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Mechanical and structural properties of archaeal hypernucleosomes

2020-12-21T00:00

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone–DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an ‘endless’ histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB–DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

Conserved DNA sequence features underlie pervasive RNA polymerase pausing

2021-03-31T00:00

Pausing of transcribing RNA polymerase is regulated and creates opportunities to control gene expression. Research in metazoans has so far mainly focused on RNA polymerase II (Pol II) promoter-proximal pausing leaving the pervasive nature of pausing and its regulatory potential in mammalian cells unclear. Here, we developed a pause detecting algorithm (PDA) for nucleotide-resolution occupancy data and a new native elongating transcript sequencing approach, termed nested NET-seq, that strongly reduces artifactual peaks commonly misinterpreted as pausing sites. Leveraging PDA and nested NET-seq reveal widespread genome-wide Pol II pausing at single-nucleotide resolution in human cells. Notably, the majority of Pol II pauses occur outside of promoter-proximal gene regions primarily along the gene-body of transcribed genes. Sequence analysis combined with machine learning modeling reveals DNA sequence properties underlying widespread transcriptional pausing including a new pause motif. Interestingly, key sequence determinants of RNA polymerase pausing are conserved between human cells and bacteria. These studies indicate pervasive sequence-induced transcriptional pausing in human cells and the knowledge of exact pause locations implies potential functional roles in gene expression.

INT-Hi-C reveals distinct chromatin architecture in endosperm and leaf tissues of Arabidopsis

2021-03-21T00:00

Higher-order chromatin structure undergoes striking changes in response to various developmental and environmental signals, causing distinct cell types to adopt specific chromatin organization. High throughput chromatin conformation capture (Hi-C) allows studying higher-order chromatin structure; however, this technique requires substantial amounts of starting material, which has limited the establishment of cell type-specific higher-order chromatin structure in plants. To overcome this limitation, we established a protocol that is applicable to a limited amount of nuclei by combining the INTACT (isolation of nuclei tagged in specific cell types) method and Hi-C (INT-Hi-C). Using this INT-Hi-C protocol, we generated Hi-C data from INTACT purified endosperm and leaf nuclei. Our INT-Hi-C data from leaf accurately reiterated chromatin interaction patterns derived from conventional leaf Hi-C data. We found that the higher-order chromatin organization of mixed leaf tissues and endosperm differs and that DNA methylation and repressive histone marks positively correlate with the chromatin compaction level. We furthermore found that self-looped interacting genes have increased expression in leaves and endosperm and that interacting intergenic regions negatively impact on gene expression in the endosperm. Last, we identified several imprinted genes involved in long-range and trans interactions exclusively in endosperm. Our study provides evidence that the endosperm adopts a distinct higher-order chromatin structure that differs from other cell types in plants and that chromatin interactions influence transcriptional activity.

Myod1 and GR coordinate myofiber-specific transcriptional enhancers

2021-04-09T00:00

Skeletal muscle is a dynamic tissue the size of which can be remodeled through the concerted actions of various cues. Here, we investigated the skeletal muscle transcriptional program and identified key tissue-specific regulatory genetic elements. Our results show that Myod1 is bound to numerous skeletal muscle enhancers in collaboration with the glucocorticoid receptor (GR) to control gene expression. Remarkably, transcriptional activation controlled by these factors occurs through direct contacts with the promoter region of target genes, via the CpG-bound transcription factor Nrf1, and the formation of Ctcf-anchored chromatin loops, in a myofiber-specific manner. Moreover, we demonstrate that GR negatively controls muscle mass and strength in mice by down-regulating anabolic pathways. Taken together, our data establish Myod1, GR and Nrf1 as key players of muscle-specific enhancer-promoter communication that orchestrate myofiber size regulation.

A conserved BAH module within mammalian BAHD1 connects H3K27me3 to Polycomb gene silencing

2021-04-06T00:00

Trimethylation of histone H3 lysine 27 (H3K27me3) is important for gene silencing and imprinting, (epi)genome organization and organismal development. In a prevalent model, the functional readout of H3K27me3 in mammalian cells is achieved through the H3K27me3-recognizing chromodomain harbored within the chromobox (CBX) component of canonical Polycomb repressive complex 1 (cPRC1), which induces chromatin compaction and gene repression. Here, we report that binding of H3K27me3 by a Bromo Adjacent Homology (BAH) domain harbored within BAH domain-containing protein 1 (BAHD1) is required for overall BAHD1 targeting to chromatin and for optimal repression of the H3K27me3-demarcated genes in mammalian cells. Disruption of direct interaction between BAHD1^BAH and H3K27me3 by point mutagenesis leads to chromatin remodeling, notably, increased histone acetylation, at its Polycomb gene targets. Mice carrying an H3K27me3-interaction-defective mutation of Bahd1^BAH causes marked embryonic lethality, showing a requirement of this pathway for normal development. Altogether, this work demonstrates an H3K27me3-initiated signaling cascade that operates through a conserved BAH ‘reader’ module within BAHD1 in mammals.

Graphical Abstract

A mammalian H3K27me3-transduction pathway operates through an H3K27me3-specific ‘reader’ module (BAH) of BAHD1, which assembles a complex with corepressors (HDACs and others) for suppressing histone acetylation and repressing expression at Polycomb target genes.

Bimodal regulation of the PRC2 complex by USP7 underlies tumorigenesis

2021-04-13T00:00

Although overexpression of EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is an eminent feature of various cancers, the regulation of its abundance and function remains insufficiently understood. We report here that the PRC2 complex is physically associated with ubiquitin-specific protease USP7 in cancer cells where USP7 acts to deubiquitinate and stabilize EZH2. Interestingly, we found that USP7-catalyzed H2BK120ub1 deubiquitination is a prerequisite for chromatin loading of PRC2 thus H3K27 trimethylation, and this process is not affected by H2AK119 ubiquitination catalyzed by PRC1. Genome-wide analysis of the transcriptional targets of the USP7/PRC2 complex identified a cohort of genes including FOXO1 that are involved in cell growth and proliferation. We demonstrated that the USP7/PRC2 complex drives cancer cell proliferation and tumorigenesis in vitro and in vivo. We showed that the expression of both USP7 and EZH2 elevates during tumor progression, corresponding to a diminished FOXO1 expression, and the level of the expression of USP7 and EZH2 strongly correlates with histological grades and prognosis of tumor patients. These results reveal a dual role for USP7 in the regulation of the abundance and function of EZH2, supporting the pursuit of USP7 as a therapeutic target for cancer intervention.

A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation

2021-04-06T00:00

The lysine specific demethylase 1 (LSD1) plays a pivotal role in cellular differentiation by regulating the expression of key developmental genes in concert with different coregulatory proteins. This process is impaired in different cancer types and incompletely understood. To comprehensively identify functional coregulators of LSD1, we established a novel tractable fluorescent reporter system to monitor LSD1 activity in living cells. Combining this reporter system with a state-of-the-art multiplexed RNAi screen, we identify the DEAD-box helicase 19A (DDX19A) as a novel coregulator and demonstrate that suppression of Ddx19a results in an increase of R-loops and reduced LSD1-mediated gene silencing. We further show that DDX19A binds to tri-methylated lysine 27 of histone 3 (H3K27me3) and it regulates gene expression through the removal of transcription promoting R-loops. Our results uncover a novel transcriptional regulatory cascade where the downregulation of genes is dependent on the LSD1 mediated demethylation of histone H3 lysine 4 (H3K4). This allows the polycomb repressive complex 2 (PRC2) to methylate H3K27, which serves as a binding site for DDX19A. Finally, the binding of DDX19A leads to the efficient removal of R-loops at active promoters, which further de-represses LSD1 and PRC2, establishing a positive feedback loop leading to a robust repression of the target gene.

Graphical Abstract

Model of the regulatory cascade for transcriptional repression, which depends on the removal of transcription-associated R-loops downstream of LSD1 activity.

Mechanism of REST/NRSF regulation of clustered protocadherin α genes

2021-04-13T00:00

Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5–1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDHα genes. Thus, REST/NRSF represses PCDHα gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

A phage satellite tunes inducing phage gene expression using a domesticated endonuclease to balance inhibition and virion hijacking

2021-04-06T00:00

Bacteria persist under constant threat of predation by bacterial viruses (phages). Bacteria-phage conflicts result in evolutionary arms races often driven by mobile genetic elements (MGEs). One such MGE, a phage satellite in Vibrio cholerae called PLE, provides specific and robust defense against a pervasive lytic phage, ICP1. The interplay between PLE and ICP1 has revealed strategies for molecular parasitism allowing PLE to hijack ICP1 processes in order to mobilize. Here, we describe the mechanism of PLE-mediated transcriptional manipulation of ICP1 structural gene transcription. PLE encodes a novel DNA binding protein, CapR, that represses ICP1’s capsid morphogenesis operon. Although CapR is sufficient for the degree of capsid repression achieved by PLE, its activity does not hinder the ICP1 lifecycle. We explore the consequences of repression of this operon, demonstrating that more stringent repression achieved through CRISPRi restricts both ICP1 and PLE. We also discover that PLE transduces in modified ICP1-like particles. Examination of CapR homologs led to the identification of a suite of ICP1-encoded homing endonucleases, providing a putative origin for the satellite-encoded repressor. This work unveils a facet of the delicate balance of satellite-mediated inhibition aimed at blocking phage production while successfully mobilizing in a phage-derived particle.

Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

2021-03-22T00:00

The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

SDC mediates DNA methylation-controlled clock pace by interacting with ZTL in Arabidopsis

2021-03-01T00:00

Molecular bases of eukaryotic circadian clocks mainly rely on transcriptional-translational feedback loops (TTFLs), while epigenetic codes also play critical roles in fine-tuning circadian rhythms. However, unlike histone modification codes that play extensive and well-known roles in the regulation of circadian clocks, whether DNA methylation (5mC) can affect the circadian clock, and the associated underlying molecular mechanisms, remains largely unexplored in many organisms. Here we demonstrate that global genome DNA hypomethylation can significantly lengthen the circadian period of Arabidopsis. Transcriptomic and genetic evidence demonstrate that SUPPRESSOR OF drm1 drm2 cmt3 (SDC), encoding an F-box containing protein, is required for the DNA hypomethylation-tuned circadian clock. Moreover, SDC can physically interact with another F-box containing protein ZEITLUPE (ZTL) to diminish its accumulation. Genetic analysis further revealed that ZTL and its substrate TIMING OF CAB EXPRESSION 1 (TOC1) likely act downstream of DNA methyltransferases to control circadian rhythm. Together, our findings support the notion that DNA methylation is important to maintain proper circadian pace in Arabidopsis, and further established that SDC links DNA hypomethylation with a proteolytic cascade to assist in tuning the circadian clock.

Methylation-directed acetylation of histone H3 regulates developmental sensitivity to histone deacetylase inhibition

2021-03-15T00:00

Hydroxamate-based lysine deacetylase inhibitors (KDACis) are approved for clinical use against certain cancers. However, intrinsic and acquired resistance presents a major problem. Treatment of cells with hydroxamates such as trichostatin A (TSA) leads to rapid preferential acetylation of histone H3 already trimethylated on lysine 4 (H3K4me3), although the importance of this H3K4me3-directed acetylation in the biological consequences of KDACi treatment is not known. We address this utilizing Dictyostelium discoideum strains lacking H3K4me3 due to disruption of the gene encoding the Set1 methyltransferase or mutations in endogenous H3 genes. Loss of H3K4me3 confers resistance to TSA-induced developmental inhibition and delays accumulation of H3K9Ac and H3K14Ac. H3K4me3-directed H3Ac is mediated by Sgf29, a subunit of the SAGA acetyltransferase complex that interacts with H3K4me3 via a tandem tudor domain (TTD). We identify an Sgf29 orthologue in Dictyostelium with a TTD that specifically recognizes the H3K4me3 modification. Disruption of the gene encoding Sgf29 delays accumulation of H3K9Ac and abrogates H3K4me3-directed H3Ac. Either loss or overexpression of Sgf29 confers developmental resistance to TSA. Our results demonstrate that rapid acetylation of H3K4me3 histones regulates developmental sensitivity to TSA. Levels of H3K4me3 or Sgf29 will provide useful biomarkers for sensitivity to this class of chemotherapeutic drug.

An RNA-binding protein complex regulates the purine-dependent expression of a nucleobase transporter in trypanosomes

2021-03-21T00:00

Post-transcriptional regulation of gene expression is particularly important in trypanosomatid protozoa. RNA-binding proteins (RBPs) regulate mRNA stability and translation, yet information about how RBPs are able to link environmental cues to post-transcriptional control is scarce. In Trypanosoma brucei, we have previously characterized a short RNA stem-loop cis-element (PuRE, Purine Responsive Element) within the 3′-UTR of the NT8 nucleobase transporter mRNA that is necessary and sufficient to confer a strong repression of gene expression in response to purines. In this study, we have identified a protein complex composed of two RNA-binding proteins (PuREBP1 and PuREBP2) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNA interference results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression.

MKRN3-mediated ubiquitination of Poly(A)-binding proteins modulates the stability and translation of GNRH1 mRNA in mammalian puberty

2021-03-21T00:00

The family of Poly(A)-binding proteins (PABPs) regulates the stability and translation of messenger RNAs (mRNAs). Here we reported that the three members of PABPs, including PABPC1, PABPC3 and PABPC4, were identified as novel substrates for MKRN3, whose deletion or loss-of-function mutations were genetically associated with human central precocious puberty (CPP). MKRN3-mediated ubiquitination was found to attenuate the binding of PABPs to the poly(A) tails of mRNA, which led to shortened poly(A) tail-length of GNRH1 mRNA and compromised the formation of translation initiation complex (TIC). Recently, we have shown that MKRN3 epigenetically regulates the transcription of GNRH1 through conjugating poly-Ub chains onto methyl-DNA bind protein 3 (MBD3). Therefore, MKRN3-mediated ubiquitin signalling could control both transcriptional and post-transcriptional switches of mammalian puberty initiation. While identifying MKRN3 as a novel tissue-specific translational regulator, our work also provided new mechanistic insights into the etiology of MKRN3 dysfunction-associated human CPP.

Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners

2021-03-21T00:00

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix α1 and loop α1α2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between the phosphorylated residues and the positively charged C-terminal helix α6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a 5000-fold loss of affinity for dsDNA. However, it does not impair binding to lamin A/C Igfold domain and emerin nucleoplasmic region, which leaves open the question of the regulation of these interactions.

Regulator-dependent temporal dynamics of a restriction-modification system's gene expression upon entering new host cells: single-cell and population studies

2021-03-21T00:00

Restriction-modification (R-M) systems represent a first line of defense against invasive DNAs, such as bacteriophage DNAs, and are widespread among bacteria and archaea. By acquiring a Type II R-M system via horizontal gene transfer, the new hosts generally become more resistant to phage infection, through the action of a restriction endonuclease (REase), which cleaves DNA at or near specific sequences. A modification methyltransferase (MTase) serves to protect the host genome against its cognate REase activity. The production of R-M system components upon entering a new host cell must be finely tuned to confer protective methylation before the REase acts, to avoid host genome damage. Some type II R-M systems rely on a third component, the controller (C) protein, which is a transcription factor that regulates the production of REase and/or MTase. Previous studies have suggested C protein effects on the dynamics of expression of an R-M system during its establishment in a new host cell. Here, we directly examine these effects. By fluorescently labelling REase and MTase, we demonstrate that lack of a C protein reduces the delay of REase production, to the point of being simultaneous with, or even preceding, production of the MTase. Single molecule tracking suggests that a REase and a MTase employ different strategies for their target search within host cells, with the MTase spending much more time diffusing in proximity to the nucleoid than does the REase. This difference may partially ameliorate the toxic effects of premature REase expression.

Human METTL18 is a histidine-specific methyltransferase that targets RPL3 and affects ribosome biogenesis and function

2021-03-08T00:00

Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance.

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation

2021-03-01T00:00

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV’s lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >10⁵ binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV’s lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV’s lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.

TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei

2021-02-28T00:00

The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more ‘metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.

BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract

2021-02-28T00:00

The tumor suppressor BRCA1 is considered a master regulator of genome integrity. Although widely recognized for its DNA repair functions, BRCA1 has also been implicated in various mechanisms of chromatin remodeling and transcription regulation. However, the precise role that BRCA1 plays in these processes has been difficult to establish due to the widespread consequences of its cellular dysfunction. Here, we use nucleoplasmic extract derived from the eggs of Xenopus laevis to investigate the role of BRCA1 in a cell-free transcription system. We report that BRCA1-BARD1 suppresses transcription initiation independent of DNA damage signaling and its established role in histone H2A ubiquitination. BRCA1-BARD1 acts through a histone intermediate, altering acetylation of histone H4K8 and recruitment of the chromatin reader and oncogene regulator BRD4. Together, these results establish a functional relationship between an established (BRCA1) and emerging (BRD4) regulator of genome integrity.

Graphical Abstract

BRCA1-BARD1 regulates transcription initiation by limiting recruitment of the chromatin reader BRD4.

Binding site profiles and N-terminal minor groove interactions of the master quorum-sensing regulator LuxR enable flexible control of gene activation and repression

2021-03-08T00:00

LuxR is a TetR family master quorum sensing (QS) regulator activating or repressing expression of hundreds of genes that control collective behaviors in Vibrios with underlying mechanism unknown. To illuminate how this regulator controls expression of various target genes, we applied ChIP-seq and DNase I-seq technologies. Vibrio alginolyticus LuxR controls expression of ∼280 genes that contain either symmetric palindrome (repDNA) or asymmetric (actDNA) binding motifs with different binding profiles. The median number of LuxR binding sites for activated genes are nearly double for that of repressed genes. Crystal structures of LuxR in complex with the respective repDNA and actDNA motifs revealed a new mode of LuxR DNA binding that involves contacts of its N-terminal extension to the minor groove. The N-terminal contacts mediated by Arginine-9 and Arginine-11 differ when LuxR binds to repDNA vs actDNA, leading to higher binding affinity at repressed targets. Moreover, modification of LuxR binding sites, binding profiles, and N-terminal extension have important consequences on QS-regulated phenotypes. These results facilitate fundamental understanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in Vibrio QS, which may facilitate to design QS inhibiting chemicals that interfere with LuxR regulation to effectively control pathogens.

Non-perfectly matching small RNAs can induce stable and heritable epigenetic modifications and can be used as molecular markers to trace the origin and fate of silencing RNAs

2021-02-01T00:00

Short non-coding RNA molecules (sRNAs) play a fundamental role in gene regulation and development in higher organisms. They act as molecular postcodes and guide AGO proteins to target nucleic acids. In plants, sRNA-targeted mRNAs are degraded, reducing gene expression. In contrast, sRNA-targeted DNA sequences undergo cytosine methylation referred to as RNA-directed DNA methylation (RdDM). Cytosine methylation can suppress transcription, thus sRNAs are potent regulators of gene expression. sRNA-mediated RdDM is involved in genome stability through transposon silencing, mobile signalling for epigenetic gene control and hybrid vigour. Since cytosine methylation can be passed on to subsequent generations, RdDM contributes to transgenerational inheritance of the epigenome. Using a novel approach, which can differentiate between primary (inducer) and secondary (amplified) sRNAs, we show that initiation of heritable RdDM does not require complete sequence complementarity between the sRNAs and their nuclear target sequences. sRNAs with up to four regularly interspaced mismatches are potent inducers of RdDM, however, the number and disruptive nature of nucleotide polymorphisms negatively correlate with their efficacy. Our findings contribute to understanding how sRNA can directly shape the epigenome and may be used in designing the next generation of RNA silencing constructs.

PAUPAR and PAX6 sequentially regulate human embryonic stem cell cortical differentiation

2021-02-05T00:00

Long noncoding RNAs (lncRNAs) play a wide range of roles in the epigenetic regulation of crucial biological processes, but the functions of lncRNAs in cortical development are poorly understood. Using human embryonic stem cell (hESC)-based 2D neural differentiation approach and 3D cerebral organoid system, we identified that the lncRNA PAUPAR, which is adjacent to PAX6, plays essential roles in cortical differentiation by interacting with PAX6 to regulate the expression of a large number of neural genes. Mechanistic studies showed that PAUPAR confers PAX6 proper binding sites on the target neural genes by directly binding the genomic regions of these genes. Moreover, PAX6 recruits the histone methyltransferase NSD1 through its C-terminal PST enrichment domain, then regulate H3K36 methylation and the expression of target genes. Collectively, our data reveal that the PAUPAR/PAX6/NSD1 complex plays a critical role in the epigenetic regulation of hESC cortical differentiation and highlight the importance of PAUPAR as an intrinsic regulator of cortical differentiation.

Repression of a large number of genes requires interplay between homologous recombination and HIRA

2021-01-28T00:00

During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51–DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.

BACH1 recruits NANOG and histone H3 lysine 4 methyltransferase MLL/SET1 complexes to regulate enhancer–promoter activity and maintains pluripotency

2021-01-27T00:00

Maintenance of stem-cell identity requires proper regulation of enhancer activity. Both transcription factors OCT4/SOX2/NANOG and histone methyltransferase complexes MLL/SET1 were shown to regulate enhancer activity, but how they are regulated in embryonic stem cells (ESCs) remains further studies. Here, we report a transcription factor BACH1, which directly interacts with OCT4/SOX2/NANOG (OSN) and MLL/SET1 methyltransferase complexes and maintains pluripotency in mouse ESCs (mESCs). BTB domain and bZIP domain of BACH1 are required for these interactions and pluripotency maintenance. Loss of BACH1 reduced the interaction between NANOG and MLL1/SET1 complexes, and decreased their occupancy on chromatin, and further decreased H3 lysine 4 trimethylation (H3K4me3) level on gene promoters and (super-) enhancers, leading to decreased enhancer activity and transcription activity, especially on stemness-related genes. Moreover, BACH1 recruited NANOG through chromatin looping and regulated remote NANOG binding, fine-tuning enhancer–promoter activity and gene expression. Collectively, these observations suggest that BACH1 maintains pluripotency in ESCs by recruiting NANOG and MLL/SET1 complexes to chromatin and maintaining the trimethylated state of H3K4 and enhancer–promoter activity, especially on stemness-related genes.

SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites

2021-02-01T00:00

Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.

The metabolite methylglyoxal-mediated gene expression is associated with histone methylglyoxalation

2021-01-21T00:00

Methylglyoxal (MG) is a byproduct of glycolysis that functions in diverse mammalian developmental processes and diseases and in plant responses to various stresses, including salt stress. However, it is unknown whether MG-regulated gene expression is associated with an epigenetic modification. Here we report that MG methylglyoxalates H3 including H3K4 and increases chromatin accessibility, consistent with the result that H3 methylglyoxalation positively correlates with gene expression. Salt stress also increases H3 methylglyoxalation at salt stress responsive genes correlated to their higher expression. Following exposure to salt stress, salt stress responsive genes were expressed at higher levels in the Arabidopsis glyI2 mutant than in wild-type plants, but at lower levels in 35S::GLYI2 35S::GLYII4 plants, consistent with the higher and lower MG accumulation and H3 methylglyoxalation of target genes in glyI2 and 35S::GLYI2 35S::GLYII4, respectively. Further, ABI3 and MYC2, regulators of salt stress responsive genes, affect the distribution of H3 methylglyoxalation at salt stress responsive genes. Thus, MG functions as a histone-modifying group associated with gene expression that links glucose metabolism and epigenetic regulation.

Development of aptamer-based inhibitors for CRISPR/Cas system

2020-10-30T00:00

The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.

Super-enhancer mediated regulation of adult β-globin gene expression: the role of eRNA and Integrator

2021-01-21T00:00

Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine β-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous β-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3′eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3′eRNA leads to a reduction in β-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult β-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.

The complex architecture of p53 binding sites

2021-01-14T00:00

Sequence-specific protein-DNA interactions are at the heart of the response of the tumor-suppressor p53 to numerous physiological and stress-related signals. Large variability has been previously reported in p53 binding to and transactivating from p53 response elements (REs) due, at least in part, to changes in direct (base) and indirect (shape) readouts of p53 REs. Here, we dissect p53 REs to decipher the mechanism by which p53 optimizes this highly regulated variable level of interaction with its DNA binding sites. We show that hemi-specific binding is more prevalent in p53 REs than previously envisioned. We reveal that sequences flanking the REs modulate p53 binding and activity and show that these effects extend to 4–5 bp from the REs. Moreover, we show here that the arrangement of p53 half-sites within its REs, relative to transcription direction, has been fine-tuned by selection pressure to optimize and regulate the response levels from p53 REs. This directionality in the REs arrangement is at least partly encoded in the structural properties of the REs. Furthermore, we show here that in the p21-5′ RE the orientation of the half-sites is such that the effect of the flanking sequences is minimized and we discuss its advantages.

Vibrio cholerae FruR facilitates binding of RNA polymerase to the fru promoter in the presence of fructose 1-phosphate

2021-01-21T00:00

In most bacteria, efficient use of carbohydrates is primarily mediated by the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), which concomitantly phosphorylates the substrates during import. Therefore, transcription of the PTS-encoding genes is precisely regulated by transcriptional regulators, depending on the availability of the substrate. Fructose is transported mainly through the fructose-specific PTS (PTS^Fru) and simultaneously converted into fructose 1-phosphate (F1P). In Gammaproteobacteria such as Escherichia coli and Pseudomonas putida, transcription of the fru operon encoding two PTS^Fru components, FruA and FruB, and the 1-phosphofructokinase FruK is repressed by FruR in the absence of the inducer F1P. Here, we show that, contrary to the case in other Gammaproteobacteria, FruR acts as a transcriptional activator of the fru operon and is indispensable for the growth of Vibrio cholerae on fructose. Several lines of evidence suggest that binding of the FruR-F1P complex to an operator which is located between the –35 and –10 promoter elements changes the DNA structure to facilitate RNA polymerase binding to the promoter. We discuss the mechanism by which the highly conserved FruR regulates the expression of its target operon encoding the highly conserved PTS^Fru and FruK in a completely opposite direction among closely related families of bacteria.

The forkhead transcription factor FOXK2 premarks lineage-specific genes in human embryonic stem cells for activation during differentiation

2021-01-12T00:00

Enhancers play important roles in controlling gene expression in a choreographed spatial and temporal manner during development. However, it is unclear how these regulatory regions are established during differentiation. Here we investigated the genome-wide binding profile of the forkhead transcription factor FOXK2 in human embryonic stem cells (ESCs) and downstream cell types. This transcription factor is bound to thousands of regulatory regions in human ESCs, and binding at many sites is maintained as cells differentiate to mesendodermal and neural precursor cell (NPC) types, alongside the emergence of new binding regions. FOXK2 binding is generally associated with active histone marks in any given cell type. Furthermore newly acquired, or retained FOXK2 binding regions show elevated levels of activating histone marks following differentiation to NPCs. In keeping with this association with activating marks, we demonstrate a role for FOXK transcription factors in gene activation during NPC differentiation. FOXK2 occupancy in ESCs is therefore an early mark for delineating the regulatory regions, which become activated in later lineages.

Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae

2020-12-24T00:00

Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1^U and Gcr1^S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1^U or Gcr1^S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1^U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1^S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1^U, inhibits dimerization of Gcr1^U and even acts in trans to inhibit Gcr1^S dimerization. The Gcr1^S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1^U and Gcr1^S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.

Graphical Abstract

Gcr1^U and Gcr1^S, generated from un-spliced and spliced forms of mRNA, respectively, act as different complexes to activate expression of glycolytic genes.

Effects of codon usage on gene expression are promoter context dependent

2021-01-07T00:00

Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. Here we show that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, we showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, our results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression.

The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

2021-01-07T00:00

Pin1 is a peptidyl-prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II (Pol II) through interaction with the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol II. We demonstrated that this function is important for cellular response to oxidative stress in the fission yeast Schizosaccharomyces pombe. In response to oxidative stress, the Atf1 transcription factor targets Sty1, the mitogen-activated protein kinase (MAPK), to specific stress-responsive promoters. Anchored Sty1 recruits Pol II through direct association with Rpb1-CTD and phosphorylates the reiterated heptad sequence at Serine 5. Pin1 binds phosphorylated CTD to promote dissociation of Sty1 from it, and directly recruits Ssu72 phosphatase to facilitate dephosphorylation of CTD for transcription elongation. In the absence of Pin1, the association of Sty1-Atf1 with Rpb1 persists on stress-responsive promoters failed to generate transcripts of the corresponding genes effectively. The identified characteristic features of the fission yeast Pin1 are conserved in humans. We demonstrated that elevated Pin1 level in cancer cells might help to sustain survival under oxidative stress generated from their altered metabolic pathways. Together, these results suggest a conserved function of Pin1 in cellular response to oxidative stress among eukaryotic cells that might have clinical implication.

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma

2021-01-07T00:00

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.

Graphical Abstract

Scheme representing how G4-ligand-induced stabilization of MDM2 P2 promoter G4 leads to accumulation of p53 and hnRNP-K proteins, resulting in stimulation of apoptosis and cell cycle arrest.

Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases

2021-01-04T00:00

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.

The nucleoid-associated protein IHF acts as a ‘transcriptional domainin’ protein coordinating the bacterial virulence traits with global transcription

2020-12-18T00:00

Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαβ heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a ‘transcriptional domainin’ protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.

IncC helper dependent plasmid-like replication of Salmonella Genomic Island 1

2021-01-06T00:00

The Salmonella genomic island 1 (SGI1) and its variants are mobilized by IncA and IncC conjugative plasmids. SGI1-family elements and their helper plasmids are effective transporters of multidrug resistance determinants. SGI1 exploits the transfer apparatus of the helper plasmid and hijacks its activator complex, AcaCD, to trigger the expression of several SGI1 genes. In this way, SGI1 times its excision from the chromosome to the helper entry and expresses mating pore components that enhance SGI1 transfer. The SGI1-encoded T4SS components and the FlhDC-family activator proved to be interchangeable with their IncC-encoded homologs, indicating multiple interactions between SGI1 and its helpers. As a new aspect of this crosstalk, we report here the helper-induced replication of SGI1, which requires both activators, AcaCD and FlhDC_SGI1, and significantly increases the stability of SGI1 when coexists with the helper plasmid. We have identified the oriV_SGI1 and shown that S004-repA operon encodes for a translationally coupled leader protein and an IncN2/N3-related RepA that are expressed under the control of the AcaCD-responsive promoter P_S004. This replicon transiently maintains SGI1 as a 4–8-copy plasmid, not only stabilizing the island but also contributing to the fast displacement of the helper plasmid.

Dynamics of TRF1 organizing a single human telomere

2020-12-21T00:00

Chromosome stability is primarily determined by telomere length. TRF1 is the core subunit of shelterin that plays a critical role in telomere organization and replication. However, the dynamics of TRF1 in scenarios of telomere-processing activities remain elusive. Using single-molecule magnetic tweezers, we here investigated the dynamics of TRF1 upon organizing a human telomere and the protein-DNA interactions at a moving telomeric fork. We first developed a method to obtain telomeres from human cells for directly measuring the telomere length by single-molecule force spectroscopy. Next, we examined the compaction and decompaction of a telomere by TRF1 dimers. TRF1 dissociates from a compacted telomere with heterogenous loops in ∼20 s. We also found a negative correlation between the number of telomeric loops and loop sizes. We further characterized the dynamics of TRF1 at a telomeric DNA fork. With binding energies of 11 k_BT, TRF1 can modulate the forward and backward steps of DNA fork movements by 2–9 s at a critical force of F_1/2, temporarily maintaining the telomeric fork open. Our results shed light on the mechanisms of how TRF1 organizes human telomeres and facilitates the efficient replication of telomeric DNA. Our work will help future research on the chemical biology of telomeres and shelterin-targeted drug discovery.

Graphical Abstract

We use single-molecule magnetic tweezers to precisely measure the absolute length of single human telomeres and reveal the dynamics of TRF1 upon organizing a telomere.

The transcriptional repressors VAL1 and VAL2 recruit PRC2 for genome-wide Polycomb silencing in Arabidopsis

2020-12-03T00:00

The Polycomb repressive complex 2 (PRC2) catalyzes histone H3 Lys27 trimethylation (H3K27me3) to repress gene transcription in multicellular eukaryotes. Despite its importance in gene silencing and cellular differentiation, how PRC2 is recruited to target loci is still not fully understood. Here, we report genome-wide evidence for the recruitment of PRC2 by the transcriptional repressors VIVIPAROUS1/ABI3-LIKE1 (VAL1) and VAL2 in Arabidopsis thaliana. We show that the val1 val2 double mutant possesses somatic embryonic phenotypes and a transcriptome strikingly similar to those of the swn clf double mutant, which lacks the PRC2 catalytic subunits SWINGER (SWN) and CURLY LEAF (CLF). We further show that VAL1 and VAL2 physically interact with SWN and CLF in vivo. Genome-wide binding profiling demonstrated that they colocalize with SWN and CLF at PRC2 target loci. Loss of VAL1/2 significantly reduces SWN and CLF enrichment at PRC2 target loci and leads to a genome-wide redistribution of H3K27me3 that strongly affects transcription. Finally, we provide evidence that the VAL1/VAL2–RY regulatory system is largely independent of previously identified modules for Polycomb silencing in plants. Together, our work demonstrates an extensive genome-wide interaction between VAL1/2 and PRC2 and provides mechanistic insights into the establishment of Polycomb silencing in plants.

Validation of human microRNA target pathways enables evaluation of target prediction tools

2020-12-10T00:00

MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting.

Identification of lysine isobutyrylation as a new histone modification mark

2020-12-11T00:00

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.

Flanking sequence preference modulates de novo DNA methylation in the mouse genome

2020-12-08T00:00

Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences result from structural differences in the catalytic domains of the two de novo DNMTs and are not a consequence of differential recruitment to the genome. Molecular dynamics simulations suggest that, in case of human DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. By exchanging arginine to a lysine, the corresponding side chain in DNMT3B, the sequence preference is reversed, confirming the requirement for arginine at this position. This context-dependent enzymatic activity provides additional insights into the complex regulation of DNA methylation patterns.

Histone methyltransferase ATX1 dynamically regulates fiber secondary cell wall biosynthesis in Arabidopsis inflorescence stem

2020-12-17T00:00

Secondary wall thickening in the sclerenchyma cells is strictly controlled by a complex network of transcription factors in vascular plants. However, little is known about the epigenetic mechanism regulating secondary wall biosynthesis. In this study, we identified that ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1), a H3K4-histone methyltransferase, mediates the regulation of fiber cell wall development in inflorescence stems of Arabidopsis thaliana. Genome-wide analysis revealed that the up-regulation of genes involved in secondary wall formation during stem development is largely coordinated by increasing level of H3K4 tri-methylation. Among all histone methyltransferases for H3K4me3 in Arabidopsis, ATX1 is markedly increased during the inflorescence stem development and loss-of-function mutant atx1 was impaired in secondary wall thickening in interfascicular fibers. Genetic analysis showed that ATX1 positively regulates secondary wall deposition through activating the expression of secondary wall NAC master switch genes, SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1). We further identified that ATX1 directly binds the loci of SND1 and NST1, and activates their expression by increasing H3K4me3 levels at these loci. Taken together, our results reveal that ATX1 plays a key role in the regulation of secondary wall biosynthesis in interfascicular fibers during inflorescence stem development of Arabidopsis.

Histone benzoylation serves as an epigenetic mark for DPF and YEATS family proteins

2020-12-08T00:00

Histone modifications and their functional readout serve as an important mechanism for gene regulation. Lysine benzoylation (Kbz) on histones is a recently identified acylation mark associated with active transcription. However, it remains to be explored whether putative readers exist to recognize this epigenetic mark. Here, our systematic binding studies demonstrated that the DPF and YEATS, but not the Bromodomain family members, are readers for histone Kbz. Co-crystal structural analyses revealed a ‘hydrophobic encapsulation’ and a ‘tip-sensor’ mechanism for Kbz readout by DPF and YEATS, respectively. Moreover, the DPF and YEATS family members display subtle yet unique features to create somewhat flexible engagements of different acylation marks. For instance, YEATS2 but not the other YEATS proteins exhibits best preference for Kbz than lysine acetylation and crotonylation due to its wider ‘tip-sensor’ pocket. The levels of histone benzoylation in cultured cells or in mice are upregulated upon sodium benzoate treatment, highlighting its dynamic regulation. In summary, our work identifies the first readers for histone Kbz and reveals the molecular basis underlying Kbz recognition, thus paving the way for further functional dissections of histone benzoylation.

A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae

2020-12-16T00:00

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5′ region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.

Genome-wide investigation of the dynamic changes of epigenome modifications after global DNA methylation editing

2020-12-09T00:00

Chromatin properties are regulated by complex networks of epigenome modifications. Currently, it is unclear how these modifications interact and if they control downstream effects such as gene expression. We employed promiscuous chromatin binding of a zinc finger fused catalytic domain of DNMT3A to introduce DNA methylation in HEK293 cells at many CpG islands (CGIs) and systematically investigated the dynamics of the introduced DNA methylation and the consequent changes of the epigenome network. We observed efficient methylation at thousands of CGIs, but it was unstable at about 90% of them, highlighting the power of genome-wide molecular processes that protect CGIs against DNA methylation. Partially stable methylation was observed at about 1000 CGIs, which showed enrichment in H3K27me3. Globally, the introduced DNA methylation strongly correlated with a decrease in gene expression indicating a direct effect. Similarly, global but transient reductions in H3K4me3 and H3K27ac were observed after DNA methylation but no changes were found for H3K9me3 and H3K36me3. Our data provide a global and time-resolved view on the network of epigenome modifications, their connections with DNA methylation and the responses triggered by artificial DNA methylation revealing a direct repressive effect of DNA methylation in CGIs on H3K4me3, histone acetylation, and gene expression.