Biology subjects

Chambers, I.

Publications and source records attributed to Chambers, I..

15 recordsLinked to original sources

bioRxiv2026

Dynamic TET1 repression coordinates activation of macroH2A.2-poised bivalent genes during neuroectoderm differentiation

Cell state transitions involve changes in gene expression programs in conjunction with epigenetic regulation occurring in chromatin. Bivalent chromatin, marked by overlapping active H3K4me3 and repressive H3K27me3, poises key developmental genes during pluripotency for cell type-specific activation during differentiation. However, the molecular mechanisms that activate bivalent genes during lineage commitment remain poorly understood. Here, we elucidate how the cytosine dioxygenase TET1 promotes neuroectoderm differentiation through dynamic chromatin binding and a non-catalytic repressor function. Through a hypothesis-free screen for TET1 chromatin complex partners, we find that TET1 physically interacts with macroH2A.2 and H2A.X on chromatin. Integrated multi-omic analyses reveal that the non-catalytic repressor function of TET1 and a novel activating function of the H2A variant macroH2A.2 together promote a poised transcriptional state within bivalent chromatin during pluripotency. Upon pluripotency exit, TET1 disengages from macroH2A.2-marked early neuroectoderm genes to permit their activation, while remaining bound as a repressor at mesoendoderm and late neural genes to prevent alternative cell state transitions. Furthermore, TET1 retention at accessible primed enhancers supports neural program activation. Together, our findings reveal a noncanonical functional interplay within bivalent chromatin between TET1 and macroH2A.2 that maintains a poised state during pluripotency and predicts selective gene activation during neuroectoderm specification.

developmental biology↗

bioRxiv2026

Evolutionary rise of a synaptic mechanism for creating and diversifying key reinforcement signals

Most neurons release either excitatory or inhibitory neurotransmitters. However, multiple inputs to the lateral habenula (LHb) co-transmit glutamate and GABA, transmitters with opposing effects on LHb output. Although the LHb has an established role in reinforcement learning, the adaptive significance of glutamate/GABA co-release remains unclear. Using experimentally informed simulations, we show that GABA co-release is sufficient to produce temporal difference (TD)-like transformations of input activity, computations commonly used for reinforcement learning and behavioral optimization. Heterogeneous GABA-to-glutamate ratios, like those found among LHb neurons ex vivo, produce diverse TD-like computations linked to higher-order decision-making. Single-cell RNA-sequencing analysis and machine-learning image analysis further indicate that glutamate/GABA co-release expanded across vertebrate evolution, from fish to mice, rats, and monkeys. Evolutionary expansion of glutamate/GABA co-release may have supported increasingly sophisticated learning and decision-making that contribute to intelligent behavior.

neuroscience↗

bioRxiv2026

NANOG assembles into self-limiting aging micelles that drive a sol-gel transition and modulate DNA dynamics

Proteins and nucleic acids form non-Newtonian liquids with complex rheological properties that contribute to their function in vivo. Here we investigate the rheology of the transcription factor NANOG, a key protein to maintain embryonic stem cell pluripotency. We find that at high concentrations, NANOG forms macroscopic aging gels that are dependent on its intrinsically disordered domain. By combining molecular dynamics simulations, mass photometry and Cryo-EM, we also discover that - in contrast with unbounded condensates formed by other intrinsically disordered proteins - NANOG forms self-limiting micelles with exposed DNA-binding domains. We show that these micelles can stabilize DNA entanglements and in turn modulate DNA dynamics. Based on our findings, we conjecture that NANOG may contribute to regulate gene expression by creating local gel-like environments that restrict genome dynamics and that its aging may ingrain mechanical memory in gene regulatory networks.

biophysics↗

bioRxiv2025

Direct recruitment of RNA Polymerase II by NANOG to activated target genes

Cell identity relies upon transcription factors (TFs). The concentration of the TF NANOG determines the efficiency of maintenance of mouse embryonic stem cell (ESC) identity. However, the mechanisms by which NANOG acts are not fully understood. Models of mammalian transcription generally propose that TFs bind DNA and connect to the transcriptional machinery indirectly via intermediary proteins. Accordingly, examples of direct contact between cell-type specific TFs and RNA synthesis enzymes in mammalian cells remain elusive. Here we show that NANOG directly contacts RNA Polymerase II (RNAPII) via aromatic residues within the low complexity domains of both proteins. NANOG can localize RNAPII to a specific DNA site, with RNAPII enhancing NANOG DNA affinity. The NANOG-RNAPII complex is dissociated by the transcriptional pause-release enzyme, CDK9. Inhibition of CDK9 enhances NANOG chromatin binding, while induction of NANOG localizes RNAPII to specific NANOG chromatin sites. Induced RNAPII localization is selective for targets activated by NANOG. A NANOG mutant that retains DNA, but not RNAPII binding, cannot stimulate RNAPII localization to chromatin, shows an impaired transcriptional response and does not drive LIF-independent ESC self-renewal. These results identify a novel, regulatable interaction between a cell-type specific TF and RNAPII that can change transcription and alter cell fate.

cell biology↗

bioRxiv2025

OTX2 controls chromatin accessibility to direct somatic versus germline differentiation

The choice between somatic and germline fates is essential for species survival. This choice occurs in embryonic epiblast cells, as these cells are competent for both somatic and germline differentiation. The transcription factor OTX2 regulates the choice between somatic and germline fates, as Otx2-null epiblast-like cells (EpiLCs) form primordial germ cell-like cells (PGCLCs) with enhanced efficiency. However, the mechanisms by which OTX2 achieves this function are not fully characterized. Here we show that OTX2 controls chromatin accessibility at specific chromatin loci to modulate gene transcription and enable somatic differentiation. By performing CUT&RUN for OTX2 and ATAC-seq in wild-type and Otx2-null embryonic stem cells and EpiLCs, we identified regions where OTX2 binding opens chromatin, either alone or in cooperation with a ZIC family protein. Enforced OTX2 expression maintains accessibility at these regions and, in addition, induces opening of [~]4,000 somatic-associated regions in cells differentiating in the presence of PGC-inducing cytokines. Once cells have acquired germline identity, these additional somatic-associated regions can no longer respond to OTX2 and remain closed. Our results indicate that OTX2 works in cells with dual competence for both somatic and germline differentiation to increase accessibility of somatic regulatory regions and induce the somatic fate at the expense of the germline.

genomics↗

bioRxiv2024

TET knockout cells transit between pluripotent states and exhibit precocious germline entry

TET1, TET2 and TET3 are DNA demethylases with critical roles in development and differentiation. To assess the contributions of TET proteins to cell function during early development, single and compound knockouts of Tet genes in mouse pluripotent embryonic stem cells (ESCs) were generated. Here we show that TET proteins are not required to transit between naive, formative and primed pluripotency. Moreover, ESCs with double-knockouts of Tet1 and Tet2 or triple-knockouts of Tet1, Tet2 and Tet3 are phenotypically indistinguishable. These TET-deficient ESCs exhibit differentiate defects; they fail to activate somatic gene expression and retain expression of pluripotency transcription factors. Therefore, TET1 and TET2, but not TET3 act redundantly to facilitate somatic differentiation. Importantly however, TET-deficient ESCs can differentiate into primordial germ cell-like cells (PGCLCs), and do so at high efficiency in the presence or absence of PGC-promoting cytokines. Moreover, acquisition of a PGCLC transcriptional programme occurs more rapidly in TET-deficient cells. These results establish that TET proteins act at the juncture between somatic and germline fates: without TET proteins, epiblast cell differentiation defaults to the germline.

developmental biology↗

bioRxiv2021

Differential repression of Otx2 underlies the capacity of NANOG and ESRRB to induce germline entry

Primordial germ cells (PGCs) are induced in the embryo by signals, including BMP emanating from extra-embryonic ectoderm, that act on cells in the post-implantation epiblast. PGC development can be recapitulated in vitro through the exposure of epiblast-like cells (EpiLCs) to appropriate cytokines, resulting in differentiation into PGC-like cells (PGCLCs). Interestingly, the requirement for cytokines to induce PGCLCs can be bypassed by enforced expression of the transcription factor (TF) NANOG. However, the underlying mechanisms are not fully elucidated. Here, we show that Otx2 downregulation is essential to enable NANOG to induce PGCLC formation. Moreover, while previous work has shown that the direct NANOG target gene Esrrb can substitute for several functions of NANOG, enforced expression of ESRRB cannot promote PGCLC specification from EpiLCs. This appears to be due to differential downregulation of Otx2 by NANOG and ESRRB, since induction of ESRRB in Otx2+/- EpiLCs activates expression of the core PGC TFs, Blimp1, Prdm14 and Ap2{gamma} and emergence of PGCLCs. This study illuminates the interplay of TFs occurring at the earliest stages of PGC specification from a state of competence.

developmental biology↗

bioRxiv2021

Resf1 supports embryonic stem cell self-renewal and effective germline entry

Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germ line specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESCs self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification.

developmental biology↗

bioRxiv2020

Ventral Tegmental Area GABA, glutamate, and glutamate-GABA neurons are heterogenous in their electrophysiological and pharmacological properties

The ventral tegmental area (VTA) contains dopamine neurons intermixed with GABA-releasing (expressing vesicular GABA transporter, VGaT), glutamate-releasing (expressing vesicular glutamate transporter, VGluT2), and co-releasing (co-expressing VGaT and VGluT2) neurons. By delivering INTRSECT viral vectors into VTA of double vglut2-Cre/vgat-Flp transgenic mice, we targeted specific VTA cell populations for ex vivo recordings. We found that VGluT2+ VGaT- and VGluT2+ VGaT+ neurons on average had relatively hyperpolarized resting membrane voltage, greater rheobase, and lower spontaneous firing frequency compared to VGluT2- VGaT+ neurons, suggesting that VTA glutamate-releasing and glutamate-GABA co-releasing neurons require stronger excitatory drive to fire than GABA-releasing neurons. In addition, we detected expression of Oprm1mRNA (encoding opioid receptors, MOR) in VGluT2+ VGaT- and VGluT2- VGaT+ neurons, and their hyperpolarization by the MOR agonist DAMGO. Collectively, we demonstrate the utility of the double transgenic mouse to access VTA glutamate, glutamate-GABA and GABA neurons, and show some electrophysiological heterogeneity among them. Impact StatementSome physiological properties of VTA glutamate-releasing and glutamate-GABA co-releasing neurons are distinct from those of VTA GABA-releasing neurons. -opioid receptor activation hyperpolarizes some VTA glutamate-releasing and some GABA-releasing neurons.

neuroscience↗

bioRxiv2020

TET1 interacts directly with NANOG via independent regions containing hydrophobic and aromatic residues

The DNA demethylase TET1 is highly expressed in embryonic stem cells. Knockout experiments indicate that TET1 is important for lineage commitment, and paradoxically, also for reprogramming to naïve pluripotency. TET1 binds to promoters through a CXXC domain which recognises unmethylated CpG dinucleotides. TET1 also binds to enhancers, presumably via interactions with partner proteins. The transcription factor NANOG interacts with TET1 and is predominantly localised at enhancers in ESCs. Therefore, NANOG may contribute to TET1 biological activity in pluripotent cells. However, the regions of TET1 involved in protein-protein interactions are mostly unknown. Here, we characterise the physical interaction between TET1 and NANOG using embryonic stem cells and bacterial expression systems. TET1 and NANOG interact through multiple binding sites that act independently. Critically, mutating conserved hydrophobic and aromatic residues within TET1 and NANOG abolishes the interaction. Comparative ChIP-seq analysis identifies genomic loci bound by both TET1 and NANOG, that correspond predominantly to pluripotency enhancers. Importantly, around half of NANOG transcriptional target genes are associated with TET1-NANOG co-bound sites. These results indicate a mechanism by which TET1 protein is targeted to specific sites of action at enhancers by direct interaction with a transcription factor.HighlightsNANOG and TET1 have regulatory roles in maintaining and reprogramming pluripotencyTET1 and NANOG interact via multiple independent binding regionsTET1 and NANOG interactions are mediated by aromatic and hydrophobic residuesTET1 residues that bind NANOG are highly conserved in mammalsCo-localisation of TET1 and NANOG on chromatin is enriched at NANOG target genesCompeting Interest StatementThe authors have declared no competing interest.AbbreviationsDSBHdouble stranded beta helix domainESCembryonic stem cellOGTO-linked N-acetylglucosamine transferaseTETTen-eleven-translocationWRtryptophan-repeatView Full Text

biochemistry↗

bioRxiv2020

Characterisation of interactions between the pluripotency transcription factors Nanog, Oct4 and Sox2

The pluripotency transcription factors (TFs) Nanog, Sox2, and Oct4 are at the centre of the gene regulatory network that controls cell identity in embryonic stem (ES) cells. However, the mechanisms by which these factors control cell fate, and their interactions with one another are not fully understood. Here we combine biophysical and novel biochemical assays to assess how these factors interact with each other quantitatively. A confocal microscopy method to detect binding of a target protein to a fluorescently labelled partner (coimmunoprecipitated bead imaging microscopy [CBIM]) is presented and used to demonstrate homotypic binding of Nanog and heterotypic binding between Nanog and Sox2 and between Nanog and Oct4. Using fluorescence correlation spectroscopy we show that in solution, Nanog but not Oct4 or Sox2 can form homomultimers. However, both Sox2 and Oct4 can form heterotypic multimers with Nanog in a manner that depends on the presence of tryptophan residues within the Nanog tryptophan repeat. Fluorescence Cross Correlation Spectroscopy shows the affinity of Nanog for dimer formation is in the order Sox2 > Nanog > Oct4. Importantly, live cell analysis demonstrates the existence of Nanog homomultimers in vivo. Together these findings extend understanding of the molecular interactions occurring between these central mediators of the pluripotency gene regulatory network at the single-molecule level.

biochemistry↗

bioRxiv2019

Competence to epithelialise coincides with competence to differentiate in pluripotent cells

Pluripotent cells reorganise themselves into an epithelium before they initiate differentiation, but it is not clear how these two events are mechanistically linked. Here we use quantitative imaging approaches to measure cellular rearrangements that accompany exit from naive pluripotency. We show that competence to epithelialise, like competence to differentiate, is a regulated process. The pro-differentiation transcription factor Tcf15 prospectively identifies cells that are competent to epithelialise. We identify early upregulation of the laminin receptor integrin alpha3 prior to differentiation and show that Tcf15 helps to regulate this change. Finally, we show that Tcf15 identifies and is required for efficient differentiation of a primed subpopulation of pluripotent cells. We conclude that competence to epithelialise is actively regulated and linked to differentiation-competence through the transcription factor Tcf15.

developmental biology↗

bioRxiv2019

G2M splits mouse embryonic stem cells into naïve and formative pluripotency states

Embryonic stem cells (ESCs) express heterogeneous levels of pluripotency and developmental transcription factors (TFs) and their cell cycle is unsynchronised when grown in the presence of serum. Here, we asked whether the cell cycle and developmental heterogeneities of ESCs are coordinated by determining the state identities of G1- and G2M-enriched mouse ESCs (mESCs) at single cell resolution. We found that G2M cells were not all the same and demonstrate their split into the naive and formative (intermediate) pluripotency states marked by high or low Esrrb expression, respectively. The naive G2M sub-state resembles ground state pluripotency of the LIF/2i cultured mESCs. The naive and formative G2M sub-states exist in the pre- and post-implantation stages of the mouse embryo, respectively, verifying developmental distinction. Moreover, the G2M sub-states partially match between the mouse and human ESCs, suggesting higher similarity of transcriptional control between these species in G2M. Our findings propose a model whereby G2M separates mESCs into naive and formative pluripotency states. This concept of G2M-diverted pluripotency states provides new framework for understanding the mechanisms of pluripotency maintenance and lineage specification in vitro and in vivo, and the development of more efficient and clinically relevant reprogramming strategies.

developmental biology↗

bioRxiv2017

Distinct SoxB1 networks are required for naïve and primed pluripotency

Deletion of Sox2 from embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naive pluripotency.

cell biology↗

bioRxiv2017

Functional dissection of the enhancer repertoire in human embryonic stem cells

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications can functionally define active enhancers remains unclear. Here we combine chromatin immunoprecipitation with a massively parallel reporter assay to identify functional enhancers in human embryonic stem cells (hESCs) genome-wide in a quantitative unbiased manner. While active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac and H3K4me1 function as enhancers, with activity changing markedly with culture conditions. Our analysis also reveals a novel enhancer set associated with housekeeping genes. Moreover, while transposable elements associate with putative enhancers only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalogue of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome.\n\nHighlightsO_LIA catalog of functional enhancers in hESCs including a novel housekeeping class\nC_LIO_LIActive enhancers feature specific transcription factors and transposable elements\nC_LIO_LIMajor shifts in enhancer activity occur during induction of naive pluripotency\nC_LIO_LISuper-enhancers consist of small units with enhancer function\nC_LI

genomics↗