Single-cell transcriptional logic of cell-fate specification and axon guidance in early-born retinal neurons

Retinal ganglion cells (RGCs), cone photoreceptors (cones), horizontal cells and amacrine cells are the first classes of neurons produced in the retina. However, an important question is how this diversity of cell states is transcriptionally produced. Here, we profiled 6067 single retinal cells to provide a comprehensive transcriptomic atlas showing the diversity of the early developing mouse retina. RNA velocities unveiled the dynamics of cell cycle coordination of early retinogenesis and define the transcriptional sequences at work during the hierarchical production of early cell-fate specification. We show that RGC maturation follows six waves of gene expression, with older-generated RGCs transcribing increasing amounts of guidance cues for young peripheral RGC axons that express the matching receptors. Spatial transcriptionally deduced features in subpopulations of RGCs allowed us to define novel molecular markers that are spatially restricted. Finally, the isolation of such a spatially restricted population, ipsilateral RGCs, allowed us to identify their molecular identity at the time they execute axon guidance decisions. Together, these data represent a valuable resource shedding light on transcription factor sequences and guidance cue dynamics during mouse retinal development.

The role of OCT4 and SOX2 in regulating chromatin accessibility and cell fate across the cell cycle in embryonic stem cells

Torgil Elias Friman

Precise spatiotemporal regulation of gene expression is essential for development and homeostasis of complex organisms. This is achieved in large part by sequence-specific transcription factors (TF) that bind to genomic regulatory elements to activate or repress transcription. DNA in eukaryotic nuclei is wrapped around histones into nucleosomes, which are inaccessible to most proteins. However, certain TFs can access their binding sites in the presence of histones and promote nucleosome eviction to increase chromatin accessibility, allowing for binding of other protein. These TFs, called pioneer factors, include OCT4 and SOX2, which are master regulators of embryonic stem (ES) cells. Dividing cells, including stem cells, must achieve proper gene regulation despite the progression of the cell cycle, which imposes several constraints. During S phase, the replication fork passes through the genome to make a copy of each chromosome, which leads to a loss of chromatin accessibility. Furthermore, the substantial chromatin compaction that occurs during mitosis to prepare for chromosome segregation is associated with eviction of most DNA-binding proteins and reduced accessibility at distal cis-regulatory elements including enhancers. How pioneer factors operate in the context of cell cycle progression is unclear. In this thesis, I will present work studying different features of OCT4 and SOX2, including how they interplay to regulate chromatin accessibility in ES cells, the impact of small endogenous fluctuations of their protein levels on cell fate and chromatin accessibility, and how their roles in regulating cell fate and chromatin accessibility is related to different stages of the cell cycle. Using live-cell imaging of fluorescently labeled proteins, we discovered that OCT4 and SOX2 were associated to mitotic chromatin. Using cell-cycle specific degradation strategies, we could show that the presence of SOX2 and OCT4 at the mitosis-G1 (M-G1) transition contributes to maintain the pluripotency of ES cells. We further showed that ES cells with high levels of OCT4 in G1, but not in S phase, are more prone to differentiate towards neuroectoderm and mesendoderm cell fates. Cells with high levels of OCT4 displayed increased chromatin accessibility at enhancers of differentiation genes, suggesting accessibility fluctuates with OCT4 levels and can prime cells for differentiation. To explore potential cell cycle-dependent regulation of chromatin accessibility, we degraded OCT4 at the M-G1 transition, which led to a reduction in accessibility at many OCT4-bound regions. When OCT4 returned later in the cell cycle, chromatin accessibility was recovered, although at many loci this recovery was incomplete, suggesting that OCT4 is required at the M-G1 transition to maintain these regions open. We then rapidly degraded OCT4 at other phases of the cell cycle. Surprisingly, loss of chromatin accessibility was highly similar at all cell cycle phases, showing that OCT4 is constantly required to maintain regulatory elements accessible. To explore the dynamic regulation of accessibility by OCT4, we performed time-course measurements of accessibility upon its degradation. This revealed that loss of accessibility was temporally correlated to the degradation of OCT4. These results suggest that chromatin accessibility is highly dynamic and continuously dependent on the presence of OCT4 across the cell cycle in ES cells.

EPFL2019

KRAB zinc-finger proteins and their transposable element targets: between antagonism and cooperation

Jonas Caspar De Tribolet-Hardy

There are 377 Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KZFPs) in the human genome, making them the largest family of transcription factors. KZFPs are defined by a N-terminal KRAB domain and several zinc-finger domains arranged in an array at the C-terminus of the proteins. The zinc-finger domains each form sequence specific interactions with double stranded DNA, allowing the zinc-finger array to target specific genomic sequences. The KRAB domain, through its interaction with the protein TRIM28 (also known as KAP1) allows for the stable silencing of transcription in a genomic region. Together these two domains allow KZFPs to bind specific regions of the genome and generally lead to the formation of heterochromatin and silencing of transcription allthough other functions for some KZFPs have been recently reported. KZFPs usually target transposable elements (TEs), mobile genomic elements that can move through the genome either by cut-and-paste or copy-paste mechanisms and make up almost half of the human genome. Different KZFPs bind to specific regions of TEs, limiting their expression and thus mitigating some of the threat they pose to human development, allowing both the TE and its host to survive. In recent years it became apparent that both certain TEs and KZFPs have roles beyond the previously described threat and mitigation of that threat. TEs harbor gene regulatory regions allowing, through their spread, for a dissemination of those regions through the genome and for new gene regulatory networks to form. KZFPs can affect the transcription of genes located in proximity to their binding sites leading to changes in gene regulation as well. A multitude of regulatory roles involving KZFPs and TEs have been described in recent years making them an exciting field of study. A major hurdle in understanding both KZFPs and TEs is to identify the binding sites of KZFPs, as they reveal both the targets of the KZFP and how, if at all, a TE is regulated by KZFPs. To do so, we expanded on previous efforts and aimed to perform chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on every member of the human KZFP family. Here we present ChIP-seq experiments for 110 new KZFPs, which together with previously published data, allow us to study the binding of almost all human KZFPs (95%). The entirety of the data was analysed together to generate a coherent dataset and results for each KZFP are made available to the public on our web portal (https://tronoapps.epfl.ch/web/krabopedia/). The identified binding sites allowed us to witness the adaptation of the KZFP family to new TEs and showed how targeted sequences shift after segmental gene duplication events. Furthermore, we could corroborate that several KZFPs target the same TE subfamilies in a seemingly redundant fashion and show that unexpectedly these KZFPs arose independently in different genomic locations. These results represent a valuable tool for anyone studying KZFPs and should aid in the pursuit of both understanding KZFPs and TEs.

EPFL2022

A Plasmodium falciparum transcriptional CDK-related kinase with a crucial role in parasite proliferation associates with histone deacetylase activity

Cyclin-dependent protein kinases (CDKs) are key regulators of the eukaryotic cell cycle and of the eukaryotic transcription machinery. Here, we report the characterisation of Pfcrk-3 (P. falciparum CDK-related kinase 3, PlasmoDB identifier PFD0740w), an unusually large CDK-related protein whose kinase domain displays maximal homology to those CDKs which are, in other eukaryotes, involved in the control of transcription. The closest enzyme in yeast is BUR1 ("Bypass Upstream activating sequence Requirement"), known to control gene expression through interaction with chromatin modification enzymes. Consistent with this, immunofluorescence data show that Pfcrk-3 colocalises with histones. We show that recombinant Pfcrk-3 associates with histone H1 kinase activity in parasite extracts, and that this association is detectable even if the catalytic domain of Pfcrk-3 is rendered inactive by site-directed mutagenesis, indicating that Pfcrk-3 is part of a complex that includes other protein kinases. Immunoprecipitates obtained from extracts of transgenic parasites expressing HA-tagged Pfcrk-3 using a hemagglutinin (HA) antibody displayed both protein kinase and histone deacetylase activities. Reverse genetics data show that the pfcrk-3 locus can be targeted only if the genetic modification does not cause loss-of-function. Taken together, our data strongly suggests that Pfcrk-3 fulfils a crucial role in the intraerythrocytic development of P. falciparum, presumably through chromatin modification-dependent regulation of gene expression.