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At least 19 recordsLinked to original sources
PubMed2002

The ETS domain transcription factor Elk-1 contains a novel class of repression domain.

The ETS domain transcription factor Elk-1 serves as an integration point for different mitogen-activated protein (MAP) kinase pathways. Phosphorylation of Elk-1 by MAP kinases triggers its activation. However, while the activation process is well understood, its downregulation-inactivation is less well characterized. The ETS DNA-binding domain plays a role in the downregulation of Elk-dependent promoter activity following mitogenic activation by recruiting the mSin3A-HDAC complex. Here we have identified a novel evolutionarily conserved repression domain in Elk-1, termed the R motif, which serves to reduce the basal transcriptional activity of Elk-1 and dampen its response to mitogenic signals. This domain is highly potent and portable and can repress transcription in trans. The R motif is related to the CRD1 repression domain in p300 and can functionally replace this domain and confer p21(waf1/cip1) inducibility on p300. However, the R motif acts in a context-dependent manner and is not p21(waf1/cip1) responsive in Elk-1. Thus, the Elk-1 R motif and the p300 CRD1 motif represent a new class of repression domains that are regulated in a context-dependent manner.

Amino Acid Motifs↗
PubMed2026

Synthetic transcriptional repression systems in plants.

Transcriptional repression is a fundamental regulatory mechanism that enables precise control of gene expression in response to developmental signals and environmental stimuli. Synthetic biology can leverage this process within plants to engineer programmable transgene repression systems. This review examines strategies for harnessing prokaryotic repressors in eukaryotic systems to develop synthetic repression systems in plants. These systems utilize modular promoter and repressor architectures that can be tuned through operator placement and repression-domain fusion, respectively, to adjust transcriptional regulation. Chemically dependent inducibility can also be introduced either through use of native derepression mechanisms of the prokaryotic repressors or the incorporation of ligand-binding domains. Finally, this review explores key challenges in designing synthetic repression systems, including kinetics constraints, balancing ON and OFF states, and differences between transient and transgenic expression contexts. Overall, this review highlights modular design frameworks for tunable transgene expression in plants.

Gene Expression Regulation, Plant↗
PubMed2025

HyperCas12a enables multiplexed CRISPRi screens.

Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. High-throughput screens using dCas9 fused to epigenome editing domains have allowed researchers to assess the impact of activation or repression of both coding and non-coding genomic regions on a phenotype of interest, but assessment of genetic interactions between those elements has been limited to pairs. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that this system is compatible with several activation and repression domains, including the P300 histone acetyltransferase domain and SIN3A interacting domain (SID). We further show that the system can be used in cultured primary immune cells and to drive differentiation of induced pluripotent stem cells. We also developed new approaches to use the dCas12a platform for simultaneous activation and repression from a single crRNA array via co-expression of multiple dCas12a orthologues. Lastly, we demonstrate that the dHyperLbCas12a effectors are highly effective for multiple modalities of high-throughput screens, namely proliferation screens and screens to dissect the independent and combinatorial contributions of CREs on gene expression. The tools and methods introduced here create new possibilities for highly multiplexed control of gene expression in a wide variety of biological systems.

Journal Article↗
PubMed2025

Unidirectional recruitment between MeCP2 and KSHV-encoded LANA revealed by CRISPR/Cas9 recruitment assay.

Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is associated with several human malignancies. During latency, the viral genomes reside in the nucleus of infected cells as large non-integrated plasmids, known as episomes. To ensure episome maintenance, the latency protein LANA tethers the viral episomes to the cell chromosomes during cell division. Directional recruitment of protein complexes is critical for the proper function of many nuclear processes. To test for recruitment directionality between LANA and cellular proteins, we directed LANA via catalytically inactive Cas9 (dCas9) to a repeat sequence to obtain easily detectable dots. Then, the recruitment of nuclear proteins to these dots can be evaluated. We termed this assay CRISPR-PITA for Protein Interaction and Telomere Recruitment Assay. Using this protein recruitment assay, we found that LANA recruits its known interactors ORC2 and SIN3A. Interestingly, LANA was unable to recruit MeCP2, but MeCP2 recruited LANA. Both LANA and histone deacetylase 1 (HDAC1) interact with the transcriptional-repression domain (TRD) and the methyl-CpG-binding domain (MBD) of MeCP2. Similar to LANA, HDAC1 was unable to recruit MeCP2. While heterochromatin protein 1 (HP1), which interacts with the N-terminal of MeCP2, can recruit MeCP2. We propose that available interacting domains force this recruitment directionality. We hypothesized that the tandem repeats in the SunTag may force MeCP2 dimerization and mimic the form of DNA-bound MeCP2. Indeed, providing only the tandem epitopes of SunTag allows LANA to recruit MeCP2 in infected cells. Therefore, CRISPR-PITA revealed the rules of unidirectional recruitment and allowed us to break this directionality.

Humans↗
PubMed1979

Interaction between mutant alleles of araC of the Escherichia coli B/r L-arabinose operon.

Strains were constructed that contain mutational alterations affecting two distinct functional domains within the araC gene protein. The araCi (catabolite repression insensitivity) and araCh (catabolite repression hypersensitivity) mutations were used to alter the catabolite repression sensitivity domain, and mutation to D-fucose resistance was used to alter the inducer binding domain. araCh, D-fucose-resistant double mutants never exhibited constitutive ara operon expression, whereas all of the araCi, D-fucose-resistant double mutants did exhibit constitutivity. When L-arabinose was used as an inducer, most of the double mutants exhibited the sensitivity to catabolite repression associated with the araCi or araCh mutation. However, when D-fucose was used as an inducer, changes in sensitivity to catabolite repression were observed that were attributed to interactions between the two protein domains. The roles of catabolite activator protein and araC gene protein in the induction of the araBAD operon were discussed.

Alleles↗
PubMed2025

Differential Crosslinking and Contractile Motors Drive Nuclear Chromatin Compaction.

During interphase, a typical cell nucleus features spatial compartmentalization of transcriptionally active euchromatin and repressed heterochromatin domains. In conventional nuclear organization, euchromatin predominantly occupies the nuclear interior, while heterochromatin, which is approximately 50% more dense than euchromatin, is positioned near the nuclear periphery. Peripheral chromatin organization can be further modulated by the nuclear lamina, which is itself a deformable structure. While a number of biophysical mechanisms for compartmentalization within rigid nuclei have been explored, we study a chromatin model consisting of an active, crosslinked polymer tethered to a deformable, polymeric lamina shell. Contractile motors, the deformability of the shell, and the spatial distribution of crosslinks all play pivotal roles in this compartmentalization. We find that a radial crosslink density distribution, even with a small linear differential of higher crosslinking density at the edge of the nucleus, combined with contractile motor activity, drives genomic segregation, in agreement with experimental observations. This arises from contractile motors preferentially drawing crosslinks into their vicinity at the nuclear periphery, forming high-density domains that promote heterochromatin formation. We also find an increased stiffness of nuclear wrinkles given the preferential heterochromatin compaction below the lamina shell, which is consistent with instantaneous nuclear stiffening under applied nanoindentation. We conclude with the potential for experimental validation of our model predictions.

Journal Article↗
PubMed2025

Differential Crosslinking and Contractile Motors Drive Nuclear Chromatin Compaction.

During interphase, a typical cell nucleus features spatial compartmentalization of transcriptionally active euchromatin and repressed heterochromatin domains. In conventional nuclear organization, euchromatin predominantly occupies the nuclear interior, while heterochromatin, which is approximately 50% more dense than euchromatin, is positioned near the nuclear periphery. Peripheral chromatin organization can be further modulated by the nuclear lamina, which is itself a deformable structure. While a number of biophysical mechanisms for compartmentalization within rigid nuclei have been explored, we study a chromatin model consisting of an active, crosslinked polymer tethered to a deformable, polymeric lamina shell. Contractile motors, the deformability of the shell, and the spatial distribution of crosslinks all play pivotal roles in this compartmentalization. We find that a radial crosslink density distribution, even with a small linear differential of higher crosslinking density at the edge of the nucleus, combined with contractile motor activity, drives genomic segregation, in agreement with experimental observations. This arises from contractile motors preferentially drawing crosslinks into their vicinity at the nuclear periphery, forming high-density domains that promote heterochromatin formation. We also find an increased stiffness of nuclear wrinkles given the preferential heterochromatin compaction below the lamina shell, which is consistent with instantaneous nuclear stiffening under applied nanoindentation. We conclude with the potential for experimental validation of our model predictions.

Journal Article↗
PubMed2025

Spatio-genetically coordinated TPR domain-containing proteins modulate c-di-GMP signaling in Vibrio vulnificus.

Vibrio species, which include several pathogens, are autochthonous to estuarine and warm coastal marine environments, where biofilm formation bolsters their ecological persistence and transmission. Here, we identify a bicistronic operon, rcbAB, whose products synergistically inhibit motility and promote biofilm maturation post-attachment by modulating intracellular c-di-GMP levels in the human and animal pathogen V. vulnificus. RcbA contains an N-terminal tetratricopeptide repeat (TPR) domain and a structured C-terminal region of unknown function, while RcbB possesses an N-terminal TPR domain and a C-terminal GGDEF domain characteristic of diguanylate cyclases. The TPR domain of RcbB represses its diguanylate cyclase activity, while RcbA's TPR domain and C-terminal region co-operatively de-repress it. Localization of both proteins to the flagellar pole is TPR-dependent but not co-dependent, although RcbA anchors RcbB to the pole in the absence of polar landmarks such as HubP and flagella. The conservation of rcbAB across diverse bacterial taxa substantiates its fundamental importance in bacterial biology. This work demonstrates how spatio-genetically coordinated TPR domain-containing proteins modulate c-di-GMP signaling, contributing to our understanding of biofilm formation in Vibrio species and potentially other bacteria. It also reveals the first evidence of inter-protein interaction via the TPR domains of both partners, challenging the conventional paradigm in which only one bears the domain.

Vibrio vulnificus↗
PubMed2026

Regulation of the histone H3K36 methyltransferase Set2 by the histone chaperone Spt6.

Histone H3 lysine 36 methylation is a conserved histone modification that is critical for maintaining eukaryotic transcriptional fidelity and genomic stability. In Saccharomyces cerevisiae, this modification is catalyzed by Set2, an ortholog of the mammalian H3K36 methyltransferase SETD2. Previous genetic, biochemical, and structural studies showed that Set2 activity is repressed by a Set2 autoinhibitory domain (AID) and that activation requires the direct binding of the histone chaperone Spt6. To study the role of Spt6 and Set2 autoinhibition in vivo, we have isolated and analyzed multiple classes of Spt6 and Set2 mutants. Our results suggest an autoinhibited form of Set2 in which the catalytic domain (CD) is bound by the AID. In strong agreement with our genetic results, biophysical experiments demonstrate that the CD and AID physically interact, and that the autoinhibition mutants disrupt this interaction. Finally, RNA sequencing and chromatin immunoprecipitation and sequencing studies show the importance of the Set2-Spt6 interaction for transcription and H3K36 methylation genome-wide. Taken together, our results support a model in which Set2 exists in an inactive, autoinhibited state in vivo through direct CD-AID interactions, with binding by Spt6 required to release the autoinhibition.

Saccharomyces cerevisiae Proteins↗
PubMed2026

Regulation of the histone H3K36 methyltransferase Set2 by the histone chaperone Spt6.

Histone H3 lysine 36 methylation is a conserved histone modification that is critical for maintaining eukaryotic transcriptional fidelity and genomic stability. In Saccharomyces cerevisiae, this modification is catalyzed by Set2, an ortholog of the mammalian H3K36 methyltransferase SETD2. Previous genetic, biochemical, and structural studies showed that Set2 activity is repressed by a Set2 autoinhibitory domain (AID) and that activation requires the direct binding of the histone chaperone Spt6. To study the role of Spt6 and Set2 autoinhibition in vivo, we have isolated and analyzed multiple classes of Spt6 and Set2 mutants. Our results suggest an autoinhibited form of Set2 in which the catalytic domain is bound by the AID. In strong agreement with our genetic results, biophysical experiments demonstrate that the catalytic domain and AID physically interact, and that the autoinhibition mutants disrupt this interaction. Finally, RNA-seq and ChIP-seq studies show the importance of the Set2-Spt6 interaction for transcription and H3K36 methylation genome-wide. Taken together, our results support a model in which Set2 exists in an inactive, autoinhibited state in vivo through direct catalytic domain-AID interactions, with binding by Spt6 required to release this autoinhibited state.

Journal Article↗
PubMed2026

ATRX Condensates as Candidate Organizers of Enhancer-Centered Nuclear Microenvironments in Neural Progenitors: A Hypothesis for Enhancer-Associated ATRX Function in Neural Progenitors.

Neural progenitor cells (NPCs) must preserve lineage identity while remaining responsive to developmental cues. Here, we discuss the hypothesis that ATRX condensates help organize enhancer-centered nuclear microenvironments in NPCs. ATRX has long been studied in heterochromatin maintenance, histone variant deposition, and chromatin remodeling; earlier work has also shown that ATRX can occupy euchromatic and active regulatory regions and contribute to transcriptional regulation. Recent evidence in human NPCs indicates that ATRX forms nuclear puncta with condensate-like properties, associates with neurogenic enhancer-rich regions, and incorporates regulatory factors such as CHD7 and p300. Perturbation of ATRX condensate formation is associated with changes in enhancer-associated ATRX occupancy, neural gene-expression programs, and neuroepithelial organization, suggesting a regulatory mode that may complement canonical heterochromatin-associated functions. We propose a dual-mode model in which folded domains contribute to chromatin anchoring at repressive regions, whereas intrinsically disordered regions support condensate-associated organization at active developmental enhancers. We emphasize that whether ATRX condensates activate enhancers de novo, stabilize pre-existing enhancer states, buffer transcriptional variability, or primarily organize cofactor localization remains unresolved. We also discuss limitations of the current evidence and outline acute, locus-specific experiments needed to test the model.

X-linked Nuclear Protein↗
PubMed2026

Dynamic balance of CRISPR-Cas immunity and resistance plasmid anti-immunity mediated by a bifunctional protein AcrIE10.

Despite targeting by CRISPR-Cas system, antimicrobial resistance plasmids are prevalent in clinical isolates of carbapenem-resistant Klebsiella pneumoniae which represent a major public health threat. A stable co-existence of plasmids and CRISPR-Cas systems is mediated by anti-CRISPR (Acr) proteins. Here, we report that previously identified AcrIE10 encoded by a resistance plasmid combines two functions: it inhibits CRISPR immunity by directly binding Cas7* subunit through its Acr domain, and acts as an Acr-associated (Aca) protein that self-represses the transcription of Acr locus. AcrIE10 is an example of an Aca protein that utilizes N-terminal ribbon-helix-helix (RHH) domain to specifically recognize the inverted repeat (IR) region in its own promoter. Crucially, a dimerization of AcrIE10 dimers is required for the effective binding to the IR and self-repression, while stoichiometry-dependent interaction with Cas7* facilitates transition to de-repressed state. These findings elucidate molecular mechanisms by which AcrIE10 operates as a dual functionAcr-Aca protein to achieve a delicate balance between host CRISPR-Cas immunity and plasmid anti-defense.

Klebsiella pneumoniae↗
PubMed2024

Implications of noncoding regulatory functions in the development of insulinomas.

Insulinomas are rare neuroendocrine tumors arising from pancreatic β cells, characterized by aberrant proliferation and altered insulin secretion, leading to glucose homeostasis failure. With the aim of uncovering the role of noncoding regulatory regions and their aberrations in the development of these tumors, we coupled epigenetic and transcriptome profiling with whole-genome sequencing. As a result, we unraveled somatic mutations associated with changes in regulatory functions. Critically, these regions impact insulin secretion, tumor development, and epigenetic modifying genes, including polycomb complex components. Chromatin remodeling is apparent in insulinoma-selective domains shared across patients, containing a specific set of regulatory sequences dominated by the SOX17 binding motif. Moreover, many of these regions are H3K27me3 repressed in β cells, suggesting that tumoral transition involves derepression of polycomb-targeted domains. Our work provides a compendium of aberrant cis-regulatory elements affecting the function and fate of β cells in their progression to insulinomas and a framework to identify coding and noncoding driver mutations.

Humans↗
PubMed2024

Structure and Methyl-lysine Binding Selectivity of the HUSH Complex Subunit MPP8.

The Human Silencing Hub (HUSH) guards the genome from the pathogenic effects of retroelement expression. Composed of MPP8, TASOR, and Periphilin-1, HUSH recognizes actively transcribed retrotransposed sequences by the presence of long (>1.5-kb) nascent transcripts without introns. HUSH recruits effectors that alter chromatin structure, degrade transcripts, and deposit transcriptionally repressive epigenetic marks. Here, we report the crystal structure of the C-terminal domain (CTD) of MPP8 necessary for HUSH activity. The MPP8 CTD consists of five ankyrin repeats followed by a domain with structural homology to the PINIT domains of Siz/PIAS-family SUMO E3 ligases. AlphaFold3 modeling of the MPP8-TASOR complex predicts that a SPOC domain and a domain with a novel fold in TASOR form extended interaction interfaces with the MPP8 CTD. Point mutations at these interfaces resulted in loss of HUSH-dependent transcriptional repression in a cell-based reporter assay, validating the AlphaFold3 model. The MPP8 chromodomain, known to bind the repressive mark H3K9me3, bound with similar or higher affinity to sequences in the H3K9 methyltransferase subunits SETDB1, ATF7IP, G9a, and GLP. Hence, MPP8 promotes heterochromatinization by recruiting H3K9 methyltransferases. Our work identifies novel structural elements in MPP8 required for HUSH complex assembly and silencing, thereby fulfilling vital functions in controlling retrotransposons.

Humans↗
PubMed2026

Strigolactones constrain rice drought acclimation by suppressing ROS scavenging through the D53-OsWRKY31-ZFP36 module.

Strigolactones (SLs) are a class of plant hormones essential for tiller development and yield under diverse environmental conditions. Drought is a major limiting factor for rice yields. Although SLs contribute to drought resistance, mechanisms and practical applications of SL pathway in drought acclimation of rice remain poorly understood. Our study shows that short-term dehydration represses SL biosynthesis in rice roots. Genetic assays indicate that disruption of SL biosynthesis or signaling elevates rice drought resistance, whereas SL signaling activation or supplementation with the SL analog GR244DO impairs drought resistance. SLs negatively regulate drought acclimation by promoting degradation of the repressor protein DWARF53 (D53). D53 interacts with the transcription factor OsWRKY31 via its N-terminal domain and suppresses the protein level of OsWRKY31, which binds to and represses transcription of the ZFP36 promoter. ZFP36 encodes a zinc-finger transcription factor that promotes H2O2 scavenging to sustain reactive oxygen species (ROS) homeostasis during drought stress. Notably, the drought-resistant upland rice variety IRAT109 exhibits lower SL levels in root exudates than the lowland rice variety Nipponbare (NP). Genome editing of key components in SL pathway enhances drought resistance in NP, Huazhan (HZ), and IRAT109. The agronomic potential of tuning SL biosynthesis is further supported by the elite D17/HTD1 allele, which weakens SL biosynthesis and improves drought resistance and grain yield in Nekken 2 (NK2) under field conditions. These findings uncover a key mechanism underlying SL-repressed drought acclimation in rice and provide an effective strategy to improve drought resistance in diverse rice varieties amid ongoing climate change.

D53↗
PubMed2005

Molecular determinants for PspA-mediated repression of the AAA transcriptional activator PspF.

The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the sigma(54) subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of sigma(54) AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal alpha-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

Bacterial Proteins↗
PubMed2026

Loss of SUMOylation drives aberrant PRC1 clustering and 3D genome rewiring independent of H3K27me3.

Polycomb repressive complex 1 (PRC1) forms nuclear condensates that organize target chromatin domains. SUMOylation modulates PRC1 clustering, but its impact on condensate properties and 3D genome architecture remains unclear. Here, we show that depletion of small ubiquitin-like modifier (SUMO) in Drosophila wing imaginal discs transforms PRC1 condensates into large structures with reduced molecular dynamics. Biophysical modeling suggests that the changes in PRC1 self-interactions are responsible for the formation of large PRC1 condensates when SUMO is depleted. Interestingly, this biophysical reorganization occurs without global loss of the H3K27me3 mark. Instead, Hi-C reveals widespread rewiring of topologically associating domain (TAD) interactions. PRC1-bound TADs lose specific long-range contacts with each other while gaining ectopic interactions with active chromatin. These topological shifts correlate with gene misregulation independently of changes in Polycomb histone modifications. Our results establish SUMOylation as a critical regulator of PRC1 condensates, demonstrating that post-translational control of biomolecular condensation modulates 3D genome architecture and transcriptional output through mechanisms separable from histone mark deposition.

Animals↗
PubMed2026

HP1α binding creates a local barrier against transcription activation and persists during chromatin decondensation.

Mouse pericentric heterochromatin forms compacted, transcriptionally silent domains, termed chromocenters, that are enriched in heterochromatin protein 1 (HP1). Whether HP1α represses chromocenters by binding locally or by maintaining a phase-separated compartment is unresolved. We investigated this question by recruiting transcriptional activators to mouse fibroblast chromocenters and quantifying repression at a transcription reporter. HP1α established a promoter-proximal barrier that suppressed weaker activators (VP16) but was overcome by stronger ones (VP64-p65-Rta [VPR] and p65). Activator-induced decondensation and transcription occurred without displacing HP1α or H3K9 trimethylation, and HP1α retained its granular distribution and stoichiometric binding dynamics. Multi-color super-resolution imaging revealed spatial segregation of transcribed and HP1α-bound regions at the nanodomain scale. A nanodomain model captured how repeat clusters independently transition between silenced and activated states. These data establish that HP1α and H3K9me3 act through independently switchable nanodomains, accounting for chromocenter regulation without invoking phase separation.

Animals↗