Aberrant epigenome in iPSC-derived dopaminergic neurons from Parkinson's disease patients

The epigenomic landscape of Parkinson's disease (PD) remains unknown. We performed a genomewide DNA methylation and a transcriptome studies in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) generated by cell reprogramming of somatic skin cells from patients with monogenic LRRK2-associated PD (L2PD) or sporadic PD (sPD), and healthy subjects. We observed extensive DNA methylation changes in PD DAn, and of RNA expression, which were common in L2PD and sPD. No significant methylation differences were present in parental skin cells, undifferentiated iPSCs nor iPSC-derived neural cultures not-enriched-in-DAn. These findings suggest the presence of molecular defects in PD somatic cells which manifest only upon differentiation into the DAn cells targeted in PD. The methylation profile from PD DAn, but not from controls, resembled that of neural cultures not-enriched-in-DAn indicating a failure to fully acquire the epigenetic identity own to healthy DAn in PD. The PD-associated hypermethylation was prominent in gene regulatory regions such as enhancers and was related to the RNA and/or protein downregulation of a network of transcription factors relevant to PD (FOXA1, NR3C1, HNF4A, and FOSL2). Using a patient-specific iPSC-based DAn model, our study provides the first evidence that epigenetic deregulation is associated with monogenic and sporadic PD

The role of genome architecture in normal and neoplastic B cells: a multi-omics approach

[eng] Nuclear organization and its impact on gene regulation have started to be elucidated thanks to the development of chromosomal conformation capture techniques. In this doctoral thesis, in situ Hi-C data and nine additional omic layers have been integrated to define and biologically characterize the dynamic changes in three-dimensional (3D) genome architecture across normal B-cell differentiation and in neoplastic cells from chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) patients. Beyond the classical genome organization into active and inactive compartments, a third intermediate 3D compartment enriched in poised and polycomb-repressed chromatin has been identified (Study 1). It has been observed that during B-cell differentiation, a 28% of the 3D genome structure changed, being an extensive activation from naive to germinal center B cells and a reversal into a naive-like 3D genome upon further maturation into memory B cells the most remarkable features. In case of neoplastic B cells, both CLL and MCL displayed entity and subtype-specific alterations in chromosome organization. Those alterations comprised large chromatin blocks containing key disease-specific genes such as EBF1 in CLL or SOX11 in MCL. Chromosomal conformation maps were also assessed to define structural variants in neoplastic cells (Study 2). The t(11;14) translocation in MCL cases leading to CCND1 deregulation was associated with a breakpoint-dependent 3D chromatin reconfiguration creating new topologically associating domain (TAD) borders. Moreover, additional rearrangements have been identified by Hi-C in MCL and have been confirmed by standard methods such as cytogenetic analyses and next generation sequencing. An integrative multi-omics approach combining 3D genome architecture with histone modifications and DNA methylation allowed the identification of candidate epigenetic drivers in MCL (Study 3). In aggressive, conventional MCLs with overexpression of the SOX11 oncogene, a distant regulatory region looping to the SOX11 promoter was uncovered promoting oncogene deregulation in a biallelic fashion. Additional experiments revealed that the PAX5 transcription factor may play a role in activating the distant SOX11 enhancer (Study 4). This doctoral thesis highlights the role of the chromatin architecture as a key epigenomic player associated with the normal differentiation and neoplastic transformation of B cells

The role of genome architecture in normal and neoplastic B cells: a multi-omics approach

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut de Investigació Biomèdica de Bellvitge (IDIBELL)[eng] Nuclear organization and its impact on gene regulation have started to be elucidated thanks to the development of chromosomal conformation capture techniques. In this doctoral thesis, in situ Hi-C data and nine additional omic layers have been integrated to define and biologically characterize the dynamic changes in three-dimensional (3D) genome architecture across normal B-cell differentiation and in neoplastic cells from chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) patients. Beyond the classical genome organization into active and inactive compartments, a third intermediate 3D compartment enriched in poised and polycomb-repressed chromatin has been identified (Study 1). It has been observed that during B-cell differentiation, a 28% of the 3D genome structure changed, being an extensive activation from naive to germinal center B cells and a reversal into a naive-like 3D genome upon further maturation into memory B cells the most remarkable features. In case of neoplastic B cells, both CLL and MCL displayed entity and subtype-specific alterations in chromosome organization. Those alterations comprised large chromatin blocks containing key disease-specific genes such as EBF1 in CLL or SOX11 in MCL. Chromosomal conformation maps were also assessed to define structural variants in neoplastic cells (Study 2). The t(11;14) translocation in MCL cases leading to CCND1 deregulation was associated with a breakpoint-dependent 3D chromatin reconfiguration creating new topologically associating domain (TAD) borders. Moreover, additional rearrangements have been identified by Hi-C in MCL and have been confirmed by standard methods such as cytogenetic analyses and next generation sequencing. An integrative multi-omics approach combining 3D genome architecture with histone modifications and DNA methylation allowed the identification of candidate epigenetic drivers in MCL (Study 3). In aggressive, conventional MCLs with overexpression of the SOX11 oncogene, a distant regulatory region looping to the SOX11 promoter was uncovered promoting oncogene deregulation in a biallelic fashion. Additional experiments revealed that the PAX5 transcription factor may play a role in activating the distant SOX11 enhancer (Study 4). This doctoral thesis highlights the role of the chromatin architecture as a key epigenomic player associated with the normal differentiation and neoplastic transformation of B cells

Dandelion uses the single-cell adaptive immune receptor repertoire to explore lymphocyte developmental origins.

Acknowledgements: We acknowledge the Cellular Genetics IT, New Pipeline Group and DNA pipelines of Sanger Institute. K.B.M. and S.A.T. are supported by Wellcome (WT211276/Z/18/Z, 108413/A/15/D, Sanger core grant WT206194 and Sanger QQ award 220540/Z/20/A). K.B.M. acknowledges funding from the MRC (MR/S035907/1). M.H. is supported by Wellcome (grant WT107931/Z/15/Z), the Lister Institute for Preventive Medicine, NIHR, and the Newcastle Biomedical Research Centre. S.A.T. is supported by an ERC Consolidator Grant ThDEFINE (646794). C.S. is supported by a Wellcome Trust Ph.D. Fellowship for Clinicians. Z.K.T. and M.R.C. are supported by a Medical Research Council Research Project Grant (MR/S035842/1). M.R.C. is supported by the National Institute of Health Research (NIHR) Research Professorship (RP-2017-08-ST2-002), a Wellcome Investigator Award (220268/Z/20/Z), the Blood and Transplant Research Unit in Organ Donation and the NIHR Cambridge Biomedical Research Centre. This publication is part of the Human Cell Atlas (www.humancellatlas.org/publications). We would like to thank the reviewers for their thoughtful comments and suggestions, which helped us to improve the quality of the manuscript.Assessment of single-cell gene expression (single-cell RNA sequencing) and adaptive immune receptor (AIR) sequencing (scVDJ-seq) has been invaluable in studying lymphocyte biology. Here we introduce Dandelion, a computational pipeline for scVDJ-seq analysis. It enables the application of standard V(D)J analysis workflows to single-cell datasets, delivering improved V(D)J contig annotation and the identification of nonproductive and partially spliced contigs. We devised a strategy to create an AIR feature space that can be used for both differential V(D)J usage analysis and pseudotime trajectory inference. The application of Dandelion improved the alignment of human thymic development trajectories of double-positive T cells to mature single-positive CD4/CD8 T cells, generating predictions of factors regulating lineage commitment. Dandelion analysis of other cell compartments provided insights into the origins of human B1 cells and ILC/NK cell development, illustrating the power of our approach. Dandelion is available at https://www.github.com/zktuong/dandelion

Robust temporal map of human in vitro myelopoiesis using single-cell genomics

We thank the Cellular Genetics wet lab support team, Cellular Genetics IT team, Sanger Sequencing operations and Sanger Cytometry Core facility for their essential help. We thank the Gene Editing team for providing iPSC knock-out lines. We would also like to thank Ruxandra Tesloianu and Luz Garcia-Alonso for their help setting up the scATAC-seq computational analysis. We thank Jana Eliasova for her help with figure design and Christina Usher and Aidan Maartens for their edits in the text. This work was mainly funded by the Open Targets consortium (OTAR026 and OTAR032 project) and the Wellcome Sanger core funding (WT206194) with additional support from Open Targets projects OTAR037, OTAR2065, OTAR2071. The authors are grateful to the funders for their support and additional care given to their members during the COVID-19 pandemic. D.A.-E. thanks CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. This study makes use of cell lines and data generated by the HiPSci Consortium, funded by The Wellcome Trust and the MRC (Medical Research Council). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This publication is part of the Human Cell Atlas- www.humancellatlas.org/publications.We thank the Cellular Genetics wet lab support team, Cellular Genetics IT team, Sanger Sequencing operations and Sanger Cytometry Core facility for their essential help. We thank the Gene Editing team for providing iPSC knock-out lines. We would also like to thank Ruxandra Tesloianu and Luz Garcia-Alonso for their help setting up the scATAC-seq computational analysis. We thank Jana Eliasova for her help with figure design and Christina Usher and Aidan Maartens for their edits in the text. This work was mainly funded by the Open Targets consortium (OTAR026 and OTAR032 project) and the Wellcome Sanger core funding (WT206194) with additional support from Open Targets projects OTAR037, OTAR2065, OTAR2071. The authors are grateful to the funders for their support and additional care given to their members during the COVID-19 pandemic. D.A.-E. thanks CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. This study makes use of cell lines and data generated by the HiPSci Consortium, funded by The Wellcome Trust and the MRC (Medical Research Council). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This publication is part of the Human Cell Atlas-www.humancellatlas.org/publications.Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities

Aberrant epigenome in iPSC-derived dopaminergic neurons from Parkinson's disease patients

The epigenomic landscape of Parkinson's disease () remains unknown. We performed a genomewide methylation and a transcriptome studies in induced pluripotent stem cell ()-derived dopaminergic neurons (n) generated by cell reprogramming of somatic skin cells from patients with monogenic 2-associated (L2) or sporadic (), and healthy subjects. We observed extensive methylation changes in n, and of expression, which were common in L2 and . No significant methylation differences were present in parental skin cells, undifferentiated s nor -derived neural cultures not-enriched-in-n. These findings suggest the presence of molecular defects in somatic cells which manifest only upon differentiation into the n cells targeted in . The methylation profile from n, but not from controls, resembled that of neural cultures not-enriched-in-n indicating a failure to fully acquire the epigenetic identity own to healthy n in . The -associated hypermethylation was prominent in gene regulatory regions such as enhancers and was related to the and/or protein downregulation of a network of transcription factors relevant to (1, 3C1, 4A, and 2). Using a patient-specific -based n model, our study provides the first evidence that epigenetic deregulation is associated with monogenic and sporadic PD

Aberrant epigenome in -derived dopaminergic neurons from Parkinson's disease patients

The epigenomic landscape of Parkinson's disease () remains unknown. We performed a genomewide methylation and a transcriptome studies in induced pluripotent stem cell ()-derived dopaminergic neurons (n) generated by cell reprogramming of somatic skin cells from patients with monogenic 2-associated (L2) or sporadic (), and healthy subjects. We observed extensive methylation changes in n, and of expression, which were common in L2 and . No significant methylation differences were present in parental skin cells, undifferentiated s nor -derived neural cultures not-enriched-in-n. These findings suggest the presence of molecular defects in somatic cells which manifest only upon differentiation into the n cells targeted in . The methylation profile from n, but not from controls, resembled that of neural cultures not-enriched-in-n indicating a failure to fully acquire the epigenetic identity own to healthy n in . The -associated hypermethylation was prominent in gene regulatory regions such as enhancers and was related to the and/or protein downregulation of a network of transcription factors relevant to (1, 3C1, 4A, and 2). Using a patient-specific -based n model, our study provides the first evidence that epigenetic deregulation is associated with monogenic and sporadic PD

Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype