Biology subjects

Brickman, J. M.

Publications and source records attributed to Brickman, J. M..

15 recordsLinked to original sources

bioRxiv2026

Tethering-mediated recruitment of pioneer factors by NuRD enables signal dependent gene activation and protects cell identity

Cell identity relies on precise control of transcription factor (TF) activity, localization and interpretation of signaling. Mitogen-activated protein kinase (MAPK) signaling regulates transcription via the ERK kinase, phosphorylating components of the Mediator complex to regulate its association with TF-bound enhancers. Here we describe two distinct forms of TF association related to signaling response, stable DNA recognition and tethering to active sites of transcription. We find that the tethering of TFs at these active sites is both essential for signaling response and cell identity. To identify tethering substrates, we focused on signaling dependent associations with the transcriptional coregulator, the Mediator, and find the NuRD complex is engaged with the Mediator at active promoters and enhancers. Here, NuRD functions as a molecular scaffold recruiting pioneer TFs like SOX2 independently of DNA sequence, enhancing ERK dependent transcription while sequestering these factors away from context inappropriate binding sites elsewhere in the genome. Balancing the recognition of a restricted spectrum of canonical DNA sites by pioneer factors while retaining them at high concentrations in the vicinity of transcription supports robust transcriptional response, ensures plasticity in differentiation and explains why these factors bind a fraction of their naturally occurring sites in any given cell type.

molecular biology↗

bioRxiv2024

Cells transit through a quiescent-like state to convert to neurons at high rates

While transcription factors (TFs) provide essential cues for directing and redirecting cell fate, TFs alone are insufficient to drive cells to adopt alternative fates. Rather, transcription factors rely on receptive cell states to induce novel identities. Cell state emerges from and is shaped by cellular history and the activity of diverse processes. Here, we define the cellular and molecular properties of a highly receptive state amenable to transcription factor-mediated direct conversion from fibroblasts to induced motor neurons. Using a well-defined model of direct conversion to a post-mitotic fate, we identify the highly proliferative, receptive state that transiently emerges during conversion. Through examining chromatin accessibility, histone marks, and nuclear features, we find that cells reprogram from a state characterized by global reductions in nuclear size and transcriptional activity. Supported by globally increased levels of H3K27me3, cells enter a quiescent-like state of reduced RNA metabolism and elevated expression of REST and p27, markers of quiescent neural stem cells. From this transient state, cells convert to neurons at high rates. Inhibition of Ezh2, the catalytic subunit of PRC2 that deposits H3K27me3, abolishes conversion. Our work offers a roadmap to identify global changes in cellular processes that define cells with different conversion potentials that may generalize to other cell-fate transitions. HighlightsO_LIProliferation drives cells to a compact nuclear state that is receptive to TF-mediated conversion. C_LIO_LIIncreased receptivity to TFs corresponds to reduced nuclear volumes. C_LIO_LIReprogrammable cells display global, genome-wide increases in H3K27me3. C_LIO_LIHigh levels of H3K27me3 support cells transits through a state of altered RNA metabolism. C_LIO_LIInhibition of Ezh2 increases nuclear size, reduces the expression of the quiescence marker p27. C_LIO_LIAcute inhibition of Ezh2 abolishes motor neuron conversion. C_LI One Sentence SummaryCells transit through a quiescent-like state characterized by global reductions in nuclear size and transcriptional activity to convert to neurons at high rates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/624928v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@ee2127org.highwire.dtl.DTLVardef@1c2a39dorg.highwire.dtl.DTLVardef@183bf46org.highwire.dtl.DTLVardef@82b98d_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2024

PRC2 Promotes Canalisation during Endodermal Differentiation

The genetic circuitry that encodes the developmental programme of mammals is regulated by transcription factors and chromatin modifiers. During early gestation, the three embryonic germ layers are established in a process termed gastrulation. The impact of deleterious mutations in chromatin modifiers such as the polycomb proteins manifests during gastrulation, leading to early developmental failure and lethality in mouse models. Embryonic stem cells have provided key insights into the molecular function of polycomb proteins, but it is impossible to fully appreciate the role of these epigenetic factors in development, or how development is perturbed due to their deficiency, in the steady-state. To address this, we have employed a tractable embryonic stem cell differentiation system to model primitive streak formation and early gastrulation. Using this approach, we find that loss of the repressive polycomb mark H3K27me3 is delayed relative to transcriptional activation, indicating a subordinate rather than instructive role in gene repression. Despite this, chemical inhibition of polycomb enhanced endodermal differentiation efficiency, but did so at the cost of lineage fidelity. These findings highlight the importance of the polycomb system in stabilising the developmental transcriptional response and, in so doing, in shoring up cellular specification.

molecular biology↗

bioRxiv2024

Deep Learning Based Models for Preimplantation Mouse and Human Development

The rapid growth of single-cell transcriptomic technology has produced an increasing number of datasets for both embryonic development and in vitro pluripotent stem cell derived models. This avalanche of data about pluripotency and the process of lineage specification has meant it has become increasingly difficult to define specific cell types or states and compare these to in vitro differentiation. Here we utilize a set of deep learning (DL) tools to integrate and classify multiple datasets. This allows for the definition of both mouse and human embryo cell types, lineages and states, thereby maximising the information one can garner from these precious experimental resources. Our approaches are built on recent initiatives for large scale human organ atlases, but here we focus on the difficult to obtain and process material that spans early mouse, and in particular, human development. Using publicly available data for these stages, we test different deep learning approaches and develop both a model to classify cell types in an unbiased fashion and define the set of genes required to identify lineages, cell types and states. We have used our predictions to probe pluripotent stem cell models for both mouse and human development, showcasing the importance of this resource as a dynamic reference for early embryogenesis.

developmental biology↗

bioRxiv2024

Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst

Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naive human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and upregulate molecular markers associated with naive pluripotency. Our functional study demonstrates for the first time the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner.

developmental biology↗

bioRxiv2023

nf-core/marsseq: systematic pre-processing pipeline for MARS-seq experiments

MotivationAs a result of advancing single sequencing technology (scRNA-seq), it has become possible to study gene regulatory mechanism(s) and their influence on evolving cell states in time at the level of individual cells. Since 2009, numerous scRNA-seq protocols have been developed, each with its own advantages, disadvantages and library preparation complexities (Ziegenhain et al. 2017). However, the interpretation of data arising from these techniques often shares similar limitations, such as the lack of a standardized pre-processing workflow and consistent data reproducibility. Here we focus on the standardization of the plate based Massively Parallel RNA Single cell Sequencing (MARS-seq, Jaitin et al. 2014) pre-processing pipeline as described in MARS-seq2.0 (Keren-Shaul et al. 2019), which was developed at the Weizmann Institute of Science. ResultsTo overcome the limitations mentioned above, we have taken the original MARS-seq2.0 pipeline and revised it to enable implementation using the nf-core framework (Ewels et al. 2020). By doing so, we have simplified pipeline execution enabling streamlined application, with increased transparency and scalability. Additionally, we have further improved the pipeline by implementing a custom workflow for RNA velocity estimation. Availability and implementationThe pipeline is part of the nf-core bioinformatics community and is freely available at https://github.com/nf-core/marsseq with data analysis at https://github.com/brickmanlab/proks-et-al-2023.

bioinformatics↗

bioRxiv2023

Enhancer status in the primitive endoderm supports unrestricted lineage plasticity in regulative development

Mammalian blastocyst formation involves the specification of trophectoderm followed by the differentiation of the inner cell mass into either epiblast or primitive endoderm. During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the primitive endoderm alone was sufficient to regenerate a complete blastocyst and continue normal postimplantation development to term. We identify an in vitro population similar to the early primitive endoderm in vivo, that exhibits the same embryonic and extra-embryonic potency, forming three dimensional embryoid structures. Commitment in early primitive endoderm is suppressed by JAK/STAT signalling, collaborating with OCT4 to safeguard enhancer status enabling multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlights the importance of primitive endoderm in perturbed development.

developmental biology↗

bioRxiv2023

Transcription Factor Co-Expression Mediates Lineage Priming for Embryonic and Extra-Embryonic Differentiation

In early mammalian development, cleavage stage blastomeres and cells of the inner cell mass (ICM) of the blastocyst co-express embryonic and extra-embryonic transcriptional determinants. Using a double protein-based reporter we identify embryonic stem cells (ESC) that co-express the extra-embryonic factor GATA6 alongside the embryonic factor SOX2 in specific conditions. Based on single cell transcriptomics we find these population resemble unsegregated ICM, exhibiting enhanced differentiation potential for endoderm while maintaining epiblast competence and suggesting they represent an ideal model to determine how GATA6 and SOX2 influence each others DNA binding. To relate this binding to future fate, we describe a complete enhancer set in both ESCs and naive extraembryonic endoderm stem cells and ask whether SOX2 and GATA6 recognize these elements in ICM-like ESC sub-population. Both factors support cooperative recognition in these lineages, with GATA6 bound alongside SOX2 on a fraction of pluripotency enhancers and SOX2 alongside GATA6 more extensively on endoderm enhancers. Our findings suggest that cooperative binding between these antagonistic factors both supports self-renewal and prepares progenitor cells for later differentiation.

developmental biology↗

bioRxiv2022

A bipartite function of ESRRB can integrate signaling over time to balance self-renewal and differentiation

Cooperative DNA binding of transcription factors (TFs) integrates external stimuli and context across tissues and time. Naive mouse embryonic stem cells are derived from early development and can sustain the pluripotent identity indefinitely. Here we ask whether TFs associated with pluripotency evolved to directly support this state, or if the state emerges from their combinatorial action. NANOG and ESRRB are key pluripotency factors that co-bind DNA. We find that when both factors are expressed, ESRRB supports pluripotency. However, when NANOG is not present, ESRRB supports a bistable culture of cells with an embryo-like primitive endoderm identity ancillary to pluripotency. The stoichiometry between NANOG and ESRRB quantitatively influences differentiation, and in silico modeling of bipartite TF activity suggests ESRRB safeguards plasticity in differentiation. Thus, the concerted activity of cooperative TFs can transform their effect to sustain intermediate cell identities and allow ex vivo expansion of highly stable stem cell models.

cell biology↗

bioRxiv2022

Differential elasticity in lineage segregation of embryonic stem cells

The question of what guides lineage segregation is central to development, where cellular differentiation leads to segregated cell populations destined for specialized functions. Here, using optical tweezers measurements of mouse embryonic stem cells (mESCs), we reveal a mechanical mechanism based on differential elasticity in the second lineage segregation of the embryonic inner cell mass into epiblast (EPI) cells - that will develop into the fetus - and primitive endoderm (PrE) - which will form extraembryonic structures such as the yolk sac. Remarkably, we find that these mechanical differences already occur during priming and not just after a cell has committed to differentiation. Specifically, we show that the mESCs are highly elastic compared to any other reported cell type and that the PrE cells are significantly more elastic than EPI-primed cells. Using a model of two cell types differing only in elasticity we show that differential elasticity alone can lead to segregation between cell types, suggesting that the mechanical attributes of the cells contribute to the segregation process. Our findings present differential elasticity as a previously unknown mechanical contributor to the lineage segregation during the embryo morphogenesis.

biophysics↗

bioRxiv2022

Evolutionary Origin of Vertebrate OCT4/POU5 Functions in Supporting Pluripotency

The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naive states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse Embryonic Stem Cells (ESCs), we found that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naive and primed pluripotency. This specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, ESC phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naive pluripotency and describe their adaptation over evolutionary time.

developmental biology↗

bioRxiv2022

Expansion of Ventral Foregut Primes the Enhancer Landscape for Organ Specific Differentiation

Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation is not seen as having a specific role. Here we exploit human embryonic stem cell derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion, it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. The use of expansion of lineage specific intermediates may therefore be an important step in high fidelity in vitro differentiation.

developmental biology↗

bioRxiv2022

Transcriptional Heterogeneity and Cell Cycle Regulation as Central Determinants of Primitive Endoderm Priming.

During embryonic development cells acquire identity at the same time as they are proliferating, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to rates of cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single cell RNA-seq in the contexts of self-renewal, priming and differentiation of embryonic stem cells (ESCs) towards the Primitive Endoderm lineage (PrE). Since ESCs are derived from the Inner Cell Mass of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing subfractions that are primed for either of the two ICM lineages, Epiblast and PrE. These subfractions represent dynamic states that can readily interconvert in culture, and the PrE subfraction is functionally primed for endoderm differentiation. Here we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naive ESC culture conditions promote Epiblast-like expansion and PrE differentiation stimulates the selective proliferation of PrE-primed cells. In endoderm differentiation, we find that this change is accompanied by a counter-intuitive increase in G1 length that also appears replicated in vivo. While FGF/ERK signalling is a known key regulator of ESCs and PrE differentiation, we find it is not just responsible for ESCs heterogeneity, but also cell cycle synchronisation, required for the inheritance of similar cell cycles between sisters and cousins. Taken together, our results point to a tight relationship between transcriptional heterogeneity and cell cycle regulation in the context of lineage priming, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage-specific fashion while allowing plasticity during early determination.

developmental biology↗

bioRxiv2021

Neighbor-specific gene expression revealed from physically interacting cells during mouse embryonic development

Development of multicellular organisms is orchestrated by persistent cell-cell communication between neighboring partners. Direct interaction between different cell types can induce molecular signals that dictate lineage specification and cell fate decisions. Current single cell RNAseq (scRNAseq) technology cannot adequately analyze cell-cell contact-dependent gene expression, mainly due to the loss of spatial information. To overcome this obstacle and resolve cell-cell contact-specific gene expression during embryogenesis, we performed RNA sequencing of physically interacting cells (PIC-seq) and assessed them alongside similar single cell transcriptomes derived from developing mouse embryos between embryonic day (E) 7.5 and E9.5. Analysis of the PIC-seq data identified novel gene expression signatures that were dependent on the presence of specific neighboring cell types. Our computational predictions, validated experimentally, demonstrated that neural progenitor (NP) cells overexpress Lhx5 and Nkx2-1 genes, when exclusively interacting with the definitive endoderm (DE) cell. Moreover, there was a reciprocal impact on the transcriptome of the DE cells, as they tend to overexpress Rax and Gsc genes when in contact with the NP cells. Using individual cell transcriptome data, we formulated a means of computationally predicting the impact of one cell type on the transcriptome of its neighboring cell types. We have further developed a distinctive spatial-tSNE to display the pseudo-spatial distribution of cells in a 2-dimensional space. In summary, we describe an innovative approach to study contact-specific gene regulation during embryogenesis with potential broader implication in other physiologically relevant processes. SignificancePhysical contact between neighboring cells is known to induce transcriptional changes in the interacting partners. Accurate measurement of these cell-cell contact based influences on the transcriptome is a very difficult experimental task. However, determining such transcriptional changes will highly enhance our understanding for the developmental processes. Current scRNAseq technology isolates the tissue into individual cells, making it hard to determine the potential transcriptomic changes due to its interacting partners. Here, we combined PIC-seq and computational algorithms to identify cell-type contact dependent transcriptional profiles focusing on endoderm development. We have computationally identified and experimentally validated specific gene expression patterns depending upon the presence of specific neighboring cell types. Our study suggests a new way to study cell-cell interactions for embryogenesis.

developmental biology↗

bioRxiv2020

Physical properties and actin organization in embryonic stem cells depend on differentiation stage

The cellular cytoskeleton provides the cell with mechanical rigidity and mediates mechanical interaction between cells and with the extracellular environment. The actin structure plays a key role in regulating cellular behaviors like motility, cell sorting, or cell polarity. From the earliest stages of development, in naive stem cells, the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. The ultrastructure of the earliest embryonic stem cells has not been investigated in living cells despite the fact that it is well-known that cells undergo morphological shape changes during the earliest stages of development. Here, we provide 3D investigations of the actin cytoskeleton of naive mouse embryonic stem cells (ESCs) in clusters of sizes relevant for early stage development using super resolution optical reconstruction microscopy (STORM). We quantitatively describe the morphological, cytoskeletal and mechanical changes appearing between cells in small clusters at the earliest stages of inner cell mass differentiation, as recapitulated by cells cultured under two media conditions, 2i and Serum/LIF, thus promoting the naive and first primed state, respectively. High resolution images of living stem cells showed that the peripheral actin structure undergoes a dramatic change between the two media conditions. The actin organization changed from being predominantly oriented parallel to the cell surface in 2i medium to a more radial orientation in Serum/LIF. Finally, using an optical trapping based technique, we detected micro-rheological differences in the cell periphery between the cells cultured in these two media, with results correlating well with the observed nano-architecture of the ESCs in the two different differentiation stages. These results pave the way for linking physical properties and cytoskeletal architecture to the development from naive stem cells to specialized cells. Statement of SignificanceCells receive mechanical signals and must provide mechanical feedback, therefore, physical properties are instrumental for cell-cell interactions. Mechanical signals mediated through the cell surface can significantly affect transport of signaling molecules and can influence biological processes like transcriptional regulation. To achieve a deeper insight into how the cytoskeletal structure is responsible for cell shape and material properties at the earliest stages of development, we employ super-resolution microscopy to image actin fibers in clusters of embryonic stem cells mimicking early development. By modification of the culturing conditions, we investigate how the actin cytoskeleton and micro-rheological properties of ESCs change between the naive ground state and the stage primed towards epiblast, thus revealing a correlation between differentiation stage and cytoskeletal structure.

biophysics↗