ResearchPad - chromosomes-and-gene-expression https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation]]> https://www.researchpad.co/article/elastic_article_6357 By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions. We identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3’UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome.

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<![CDATA[Controlling gene activation by enhancers through a drug-inducible topological insulator]]> https://www.researchpad.co/article/N2bbf1e8f-ab02-4251-b534-23ad351c1342 While regulation of gene-enhancer interaction is intensively studied, its application remains limited. Here, we reconstituted arrays of CTCF-binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. In human induced pluripotent stem cells, STITCH inserted between MYC and the enhancer down-regulated MYC. Progressive mutagenesis of STITCH led to a preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. STITCH also altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Finally, STITCH inserted near NEUROG2 impaired the gene activation in differentiating neural progenitor cells. Thus, STITCH should be broadly useful for functional genetic studies.

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<![CDATA[Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex]]> https://www.researchpad.co/article/Nbfc8d5d1-da72-4c2a-9797-ce3b29738b07

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe.

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<![CDATA[Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides]]> https://www.researchpad.co/article/Nd4d9dc37-38a0-4d2f-beaa-88139ecf1e32

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418-induced miscoding alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

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<![CDATA[Finding sense in the context]]> https://www.researchpad.co/article/N950796ad-a816-4ab6-a8d0-a9d44ecfe049

Ribosomal profiling has shed new light on how ribosomes can ignore stop codons in messenger RNA.

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<![CDATA[Losing Dnmt3a dependent methylation in inhibitory neurons impairs neural function by a mechanism impacting Rett syndrome]]> https://www.researchpad.co/article/Nba239759-6617-4cf9-a29c-0a5b7fa9fa31

Methylated cytosine is an effector of epigenetic gene regulation. In the brain, Dnmt3a is the sole ‘writer’ of atypical non-CpG methylation (mCH), and MeCP2 is the only known ‘reader’ for mCH. We asked if MeCP2 is the sole reader for Dnmt3a dependent methylation by comparing mice lacking either protein in GABAergic inhibitory neurons. Loss of either protein causes overlapping and distinct features from the behavioral to molecular level. Loss of Dnmt3a causes global loss of mCH and a subset of mCG sites resulting in more widespread transcriptional alterations and severe neurological dysfunction than MeCP2 loss. These data suggest that MeCP2 is responsible for reading only part of the Dnmt3a dependent methylation in the brain. Importantly, the impact of MeCP2 on genes differentially expressed in both models shows a strong dependence on mCH, but not Dnmt3a dependent mCG, consistent with mCH playing a central role in the pathogenesis of Rett Syndrome.

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<![CDATA[Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells]]> https://www.researchpad.co/article/N70fe9b6a-0774-42da-87db-cc50e4bf8ef1

Replication checkpoint is essential for maintaining genome integrity in response to various replication stresses as well as during the normal growth. The evolutionally conserved ATR-Claspin-Chk1 pathway is induced during replication checkpoint activation. Cdc7 kinase, required for initiation of DNA replication at replication origins, has been implicated in checkpoint activation but how it is involved in this pathway has not been known. Here, we show that Cdc7 is required for Claspin-Chk1 interaction in human cancer cells by phosphorylating CKBD (Chk1-binding-domain) of Claspin. The residual Chk1 activation in Cdc7-depleted cells is lost upon further depletion of casein kinase1 (CK1γ1), previously reported to phosphorylate CKBD. Thus, Cdc7, in conjunction with CK1γ1, facilitates the interaction between Claspin and Chk1 through phosphorylating CKBD. We also show that, whereas Cdc7 is predominantly responsible for CKBD phosphorylation in cancer cells, CK1γ1 plays a major role in non-cancer cells, providing rationale for targeting Cdc7 for cancer cell-specific cell killing.

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<![CDATA[TRF1 averts chromatin remodelling, recombination and replication dependent-break induced replication at mouse telomeres]]> https://www.researchpad.co/article/N740ed838-f073-4679-bddc-320f03f30b53

Telomeres are a significant challenge to DNA replication and are prone to replication stress and telomere fragility. The shelterin component TRF1 facilitates telomere replication but the molecular mechanism remains uncertain. By interrogating the proteomic composition of telomeres, we show that mouse telomeres lacking TRF1 undergo protein composition reorganisation associated with the recruitment of DNA damage response and chromatin remodellers. Surprisingly, mTRF1 suppresses the accumulation of promyelocytic leukemia (PML) protein, BRCA1 and the SMC5/6 complex at telomeres, which is associated with increased Homologous Recombination (HR) and TERRA transcription. We uncovered a previously unappreciated role for mTRF1 in the suppression of telomere recombination, dependent on SMC5 and also POLD3 dependent Break Induced Replication at telomeres. We propose that TRF1 facilitates S-phase telomeric DNA synthesis to prevent illegitimate mitotic DNA recombination and chromatin rearrangement.

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<![CDATA[Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle]]> https://www.researchpad.co/article/N246eade3-db95-48ed-9ccd-534b1049712e

The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.

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<![CDATA[RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster]]> https://www.researchpad.co/article/Nfb060a59-7316-4a6c-b040-c414dc726d32

Tandemly-repeated DNAs, or satellites, are enriched in heterochromatic regions of eukaryotic genomes and contribute to nuclear structure and function. Some satellites are transcribed, but we lack direct evidence that specific satellite RNAs are required for normal organismal functions. Here, we show satellite RNAs derived from AAGAG tandem repeats are transcribed in many cells throughout Drosophila melanogaster development, enriched in neurons and testes, often localized within heterochromatic regions, and important for viability. Strikingly, we find AAGAG transcripts are necessary for male fertility, and that AAGAG RNA depletion results in defective histone-protamine exchange, sperm maturation and chromatin organization. Since these events happen late in spermatogenesis when the transcripts are not detected, we speculate that AAGAG RNA in primary spermatocytes ‘primes’ post-meiosis steps for sperm maturation. In addition to demonstrating essential functions for AAGAG RNAs, comparisons between closely related Drosophila species suggest that satellites and their transcription evolve quickly to generate new functions.

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<![CDATA[The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog]]> https://www.researchpad.co/article/N78002389-e155-4f2e-899c-1d0d6f578429

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

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<![CDATA[Soluble PD-L1 generated by endogenous retroelement exaptation is a receptor antagonist]]> https://www.researchpad.co/article/N6393fb74-cb81-4160-ad2c-484f2e0be439

Immune regulation is a finely balanced process of positive and negative signals. PD-L1 and its receptor PD-1 are critical regulators of autoimmune, antiviral and antitumoural T cell responses. Although the function of its predominant membrane-bound form is well established, the source and biological activity of soluble PD-L1 (sPD-L1) remain incompletely understood. Here, we show that sPD-L1 in human healthy tissues and tumours is produced by exaptation of an intronic LINE-2A (L2A) endogenous retroelement in the CD274 gene, encoding PD-L1, which causes omission of the transmembrane domain and the regulatory sequence in the canonical 3’ untranslated region. The alternatively spliced CD274-L2A transcript forms the major source of sPD-L1 and is highly conserved in hominids, but lost in mice and a few related species. Importantly, CD274-L2A-encoded sPD-L1 lacks measurable T cell inhibitory activity. Instead, it functions as a receptor antagonist, blocking the inhibitory activity of PD-L1 bound on cellular or exosomal membranes.

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<![CDATA[Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells]]> https://www.researchpad.co/article/N9a9620af-2298-461c-97a9-5a2ed58881f8

We previously demonstrated that CRM1, a major nuclear export factor, accumulates at Hox cluster regions to recruit nucleoporin-fusion protein Nup98HoxA9, resulting in robust activation of Hox genes (Oka et al., 2016). However, whether this phenomenon is general to other leukemogenic proteins remains unknown. Here, we show that two other leukemogenic proteins, nucleoporin-fusion SET-Nup214 and the NPM1 mutant, NPM1c, which contains a nuclear export signal (NES) at its C-terminus and is one of the most frequent mutations in acute myeloid leukemia, are recruited to the HOX cluster region via chromatin-bound CRM1, leading to HOX gene activation in human leukemia cells. Furthermore, we demonstrate that this mechanism is highly sensitive to a CRM1 inhibitor in leukemia cell line. Together, these findings indicate that CRM1 acts as a key molecule that connects leukemogenic proteins to aberrant HOX gene regulation either via nucleoporin-CRM1 interaction (for SET-Nup214) or NES-CRM1 interaction (for NPM1c).

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<![CDATA[Chromatinization of Escherichia coli with archaeal histones]]> https://www.researchpad.co/article/N67ba61d0-6e51-41df-96d2-842abdab39a6

Nucleosomes restrict DNA accessibility throughout eukaryotic genomes, with repercussions for replication, transcription, and other DNA-templated processes. How this globally restrictive organization emerged during evolution remains poorly understood. Here, to better understand the challenges associated with establishing globally restrictive chromatin, we express histones in a naive system that has not evolved to deal with nucleosomal structures: Escherichia coli. We find that histone proteins from the archaeon Methanothermus fervidus assemble on the E. coli chromosome in vivo and protect DNA from micrococcal nuclease digestion, allowing us to map binding footprints genome-wide. We show that higher nucleosome occupancy at promoters is associated with lower transcript levels, consistent with local repressive effects. Surprisingly, however, this sudden enforced chromatinization has only mild repercussions for growth unless cells experience topological stress. Our results suggest that histones can become established as ubiquitous chromatin proteins without interfering critically with key DNA-templated processes.

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<![CDATA[Mechanism of environmentally driven conformational changes that modulate H-NS DNA-bridging activity]]> https://www.researchpad.co/article/5b446d88463d7e3b3b9b1bba

Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria and is believed to be a crucial player in bacterial chromatin organization via its DNA-bridging activity. H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically, it is unclear how functional modulation of H-NS by such factors is achieved. Here, we show that a diverse spectrum of H-NS modulators alter the DNA-bridging activity of H-NS. Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA-binding mode. Similarly, synergistic and antagonistic co-regulators modulate the DNA-bridging efficiency. Structural studies suggest a conserved mechanism: H-NS switches between a ‘closed’ and an ‘open’, bridging competent, conformation driven by environmental cues and interaction partners.

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<![CDATA[A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolution]]> https://www.researchpad.co/article/5c8015abd5eed0c484a9f8cb

In eukaryotes, ribosome profiling provides insight into the mechanism of protein synthesis at the codon level. In bacteria, however, the method has been more problematic and no consensus has emerged for how to best prepare profiling samples. Here, we identify the sources of these problems and describe new solutions for arresting translation and harvesting cells in order to overcome them. These improvements remove confounding artifacts and improve the resolution to allow analyses of ribosome behavior at the codon level. With a clearer view of the translational landscape in vivo, we observe that filtering cultures leads to translational pauses at serine and glycine codons through the reduction of tRNA aminoacylation levels. This observation illustrates how bacterial ribosome profiling studies can yield insight into the mechanism of protein synthesis at the codon level and how these mechanisms are regulated in response to changes in the physiology of the cell.

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<![CDATA[Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells]]> https://www.researchpad.co/article/5c09a50ed5eed0c4842cbaa6

Genome-wide screens in Drosophila cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for Drosophila, including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells.

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<![CDATA[Species and cell-type properties of classically defined human and rodent neurons and glia]]> https://www.researchpad.co/article/5c103238d5eed0c4842639f4

Determination of the molecular properties of genetically targeted cell types has led to fundamental insights into mouse brain function and dysfunction. Here, we report an efficient strategy for precise exploration of gene expression and epigenetic events in specific cell types in a range of species, including postmortem human brain. We demonstrate that classically defined, homologous neuronal and glial cell types differ between rodent and human by the expression of hundreds of orthologous, cell specific genes. Confirmation that these genes are differentially active was obtained using epigenetic mapping and immunofluorescence localization. Studies of sixteen human postmortem brains revealed gender specific transcriptional differences, cell-specific molecular responses to aging, and the induction of a shared, robust response to an unknown external event evident in three donor samples. Our data establish a comprehensive approach for analysis of molecular events associated with specific circuits and cell types in a wide variety of human conditions.

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<![CDATA[Multiple serine transposase dimers assemble the transposon-end synaptic complex during IS607-family transposition]]> https://www.researchpad.co/article/5c103220d5eed0c4842636b6

IS607-family transposons are unusual because they do not have terminal inverted repeats or generate target site duplications. They encode two protein-coding genes, but only tnpA is required for transposition. Our X-ray structures confirm that TnpA is a member of the serine recombinase (SR) family, but the chemically-inactive quaternary structure of the dimer, along with the N-terminal location of the DNA binding domain, are different from other SRs. TnpA dimers from IS1535 cooperatively associate with multiple subterminal repeats, which together with additional nonspecific binding, form a nucleoprotein filament on one transposon end that efficiently captures a second unbound end to generate the paired-end complex (PEC). Formation of the PEC does not require a change in the dimeric structure of the catalytic domain, but remodeling of the C-terminal α-helical region is involved. We posit that the PEC recruits a chemically-active conformer of TnpA to the transposon end to initiate DNA chemistry.

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