scholarly journals OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xin Huang ◽  
Kyoung-mi Park ◽  
Paul Gontarz ◽  
Bo Zhang ◽  
Joshua Pan ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Protein Protein Interactions ◽  
Human Escs ◽  
Nucleosome Remodeling ◽  
Chromatin Remodelers ◽  
Baf Complex ◽  
Common Transcription Factor ◽  
Optimal Maintenance

AbstractUnderstanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.

scholarly journals A Specificity and Targeting Subunit of a Human SWI/SNF Family-Related Chromatin-Remodeling Complex

2000 ◽  
Vol 20 (23) ◽  
pp. 8879-8888 ◽  
Author(s):  
Zuqin Nie ◽  
Yutong Xue ◽  
Dafeng Yang ◽  
Sharleen Zhou ◽  
Bonnie J. Deroo ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Dna Interaction ◽  
Protein Protein Interactions ◽  
Baf Complex ◽  
Chromatin Remodeling Complexes

ABSTRACT The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc fromSaccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.

scholarly journals Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ava C Carter ◽  
Jin Xu ◽  
Meagan Y Nakamoto ◽  
Yuning Wei ◽  
Brian J Zarnegar ◽  
...  
Keyword(s):  
Gene Silencing ◽  
X Chromosome ◽  
Protein Interactions ◽  
Sex Chromosome ◽  
Embryonic Stem ◽  
Structural Similarity ◽  
Chromatin Accessibility ◽  
Endogenous Retrovirus ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation

The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

scholarly journals Inhibition of β-catenin–TCF1 interaction delays differentiation of mouse embryonic stem cells

2015 ◽  
Vol 211 (1) ◽  
pp. 39-51 ◽  
Author(s):  
Sujash S. Chatterjee ◽  
Abil Saj ◽  
Tenzin Gocha ◽  
Matthew Murphy ◽  
Foster C. Gonsalves ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Protein Interactions ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Protein Protein Interactions ◽  
Nanog Expression ◽  
Context Specific

The ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/β-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how β-catenin’s function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of β-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that β-catenin’s function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein–protein interactions drive stem cell fate.

scholarly journals Tracking chromatin state changes using μMap photo-proximity labeling

2021 ◽  
Author(s):  
Ciaran P Seath ◽  
Antony J Burton ◽  
David W. C. MacMillan ◽  
Tom W Muir
Keyword(s):  
Cell Fate ◽  
Small Molecule ◽  
Protein Interactions ◽  
Control Cell ◽  
Global Changes ◽  
Therapeutic Effects ◽  
Protein Protein Interactions ◽  
Chromatin Remodelers ◽  
Expression Levels ◽  
Epigenetic Drug

Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health. However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study. Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-μMap method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics. We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-μMap improves our fundamental understanding of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

scholarly journals Engineered illumination devices for optogenetic control of cellular signaling dynamics

2019 ◽  
Author(s):  
Nicole A. Repina ◽  
Thomas McClave ◽  
Xiaoping Bao ◽  
Ravi S. Kane ◽  
David V. Schaffer
Keyword(s):  
Cell Fate ◽  
High Throughput ◽  
Protein Interactions ◽  
Cell Biology ◽  
Low Cost ◽  
Optical Design ◽  
Embryonic Stem ◽  
Protein Protein Interactions ◽  
Optogenetic Control

ABSTRACTSpatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, currentin vitromethods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. Here we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). The optical design of LAVA devices was optimized for uniform illumination of multi-well cell culture plates to enable high-throughput, spatiotemporal optogenetic activation of signaling pathways and protein-protein interactions. Using the LAVA devices, variation in light intensity induced a dose-dependent response in optoWnt activation and downstream Brachyury expression in human embryonic stem cells (hESCs). Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models embryonic presentation of Wnt signalsin vitro. The engineered LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.

scholarly journals Tracking chromatin state changes using µMap photo-proximity labeling

2021 ◽  
Author(s):  
Tom Muir ◽  
Ciaran Seath ◽  
David MacMillan ◽  
Antony Burton
Keyword(s):  
Cell Fate ◽  
Small Molecule ◽  
Protein Interactions ◽  
Control Cell ◽  
Global Changes ◽  
Therapeutic Effects ◽  
Protein Protein Interactions ◽  
Chromatin Remodelers ◽  
Expression Levels ◽  
Epigenetic Drug

Abstract Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health. However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study. Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-µMap method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics. We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labeling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-µMap improves our fundamental understand-ing of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

Regulation of ATP-dependent chromatin remodelers: accelerators/brakes, anchors and sensors

2018 ◽  
Vol 46 (6) ◽  
pp. 1423-1430 ◽  
Author(s):  
Somnath Paul ◽  
Blaine Bartholomew
Keyword(s):  
Protein Interactions ◽  
Atp Hydrolysis ◽  
Self Regulation ◽  
Protein Protein Interactions ◽  
Dna Translocation ◽  
Chromatin Remodelers ◽  
Histone Octamer ◽  
Double Stranded Dna ◽  
Accessory Subunits ◽  
Dna Translocase

All ATP-dependent chromatin remodelers have a DNA translocase domain that moves along double-stranded DNA when hydrolyzing ATP, which is the key action leading to DNA moving through nucleosomes. Recent structural and biochemical data from a variety of different chromatin remodelers have revealed that there are three basic ways in which these remodelers self-regulate their chromatin remodeling activity. In several instances, different domains within the catalytic subunit or accessory subunits through direct protein–protein interactions can modulate the ATPase and DNA translocation properties of the DNA translocase domain. These domains or subunits can stabilize conformations that either promote or interfere with the ability of the translocase domain to bind or retain DNA during translocation or alter the ability of the enzyme to hydrolyze ATP. Second, other domains or subunits are often necessary to anchor the remodeler to nucleosomes to couple DNA translocation and ATP hydrolysis to DNA movement around the histone octamer. These anchors provide a fixed point by which remodelers can generate sufficient torque to disrupt histone–DNA interactions and mobilize nucleosomes. The third type of self-regulation is in those chromatin remodelers that space nucleosomes or stop moving nucleosomes when a particular length of linker DNA has been reached. We refer to this third class as DNA sensors that can allosterically regulate nucleosome mobilization. In this review, we will show examples of these from primarily the INO80/SWR1, SWI/SNF and ISWI/CHD families of remodelers.

scholarly journals Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability

2019 ◽  
Vol 116 (30) ◽  
pp. 14961-14970 ◽  
Author(s):  
Veronica M. Pravata ◽  
Villo Muha ◽  
Mehmet Gundogdu ◽  
Andrew T. Ferenbach ◽  
Poonam S. Kakade ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Protein Interactions ◽  
Catalytic Domain ◽  
Embryonic Stem ◽  
Global Changes ◽  
Missense Mutations ◽  
Protein Protein Interactions ◽  
Delayed Differentiation ◽  
Nucleocytoplasmic Proteins ◽  
Glcnac Transferase

O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein–protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.

scholarly journals SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Samuel A Myers ◽  
Sailaja Peddada ◽  
Nilanjana Chatterjee ◽  
Tara Friedrich ◽  
Kiichrio Tomoda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Protein Interactions ◽  
Regulatory Mechanism ◽  
Embryonic Stem ◽  
Transactivation Domain ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Reprogramming Efficiency ◽  
Induction Of Differentiation

The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor.

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