Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity.

Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.

CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization.

Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.

Lamina-Associated Domains: Links with Chromosome Architecture, Heterochromatin, and Gene Repression.

In metazoan cell nuclei, hundreds of large chromatin domains are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to help organize chromosomes inside the nucleus and have been associated with gene repression. Here, we discuss the properties of LADs, the molecular mechanisms that determine their association with the nuclear lamina, their dynamic links with other nuclear compartments, and their proposed roles in gene regulation.

Swi/Snf chromatin remodeling regulates transcriptional interference and gene repression.

Alternative transcription start sites can affect transcript isoform diversity and translation levels. In a recently described form of gene regulation, coordinated transcriptional and translational interference results in transcript isoform-dependent changes in protein expression. Specifically, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, interfering with expression of the gene-proximal promoter. Although transcriptional and chromatin features associated with LUTI expression have been described, the mechanism underlying LUTI-based transcriptional interference is not well understood. Using an unbiased genetic approach followed by functional genomics, we uncovered that the Swi/Snf chromatin remodeling complex is required for co-transcriptional nucleosome remodeling that leads to LUTI-based repression. We identified genes with tandem promoters that rely on Swi/Snf function for transcriptional interference during protein folding stress, including LUTI-regulated genes. This study provides clear evidence for Swi/Snf playing a direct role in gene repression via a cis transcriptional interference mechanism.

Modularity of PRC1 composition and chromatin interaction define condensate properties.

Polycomb repressive complexes (PRCs) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells that are proposed to contribute to the maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using a reconstitution approach and single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. We find that the exact combination of PHC and CBX subunits determines condensate initiation, morphology, stability, and dynamics. Particularly, PHC2's polymerization activity influences condensate dynamics by promoting the formation of distinct domains that adhere to each other but do not coalesce. Live-cell imaging confirms CBX's role in condensate initiation and highlights PHC's importance for condensate stability. We propose that PRC1 composition can modulate condensate properties, providing crucial regulatory flexibility across developmental stages.

Dynamic BTB-domain filaments promote clustering of ZBTB proteins.

The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.

A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells.

The CBX family of proteins is central to proper mammalian development via key roles in Polycomb-mediated maintenance of repression. CBX proteins in differentiated lineages have chromatin compaction and phase separation activities that might contribute to maintaining repressed chromatin. The predominant CBX protein in pluripotent cells, CBX7, lacks the domain required for these activities. We inserted this functional domain into CBX7 in embryonic stem cells (ESCs) to test the hypothesis that it contributes a key epigenetic function. ESCs expressing this chimeric CBX7 were impaired in their ability to properly form embryoid bodies and neural progenitor cells and showed reduced activation of lineage-specific genes across differentiation. Neural progenitors exhibited a corresponding inappropriate maintenance of Polycomb binding at neural-specific loci over the course of differentiation. We propose that a switch in the ability to compact and phase separate is a central aspect of Polycomb group function during the transition from pluripotency to differentiated lineages.

Chromatin protein complexes involved in gene repression in lamina-associated domains.

Lamina-associated domains (LADs) are large chromatin regions that are associated with the nuclear lamina (NL) and form a repressive environment for transcription. The molecular players that mediate gene repression in LADs are currently unknown. Here, we performed FACS-based whole-genome genetic screens in human cells using LAD-integrated fluorescent reporters to identify such regulators. Surprisingly, the screen identified very few NL proteins, but revealed roles for dozens of known chromatin regulators. Among these are the negative elongation factor (NELF) complex and interacting factors involved in RNA polymerase pausing, suggesting that regulation of transcription elongation is a mechanism to repress transcription in LADs. Furthermore, the chromatin remodeler complex BAF and the activation complex Mediator can work both as activators and repressors in LADs, depending on the local context and possibly by rewiring heterochromatin. Our data indicate that the fundamental regulators of transcription and chromatin remodeling, rather than interaction with NL proteins, play a major role in transcription regulation within LADs.

Replicational Dilution of H3K27me3 in Mammalian Cells and the Role of Poised Promoters.

Polycomb repressive complex 2 (PRC2) places H3K27me3 at developmental genes and is causally implicated in keeping bivalent genes silent. It is unclear if that silence requires minimum H3K27me3 levels and how the mark transmits faithfully across mammalian somatic cell generations. Mouse intestinal cells lacking EZH2 methyltransferase reduce H3K27me3 proportionately at all PRC2 target sites, but ∼40% uniform residual levels keep target genes inactive. These genes, derepressed in PRC2-null villus cells, remain silent in intestinal stem cells (ISCs). Quantitative chromatin immunoprecipitation and computational modeling indicate that because unmodified histones dilute H3K27me3 by 50% each time DNA replicates, PRC2-deficient ISCs initially retain sufficient H3K27me3 to avoid gene derepression. EZH2 mutant human lymphoma cells also require multiple divisions before H3K27me3 dilution relieves gene silencing. In both cell types, promoters with high basal H3K4me2/3 activate in spite of some residual H3K27me3, compared to less-poised promoters. These findings have implications for PRC2 inhibition in cancer therapy.

A central role for canonical PRC1 in shaping the 3D nuclear landscape.

Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system.

BRD4-PRC2 represses transcription of T-helper 2-specific negative regulators during T-cell differentiation.

BRD4 is a well-recognized transcriptional activator, but how it regulates gene transcriptional repression in a cell type-specific manner has remained elusive. In this study, we report that BRD4 works with Polycomb repressive complex 2 (PRC2) to repress transcriptional expression of the T-helper 2 (Th2)-negative regulators Foxp3 and E3-ubiqutin ligase Fbxw7 during lineage-specific differentiation of Th2 cells from mouse primary naïve CD4+ T cells. Brd4 binds to the lysine-acetylated-EED subunit of the PRC2 complex via its second bromodomain (BD2) to facilitate histone H3 lysine 27 trimethylation (H3K27me3) at target gene loci and thereby transcriptional repression. We found that Foxp3 represses transcription of Th2-specific transcription factor Gata3, while Fbxw7 promotes its ubiquitination-directed protein degradation. BRD4-mediated repression of Foxp3 and Fbxw7 in turn promotes BRD4- and Gata3-mediated transcriptional activation of Th2 cytokines including Il4, Il5, and Il13. Chemical inhibition of the BRD4 BD2 induces transcriptional de-repression of Foxp3 and Fbxw7, and thus transcriptional downregulation of Il4, Il5, and Il13, resulting in inhibition of Th2 cell lineage differentiation. Our study presents a previously unappreciated mechanism of BRD4's role in orchestrating a Th2-specific transcriptional program that coordinates gene repression and activation, and safeguards cell lineage differentiation.

Phase separation of Polycomb-repressive complex 1 is governed by a charged disordered region of CBX2.

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb-repressive complex 1 (PRC1) family complexes are central to maintaining this repression. These include a set of canonical PRC1 complexes, each of which contains four core proteins, including one from the CBX family. These complexes have been shown previously to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus. We show here that reconstituted PRC1 readily phase-separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of Cbx2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells and have been shown previously to eliminate nucleosome compaction in vitro and generate axial patterning defects in mice. Thus, the domain of CBX2 that is important for phase separation is the same domain shown previously to be important for chromatin compaction and proper development, raising the possibility of a mechanistic or evolutionary link between these activities.

The yin and yang of pioneer transcription factors: Dual roles in repression and activation.

Pioneer transcription factors, by virtue of their ability to target nucleosomal DNA in silent chromatin, play crucial roles in eliciting cell fate decisions during development and cellular reprogramming. In addition to their well-established role in chromatin opening to activate gene expression programs, recent studies have demonstrated that pioneer factors have the complementary function of being able to silence the starting cell identity through targeted chromatin repression. Given recent discoveries regarding the repressive aspect of pioneer function, we discuss the basis by which pioneer factors can suppress alternative lineage programs in the context of cell fate control.

Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes.

Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin. These classifications are more accurate than the histone marks alone in predicting transcriptional silence and resistance of alternate fate genes to activation during direct cell conversion. Our proteomics of H3K9me3-marked srHC and functional screens revealed diverse proteins, including RBMX and RBMXL1, that impede gene induction during cellular reprogramming. Isolation of srHC with gradient-seq provides a genome-wide map of chromatin structure, elucidating subtypes of repressed domains that are uniquely predictive of diverse other chromatin properties.

Polycomb Repressive Complex 2 Methylates Elongin A to Regulate Transcription.

Polycomb repressive complex 2 (PRC2-EZH2) methylates histone H3 at lysine 27 (H3K27) and is required to maintain gene repression during development. Misregulation of PRC2 is linked to a range of neoplastic malignancies, which is believed to involve methylation of H3K27. However, the full spectrum of non-histone substrates of PRC2 that might also contribute to PRC2 function is not known. We characterized the target recognition specificity of the PRC2 active site and used the resultant data to screen for uncharacterized potential targets. The RNA polymerase II (Pol II) transcription elongation factor, Elongin A (EloA), is methylated by PRC2 in vivo. Mutation of the methylated EloA residue decreased repression of a subset of PRC2 target genes as measured by both steady-state and nascent RNA levels and perturbed embryonic stem cell differentiation. We propose that PRC2 modulates transcription of a subset of low expression target genes in part via methylation of EloA.

Direct and indirect gene repression by the ecdysone cascade during mosquito reproductive cycle.

SignificanceHematophagous Aedes aegypti mosquitoes spread devastating viral diseases. Upon blood feeding, a steroid hormone, 20-hydroxyecdysone (20E), initiates a reproductive program during which thousands of genes are differentially expressed. While 20E-mediated gene activation is well known, repressive action by this hormone remains poorly understood. Using bioinformatics and molecular biological approaches, we have identified the mechanisms of 20E-dependent direct and indirect transcriptional repression by the ecdysone receptor (EcR). While indirect repression involves E74, EcR binds to an ecdysone response element different from those utilized in 20E-mediated gene activation to exert direct repressive action. Moreover, liganded EcR recruits a corepressor Mi2, initiating chromatin compaction. This study advances our understanding of the 20E-EcR repression mechanism and could lead to improved vector control approaches.

Repression of Acinetobacter baumannii DNA damage response requires DdrR-assisted binding of UmuDAb dimers to atypical SOS box.

The DNA damage response of the multi-drug-resistant nosocomial pathogen Acinetobacter baumannii possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to Acinetobacter (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb's binding to its proposed operator present upstream of the six co-repressed umuDC or umuC genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCEAcinetobacter baumannii is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.

Sox2-Evf2 lncRNA-mediated mechanisms of chromosome topological control in developing forebrain.

The Evf2 long non-coding RNA directs Dlx5/6 ultraconserved enhancer(UCE)-intrachromosomal interactions, regulating genes across a 27 Mb region on chromosome 6 in mouse developing forebrain. Here, we show that Evf2 long-range gene repression occurs through multi-step mechanisms involving the transcription factor Sox2. Evf2 directly interacts with Sox2, antagonizing Sox2 activation of Dlx5/6UCE, and recruits Sox2 to the Dlx5/6eii shadow enhancer and key Dlx5/6UCE interaction sites. Sox2 directly interacts with Dlx1 and Smarca4, as part of the Evf2 ribonucleoprotein complex, forming spherical subnuclear domains (protein pools, PPs). Evf2 targets Sox2 PPs to one long-range repressed target gene (Rbm28), at the expense of another (Akr1b8). Evf2 and Sox2 shift Dlx5/6UCE interactions towards Rbm28, linking Evf2/Sox2 co-regulated topological control and gene repression. We propose a model that distinguishes Evf2 gene repression mechanisms at Rbm28 (Dlx5/6UCE position) and Akr1b8 (limited Sox2 availability). Genome-wide control of RNPs (Sox2, Dlx and Smarca4) shows that co-recruitment influences Sox2 DNA binding. Together, these data suggest that Evf2 organizes a Sox2 PP subnuclear domain and, through Sox2-RNP sequestration and recruitment, regulates chromosome 6 long-range UCE targeting and activity with genome-wide consequences.

Native CRISPR-Cas-based programmable multiplex gene repression in Klebsiella variicola.

Klebsiella variicola is a Gram-negative bacterium that is frequently isolated from a wide variety of natural niches. It is a ubiquitous opportunistic pathogen that can cause diverse infections in plants, animals, and humans. It also has significant biotechnological potential. However, due to the lack of efficient genetic tools, the molecular basis contributing to the pathogenesis and beneficial activities of K. variicola remains poorly understood. In this study, we found and characterized a native type I-E CRISPR-Cas system in a recently isolated K. variicola strain KV-1. The system cannot cleave target DNA sequences due to the inactivation of the Cas3 nuclease by a transposable element but retains the activity of the crRNA-guided Cascade binding to the target DNA sequence. A targeting plasmid carrying a mini-CRISPR to encode a crRNA was designed and introduced into the KV-1 strain, which successfully repurposed the native type I-E CRISPR-Cas system to inhibit the expression of the target gene efficiently and specifically. Moreover, by creating a mini-CRISPR to encode multiple crRNAs, multiplex gene repression was achieved by providing a single targeting plasmid. This work provides the first native CRISPR-Cas-based tool for programmable multiplex gene repression in K. variicola, which will facilitate studying the pathogenic mechanism of K. variicola and enable metabolic engineering to produce valuable bioproducts.

CaMKIV-Mediated Phosphorylation Inactivates Freud-1/CC2D1A Repression for Calcium-Dependent 5-HT1A Receptor Gene Induction.

Calcium calmodulin-dependent protein kinase (CaMK) mediates calcium-induced neural gene activation. CaMK also inhibits the non-syndromic intellectual disability gene, Freud-1/CC2D1A, a transcriptional repressor of human serotonin-1A (5-HT1A) and dopamine-D2 receptor genes. The altered expression of these Freud-1-regulated genes is implicated in mental illnesses such as major depression and schizophrenia. We hypothesized that Freud-1 is blocked by CaMK-induced phosphorylation. The incubation of purified Freud-1 with either CaMKIIα or CaMKIV increased Freud-1 phosphorylation that was partly prevented in Freud-1-Ser644Ala and Freud-1-Thr780Ala CaMK site mutants. In human SK-N-SH neuroblastoma cells, active CaMKIV induced the serine and threonine phosphorylation of Freud-1, and specifically increased Freud-1-Thr780 phosphorylation in transfected HEK-293 cells. The activation of purified CaMKIIα or CaMKIV reduced Freud-1 binding to its DNA element on the 5-HT1A and dopamine-D2 receptor genes. In SK-N-SH cells, active CaMKIV but not CaMKIIα blocked the Freud-1 repressor activity, while Freud-1 Ser644Ala, Thr780Ala or dual mutants were resistant to inhibition by activated CaMKIV or calcium mobilization. These results indicate that the Freud-1 repressor activity is blocked by CaMKIV-induced phosphorylation at Thr780, resulting in the up-regulation of the target genes, such as the 5-HT1A receptor gene. The CaMKIV-mediated inhibition of Freud-1 provides a novel de-repression mechanism to induce 5-HT1A receptor expression for the regulation of cognitive development, behavior and antidepressant response.