis of cohesins during meiosis" Comparative Biochemistry and Physiology Part A 132 : S132-S133 (Society for Experimental Biology Annual Meeting, Swansea , U.K. , 8th - 12th April 2002)

A.M. Bhatt, J. Costa-Nunes , Y. Li & H.G. Dickinson (2001) "Functional analysis of Arabidopsis cohesins" (Chromosome Dynamics & Expression, the Fourteenth John Innes Symposium, 5th - 8th September 2001)

A.M. Bhatt, J. Costa-Nunes , Y. Li & H.G. Dickinson (2001) "Functional analysis of Arabidopsis cohesins" Journal of Experimental Botany 52 (March Supplement): 102 (5th European Meiosis Meeting, 2nd - 6th April 2001)

A.M. Bhatt, J. Costa-Nunes , F. Osman, S. O'Shea, Y. Li & H.G. Dickinson (1999). " DIF1 is a homologue of the S. pombe REC8 / RAD21 cohesins, and is essential for meiosis in Arabidopsis thaliana " (Genetics of Growth & Cell Division, The Genetical Society Spring Meeting - 24th - 26th March 1999)

J. Costa-Nunes , A. M. Bhatt, M. Spielman, C. Canales, R. Scott & H.G. Dickinson. (1998) "Genes Essential for Meiosis in Arabidopsis" (Meiosis Gordon Conference, Colby-Sawyer College, New Hampshire, U.S.A. - 14th - 19th June 1998)

J. Costa-Nunes , A.M. Bhatt, S.F. O'Shea & H.G. Dickinson (1998) " DIF1 , a gene essential for meiosis, and its homologue." Journal of Experimental Biology 49 (May Supplement): 85 (Society for Experimental Biology Annual Meeting, University of York, York , U.K., 23rd - 27th March 1998)

A.M. Bhatt, J. C. Nunes , S. O'Shea, F. Osman, S. McCready & H.G. Dickinson (1998) "Functional analysis of DIF1 , an Arabidopsis gene essential for meiosis." Journal of Experimental Biology 49 (May Supplement): 81 (Society for Experimental Biology Annual Meeting, University of York, York, U.K., 23rd - 27th March 1998).

Sequence submission to public databases :

- da Costa-Nunes JA, Gierlinski M, Sasaki T, Haagensen EJ, Gilbert DM, Blow JJ (2023) The location and development of Replicon Cluster Domains in early replicating DNA. GitHub. https://github.com/bartongroup/MG_EarlyReplication

- Costa Nunes, J.A., Bhatt, A.M. and Dickinson, H.G. (2001); Database Direct Sequence Submission AF400129 Arabidopsis thaliana RAD21-3 mRNA, complete cds gi|18157648|gb|AF400129.1|AF400129[18157648]

- Costa Nunes, J.A., Bhatt, A.M. and Dickinson, H.G. (2001); Database Direct Sequence Submission AF400128 Arabidopsis thaliana RAD21-2 mRNA, complete cds gi|18157646|gb|AF400128.1|AF400128[18157646]

- Costa Nunes, J.A., Bhatt, A.M. and Dickinson, H.G. (2001); Database Direct Sequence Submission AF400127 Arabidopsis thaliana RAD21-1 variant 2 mRNA, complete cds gi|18157644|gb|AF400127.1|AF400127[18157644]

- Costa Nunes, J.A., Bhatt, A.M. and Dickinson, H.G. (2001); Database Direct Sequence Submission AF400126 Arabidopsis thaliana RAD21-1 variant 1 mRNA, complete cds gi|18157642|gb|AF400126.1|AF400126[18157642]

2008 - 2011 - Research project

(funded by the Portuguese Research Funding Agency - FCT)

PTDC/BIA-BCM/64192/2006 - Chromosome Cohesion and DNA repair in Arabidopsis Funding: €52.000

(one of the 19% top proposals selected for funding, out of a total of 163 proposals - on Molecular and Cellular Biology - external funding)

Principal Investigator of the Project - José A. M. da Costa Nunes

Graduate Education and some relevant traineeships

1994-1998 Undergraduate degree (Agronomy Engineering; Plant breeding) (a 5 years degree),

Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Portugal

1994-1998 MSc degree (Plant Biotechnology),

Faculdade de Ciências, Universidade de Lisboa, Portugal

1996 Traineeship at I.G.E.R.

Institute for Grassland and Environmental Research, Aberystwyth, Wales, UK

1997-2001 DPhil (Oxon) (PhD degree),

Department of Plant Sciences, University of Oxford, UK

Supervisor: Professor Hugh Dickinson

1999 Course: "Arabidopsis Molecular Genetics" Cold Spring Harbour Course

Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y., USA

(a 21 days course)

Relevant Post-Doc Research training (Plant Sciences) and some other relevant courses and workshops attended :

2001 Short-term Post-Doc,