ESR 1: Patrick Lim
The role of SET in ESCs and search of novel chromatin regulators of pluripotency

The Hebrew University of Jerusalem, Jerusalem, Israel

(supervisor: Eran Meshorer)

The nuclear oncogene SET (TAF1) was shown by the Meshorer lab to undergo alternative promoter switching whereby the expression of one isoform, SETɑ, decreases and another isoform, SETβ, increases upon differentiation in mouse embryonic stem cells (mESCs) (Harikumar et al, 2017). In the Meshorer lab, I will pursue this line of inquiry further by characterising the interactions between SET and its binding partners. By performing ChIP-seq for P53, I hope to uncover a change in P53 promoter target sites that may be responsible for altered lineage choice in SET KOs. In the lab, I will learn to conduct experiments in tissue culture and how to collect data from sequencing experiments. During the first EpiSyStem workshop, I was exposed to bioinformatics methods and techniques which I will apply to research and analyses of data I collect in future experiments.

ESR 2: Juliane Viegas
The Role of Caprin1 in Pluripotency and ESC Differentiation

The Hebrew University of Jerusalem, Jerusalem, Israel

(supervisor: Eran Meshorer)

Embryonic stem cells (ESC) retain a dual capacity to maintain in a self-renewal stage and to differentiate into any cell type in the body. ESCs are regulated by a set of transcription factors (TFs) that are responsible for their function. Currently, several nuclear pluripotency regulators of ESCs have been identified and studied, however cytoplasmic regulators of pluripotency are scarce. The Meshorer Lab has previously generated an endogenously-labelled fluorescent fusion-protein library in mouse ESCs (Harikumar et al., 2017). To reveal novel non-nuclear regulators of ESCs, the library was used to search for factors that are down-regulated during differentiation. As a result, CAPRIN1 (CAPR1) was identified as a potential novel regulator of stem cell function.

My research aims to reveal the function of CAPR1 in the maintenance of pluripotency and ESC early differentiation. CAPR1 is a cell cycle associated, conserved, cytoplasmic protein and a major RNA binding component in RNA granules.

ESR 3: Joe Verity-Legg
Developing a method for simultaneous, single cell whole genome bisulphite sequencing and histone modification localisation

Hubrecht Institut, Utrecht, the Netherlands

(Supervisor: Alexander van Oudernaaden)

It is well known that all cells in multi-cellular organisms carry largely identical genomes. However, in complex organisms a wide breadth and complexity of cellular functions exists. This variability, established during development of the organism from a single zygotic cell to a multicellular adult, is thought to be controlled via epigenetic mechanisms. These mechanisms include the establishing of genome wide DNA methylation patterns, a chemical modification of nucleotide bases themselves, and modifications of the histones (by the histone tails) that comprise the nucleosome and form the foundation of the supramolecular structure of the genome.

At current, I am working on establishing a method for assessing the spatial relationship between both histone modifications and methylation in single cells. The method proposed targets antibodies to a given histone modification before tethering a GpC Methyltransferase fusion protein to this antibody. When activated the enzyme will methylate DNA in a context not normally present in humans (GpC). This may be achieved in bulk, a key potential advantage of the method both in terms of throughput and sample loss prevention, before sorting single cells and following established single cell, whole genome bisulphite sequencing protocols.

ESR 4: Helena Viñas Gaza
Analysis of the methylome, the single nucleotide variations and the chromatin modifications along the mouse intestine

Hubrecht Institut Utrecht, the Netherlands

(Supervisor: Alexander van Oudernaaden)

The small intestinal epithelium has a characteristic organization with crypt-villus units. The crypts are invaginations containing several different types of epithelial cells. At the bottom of these crypts, stem cells rapidly proliferate to produce the progenitors that will finally give rise to clonal populations of differentiated cells. This epithelium is replaced every 3 to 5 days in adult mammals. The process of differentiation is tightly regulated and many elements are involved. Epigenetic factors, such as DNA methylation and chromatin modifications, may be associated with the differentiation process. The correlation between the methylation state of genes and changes in their expression during differentiation is still elusive. We hypothesise that the changes in methylation play a role in the cell differentiation into one of the lineages.

In the context of chromatin, DNA methylation is not the only epigenetic factors to take into consideration. There is a complex relationship between DNA methylation and histone modifications, such as ubiquitylation, methylation and acetylation. DNA methylation is usually found in heterochromatin regions but decreased at enhancers and active promoters. Whether or not we can use DNA methylation as a good indicator of high nucleosome occupancy is still debatable. In order to establish a correlation between the methylation and chromatin accessibility we are going to use a modified technique of the chromatin immunocleavage (ChIC) method established by Schmid et al. to investigate and compare different chromatin modifications, such as H3K27me and H3K9me, in the duodenum, jejunum and ilium of mouse intestine. The aim of the project is to be able to characterize the epigenetic profile of the mouse intestine to better understand its clonality and development at a single cell level.

ESR 5: Velin Sequiera
Identifying the epigenetic readers in mouse

Radboud University, Neimijen, the Netherlands

(Supervisor: Michiel Vermeulen)

Epigenetic modifications, such as DNA methylation and histone modifications, alter DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. DNA methylation mostly occurs in a CpG dinucleotide context and involves the covalent addition of a methyl group at the 5-carbon position of cytosine (5mC), catalyzed by DNA methyltransferases (DNMTs). DNA demethylation is mediated by Ten-eleven translocation (TET) enzymes that successively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other derivatives, to eventually derive an unmodified cytosine. Dynamic changes in DNA methylation and demethylation orchestrate the transcriptional network during developmental processes like lineage specification. Alterations in these pathways are implicated in cancer. Cytosine methylation. However, CpG islands (CGIs), characterized by a high CpG density, are often found in promoter regions of genes and are typically hypomethylated. Although methylation of CGIs is canonically associated with transcriptional silencing, recent research has provided evidences refuting this claim. Therefore, molecular mechanisms underlying the association between DNA methylation and regulation of gene expression have proven difficult to decipher.

I will employ quantitative interaction proteomics assay to identify readers of developmentally regulated methylated regions in mouse ESCs. Identified high affinity readers will then be functionally validated using CUT&RUN sequencing. Their potential importance for stem cell identity and lineage commitment will be investigated using perturbation experiments (over-expression and/or CRISPR/Cas9 mediated knockout). This workflow will also be applied to identify and functionally analyze methylation readers in differentiated cells (e.g., neural progenitor cells, intestinal stem cells) which will reveal molecular players pivotal for cellular identity and function. Furthermore, this technique will be employed to highlight methylation readers of cancer-specific methylated promoters to unravel dysregulated pathways in cancer. This technique will be potentially combined with single-locus proteomics (on-going Vermeulen Lab) for studying molecular components involved in dynamic regulation of in vivo activity at a given chromosomal location. Eventually, this protein-DNA binding assay will be optimized to identify readers of oxidized derivatives like hydroxymethylated regions. Thus, the project overall aims to molecularly dissect how ‘readers’ of the epigenome instruct the transcriptional program, thereby coordinating cell-fate dynamics.

ESR 6: Adrianos Skaros
iPSCs in Self-Domestication and neurodevelopmental disorders

University of Milan, Milan, Italy

(Supervisor: Giuseppe Testa)

My aim is probing experimentally the human self-domestication syndrome of Williams-Beuren syndrome and 7q11.23 duplication syndrome Starting point: Detect highly significant overlaps between 7q11.23 duplication syndrome CNV-dependent dysregulation in cortical neuronal and neural crest lineages, and the genetic signature distinguishing AMH from archaic hominins.

Part A

  1. Computational and literature-based prioritization of key targets 
  2. Investigate the circuitries distinguishing Anatomically Modern Humans (AMH) from Archaic hominins at the genetic level: Cas9-based Neanderthalisation of human pluripotent lineages carrying top-ranking individual changes (N-iPSC)
  3. Differentiation of aforementioned N-iPSCs into neural crest stem cells and brain cortical organoids
  4. Deconvolution of self-domestication-relevant developmental trajectories through single-cell transcriptomics

Part B

  1. Computational and literature-based prioritization of key targets
  2. Utilise specific KD- and KO-cell lines derived from neurodevelopmental patients (7dup) and differentiate them into NCSCs and Brain organoids
ESR 7: Marlene Pereira
Patient-derived disease modelling as a tool to uncover the molecular pathogenesis of neurodevelopmental disorders

University of Milan, Milan, Italy

(Supervisor: Giuseppe Testa)

Being multifaceted conditions characterized by impairments in cognition, communication, behaviour and motor skills, neurodevelopmental disorders (NDDs) result from abnormal CNS development. Our poor understanding of the molecular pathogenesis of NDDs led researchers to look for alternative methodologies to study the mechanisms underlying these disorders. A breakthrough in human disease research over the last decade has emerged from cellular reprogramming technologies, using reprogrammed patient somatic cells because it captures a patient’s genome in a pluripotent stage allowing us to study, for the first time, the initial development and progression of pathology in live human cells in a controlled environment.

I aim to dissect the molecular pathogenesis of a group of NDDs using patient-specific induced pluripotent stem cells (iPSCs) generated from skin biopsies of affected patients and matched unaffected relatives as well as 3D cortical organoids to functionally dissect the impact and variability of these disorders. We will use a large cohort of iPSC lines from two different paradigmatic neurodevelopmental disorders such as Weaver Syndrome (WS) and Kabuki Syndrome (KS) caused by mutations, respectively, in EZH2 (catalysing H2K27 tri-methylation) and KMT2D or KDM6A (catalysing H3K4 mono-methylation and di- and trimethyl H3K27 demethylation, respectively), as well as Gabriele-DeVries syndrome (GADVES) characterized by mutations in YY1 (mediates gene activation interacting with the chromatin remodelling complex INO80).

By using disease-modelling as a tool to investigate the pathogenesis of NDDs across different time points, tissues and preparations will allow us to gather new insights about the molecular pathogenesis of these disorders.

ESR 9: Marion Gennet
Characterization of early-embryonic like cells in vitro

Helmholz Centrum, Munich Germany

(Supervisor: Maria-Elena Torres-Padilla)

From the zygote to the adult organism, one totipotent cell divides until its daughters differentiate into the hundreds of cell types composing the body. In the first days of embryonic development a crucial step occurs: the transition from a totipotent to a pluripotent state. In vivo, this transition is one-way only and our current understanding of this process is still incomplete.

Currently, there is no good model to characterize the totipotent state in vitro, while pluripotency is extensively studied in embryonic and induced pluripotent stem cells. During my PhD project, I will investigate the occurrence and mechanisms underlying the reprogramming of such 2C-like cells in mouse embryonic stem cells (ES) as well as in mouse iPSCs, which should help establish new culture models for studying totipotency in vitro. This can have applications in both our basic understanding of the early embryo development and the improvement of current regenerative strategies.

ESR 11: Stefano Arfè
Dynamics of histone chaperones & histone variants in defining chromosomal landmarks.

Institut Curie, Paris, France

(Supervisor: Geneviève Almouzni)

The aim of my work is to investigate histone dynamics using ChIP-seq, and state-of-the-art imaging approaches, including single molecule tracking by photo-activated localization microscopy (smtPALM) and stochastic optical reconstruction microscopy (STORM).

These mechanisms will be elucidated in physiological conditions and following differentiation during DNA replication and DNA repair. I will characterize how dosage imbalances of histone chaperones and histone variants impinge on embryonic stem (ES) cell fate transition.

I will perform these studies in engineered mouse ES cells lines expressing modified version of histone H3 variants conjugated with the SNAP-tag system. The SNAP-tag system is a powerful tool that allows to track at single molecule level the distribution of histone variants. With this technology, detection of old histone recycling and new histone deposition could be detected with a temporal resolution of less than one second and over several cell divisions. We can follow these dynamics with two different approaches: combining SNAP-tag system with high-throughput sequencing (SNAP-ChIP-seq) and with the STORM.

ESR 12: Pinar Accor
Understanding the role of H3K27 tri- and di-methylation in ESCs

Diagenode, Liège, Belgium

(Supervisor: Michiel Vermeulen)

I aim to develop an optimized method compatible with a kit format for the isolation of chromatin from fixed cells for mass-spectrometry analysis of chromatin-associated proteins (histones PTMs included). A few pilot experiments were performed. Initial tests were carried out on HeLa cells using H9K9me3 antibody (Diagenode, cat# C15410193) and iDeal ChIP-seq kit for transcriptional factors (with minor modifications) (Diagenode, cat# C01010055) followed by PAGE and in-gel trypsin digestion (similar to ChroP approach, T. Bonaldi , 2014) and mass-spectrometry analysis (GIGA MS-facility). We concluded that this workflow (PAGE + in gel digestion) is barely compatible with a commercial product (long, not a user-friendly, loss of material etc). Second, this approach does not allow screening for all proteins associated with chromatin. Moreover, identification of PTMs requires advanced skills.

We have identified ~1400 proteins. We were able to identify 365 from 543 known CTCF interactors recorded in the CORUM database (fig.2 and 3). However, it is difficult to assess which proteins might be potential interactors and which ones are just background binders. We believe that ChIP protocol has to be optimized further in order to reduce the background. The data analysis and interpretation will be done with mass spectrometry in collaboration with Dr Michiel Vermeulen’s lab and Radboud University.

ESR 14: Xue Sun
Regulation and Heterogeneity Analysis of Epigenomic States in Human Cellular Differentiation

The Hebrew University of Jerusalem, Jerusalem, Israel

(Supervisor: Oren Ram)

Chromatin modifications are key players of cell state regulation, some of which may be epigenetically inherited. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used method for mapping histone modifications, transcription factors and chromatin regulators interactions genome-wide. In previous study, a single-cell ChIP-seq platform has been developed for profiling chromatin at single-cell resolution, which combines drop-based microfluidics with genomic barcoding. This platform has overcome the limitation of the conventional methods, which yields averaged ‘ensemble’ profiles that are insensitive to internal heterogeneity. However, this method has a major shortcoming as single-cell chromatin maps are highly sparse, with only about a thousand peaks detected in each individual cell. As a result, single cell ChIP-seq data are highly noisy and difficult to interpret with low sensitivity.

My aim is to develop the Clone ChIP-seq to expand our technological toolbox and to overcome the sensitivity limitation of single cell assays. Before the setup of Clone ChIP-seq, we will first need to study the preservation of clonal similarity at early stage of cell expansion. Then we will move on to the setup of Clone ChIP-seq technology. Eventually, we could investigate numbers of unsolved biological questions with existing methods using our new method. It will enable a much better sensitivity with a small number of cells input, so we could identify active regulatory networks in rare cell populations4. We can also get a clearer chromatin markers map across different tissues, cell types, and developmental stages.