Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity

Regulatory T cell (Treg) therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knockin in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, although FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knockin with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies

A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His(6)-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni(2+)-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs

Functional Maps of Protein Complexes from Quantitative Genetic Interaction Data

Recently, a number of advanced screening technologies have allowed for the comprehensive quantification of aggravating and alleviating genetic interactions among gene pairs. In parallel, TAP-MS studies (tandem affinity purification followed by mass spectroscopy) have been successful at identifying physical protein interactions that can indicate proteins participating in the same molecular complex. Here, we propose a method for the joint learning of protein complexes and their functional relationships by integration of quantitative genetic interactions and TAP-MS data. Using 3 independent benchmark datasets, we demonstrate that this method is >50% more accurate at identifying functionally related protein pairs than previous approaches. Application to genes involved in yeast chromosome organization identifies a functional map of 91 multimeric complexes, a number of which are novel or have been substantially expanded by addition of new subunits. Interestingly, we find that complexes that are enriched for aggravating genetic interactions (i.e., synthetic lethality) are more likely to contain essential genes, linking each of these interactions to an underlying mechanism. These results demonstrate the importance of both large-scale genetic and physical interaction data in mapping pathway architecture and function

The SWR1 Histone Replacement Complex Causes Genetic Instability and Genome-Wide Transcription Misregulation in the Absence of H2A.Z

The SWR1 complex replaces the canonical histone H2A with the variant H2A.Z (Htz1 in yeast) at specific chromatin regions. This dynamic alteration in nucleosome structure provides a molecular mechanism to regulate transcription, gene silencing, chromosome segregation and DNA repair. Here we show that genetic instability, sensitivity to drugs impairing different cellular processes and genome-wide transcriptional misregulation in htz1Δ can be partially or totally suppressed if SWR1 is not formed (swr1Δ), if it forms but cannot bind to chromatin (swc2Δ) or if it binds to chromatin but lacks histone replacement activity (swc5Δ and the ATPase-dead swr1-K727G). These results suggest that in htz1Δ the nucleosome remodelling activity of SWR1 affects chromatin integrity because of an attempt to replace H2A with Htz1 in the absence of the latter. This would impair transcription and, either directly or indirectly, other cellular processes. Specifically, we show that in htz1Δ, the SWR1 complex causes an accumulation of recombinogenic DNA damage by a mechanism dependent on phosphorylation of H2A at Ser129, a modification that occurs in response to DNA damage, suggesting that the SWR1 complex impairs the repair of spontaneous DNA damage in htz1Δ. In addition, SWR1 causes DSBs sensitivity in htz1Δ; consistently, in the absence of Htz1 the SWR1 complex bound near an endonuclease HO-induced DSB at the mating-type (MAT) locus impairs DSB-induced checkpoint activation. Our results support a stepwise mechanism for the replacement of H2A with Htz1 and demonstrate that a tight control of this mechanism is essential to regulate chromatin dynamics but also to prevent the deleterious consequences of an incomplete nucleosome remodelling

Genome-Wide Interaction Analysis of Air Pollution Exposure and Childhood Asthma with Functional Follow-up

Rationale: The evidence supporting an association between traffic-related air pollution exposure and incident childhood asthma is inconsistent and may depend on genetic factors. Objectives: To identify gene–environment interaction effects on childhood asthma using genome-wide single-nucleotide polymorphism (SNP) data and air pollution exposure. Identified loci were further analyzed at epigenetic and transcriptomic levels. Methods: We used land use regression models to estimate individual air pollution exposure (represented by outdoor NO2 levels) at the birth address and performed a genome-wide interaction study for doctors’ diagnoses of asthma up to 8 years in three European birth cohorts (n = 1,534) with look-up for interaction in two separate North American cohorts, CHS (Children’s Health Study) and CAPPS/SAGE (Canadian Asthma Primary Prevention Study/Study of Asthma, Genetics and Environment) (n = 1,602 and 186 subjects, respectively). We assessed expression quantitative trait locus effects in human lung specimens and blood, as well as associations among air pollution exposure, methylation, and transcriptomic patterns. Measurements and Main Results: In the European cohorts, 186 SNPs had an interaction P < 1 × 10−4 and a look-up evaluation of these disclosed 8 SNPs in 4 loci, with an interaction P < 0.05 in the large CHS study, but not in CAPPS/SAGE. Three SNPs within adenylate cyclase 2 (ADCY2) showed the same direction of the interaction effect and were found to influence ADCY2 gene expression in peripheral blood (P = 4.50 × 10−4). One other SNP with P < 0.05 for interaction in CHS, rs686237, strongly influenced UDP-Gal:betaGlcNAc β-1,4-galactosyltransferase, polypeptide 5 (B4GALT5) expression in lung tissue (P = 1.18 × 10−17). Air pollution exposure was associated with differential discs, large homolog 2 (DLG2) methylation and expression. Conclusions: Our results indicated that gene–environment interactions are important for asthma development and provided supportive evidence for interaction with air pollution for ADCY2, B4GALT5, and DLG2

Triptolide (TPL) Inhibits Global Transcription by Inducing Proteasome-Dependent Degradation of RNA Polymerase II (Pol II)

Triptolide (TPL), a key biologically active component of the Chinese medicinal herb Tripterygium wilfordii Hook. f., has potent anti-inflammation and anti-cancer activities. Its anti-proliferative and pro-apoptotic effects have been reported to be related to the inhibition of Nuclear Factor κB (NF-κB) and Nuclear Factor of Activated T-cells (NFAT) mediated transcription and suppression of HSP70 expression. The direct targets and precise mechanisms that are responsible for the gene expression inhibition, however, remain unknown. Here, we report that TPL inhibits global gene transcription by inducing proteasome-dependent degradation of the largest subunit of RNA polymerase II (Rpb1) in cancer cells. In the presence of proteosome inhibitor MG132, TPL treatment causes hyperphosphorylation of Rpb1 by activation of upstream protein kinases such as Positive Transcription Elongation Factor b (P-TEFb) in a time and dose dependent manner. Also, we observe that short time incubation of TPL with cancer cells induces DNA damage. In conclusion, we propose a new mechanism of how TPL works in killing cancer. TPL inhibits global transcription in cancer cells by induction of phosphorylation and subsequent proteasome-dependent degradation of Rpb1 resulting in global gene transcription, which may explain the high potency of TPL in killing cancer

SWI/SNF and Asf1 Independently Promote Derepression of the DNA Damage Response Genes under Conditions of Replication Stress

The histone chaperone Asf1 and the chromatin remodeler SWI/SNF have been separately implicated in derepression of the DNA damage response (DDR) genes in yeast cells treated with genotoxins that cause replication interference. Using genetic and biochemical approaches, we have tested if derepression of the DDR genes in budding yeast involves functional interplay between Asf1 and SWI/SNF. We find that Asf1 and SWI/SNF are both recruited to DDR genes under replication stress triggered by hydroxyurea, and have detected a soluble complex that contains Asf1 and the Snf2 subunit of SWI/SNF. SWI/SNF recruitment to DDR genes however does not require Asf1, and deletion of Snf2 does not affect Asf1 occupancy of DDR gene promoters. A checkpoint engagement defect is sufficient to explain the synthetic effect of deletion of ASF1 and SNF2 on derepression of the DDR genes in hydroxyurea-treated cells. Collectively, our results show that the DDR genes fall into a class in which Asf1 and SWI/SNF independently control transcriptional induction

The E1A-Associated p400 Protein Modulates Cell Fate Decisions by the Regulation of ROS Homeostasis

The p400 E1A-associated protein, which mediates H2A.Z incorporation at specific promoters, plays a major role in cell fate decisions: it promotes cell cycle progression and inhibits induction of apoptosis or senescence. Here, we show that p400 expression is required for the correct control of ROS metabolism. Depletion of p400 indeed increases intracellular ROS levels and causes the appearance of DNA damage, indicating that p400 maintains oxidative stress below a threshold at which DNA damages occur. Suppression of the DNA damage response using a siRNA against ATM inhibits the effects of p400 on cell cycle progression, apoptosis, or senescence, demonstrating the importance of ATM–dependent DDR pathways in cell fates control by p400. Finally, we show that these effects of p400 are dependent on direct transcriptional regulation of specific promoters and may also involve a positive feedback loop between oxidative stress and DNA breaks since we found that persistent DNA breaks are sufficient to increase ROS levels. Altogether, our results uncover an unexpected link between p400 and ROS metabolism and allow deciphering the molecular mechanisms largely responsible for cell proliferation control by p400

Missing value imputation for microarray gene expression data using histone acetylation information

<p>Abstract</p> <p>Background</p> <p>It is an important pre-processing step to accurately estimate missing values in microarray data, because complete datasets are required in numerous expression profile analysis in bioinformatics. Although several methods have been suggested, their performances are not satisfactory for datasets with high missing percentages.</p> <p>Results</p> <p>The paper explores the feasibility of doing missing value imputation with the help of gene regulatory mechanism. An imputation framework called histone acetylation information aided imputation method (HAIimpute method) is presented. It incorporates the histone acetylation information into the conventional KNN(<it>k</it>-nearest neighbor) and LLS(local least square) imputation algorithms for final prediction of the missing values. The experimental results indicated that the use of acetylation information can provide significant improvements in microarray imputation accuracy. The HAIimpute methods consistently improve the widely used methods such as KNN and LLS in terms of normalized root mean squared error (NRMSE). Meanwhile, the genes imputed by HAIimpute methods are more correlated with the original complete genes in terms of Pearson correlation coefficients. Furthermore, the proposed methods also outperform GOimpute, which is one of the existing related methods that use the functional similarity as the external information.</p> <p>Conclusion</p> <p>We demonstrated that the using of histone acetylation information could greatly improve the performance of the imputation especially at high missing percentages. This idea can be generalized to various imputation methods to facilitate the performance. Moreover, with more knowledge accumulated on gene regulatory mechanism in addition to histone acetylation, the performance of our approach can be further improved and verified.</p

Actin-Related Protein Arp6 Influences H2A.Z-Dependent and -Independent Gene Expression and Links Ribosomal Protein Genes to Nuclear Pores

Actin-related proteins are ubiquitous components of chromatin remodelers and are conserved from yeast to man. We have examined the role of the budding yeast actin-related protein Arp6 in gene expression, both as a component of the SWR1 complex (SWR-C) and in its absence. We mapped Arp6 binding sites along four yeast chromosomes using chromatin immunoprecipitation from wild-type and swr1 deleted (swr1Δ) cells. We find that a majority of Arp6 binding sites coincide with binding sites of Swr1, the catalytic subunit of SWR-C, and with the histone H2A variant Htz1 (H2A.Z) deposited by SWR-C. However, Arp6 binding detected at centromeres, the promoters of ribosomal protein (RP) genes, and some telomeres is independent of Swr1 and Htz1 deposition. Given that RP genes and telomeres both show association with the nuclear periphery, we monitored the ability of Arp6 to mediate the localization of chromatin to nuclear pores. Arp6 binding is sufficient to shift a randomly positioned locus to nuclear periphery, even in a swr1Δ strain. Arp6 is also necessary for the pore association of its targeted RP promoters possibly through cell cycle-dependent factors. Loss of Arp6, but not Htz1, leads to an up-regulation of these RP genes. In contrast, the pore-association of GAL1 correlates with Htz1 deposition, and loss of Arp6 reduces both GAL1 activation and peripheral localization. We conclude that Arp6 functions both together with the nucleosome remodeler Swr1 and also without it, to mediate Htz1-dependent and Htz1-independent binding of chromatin domains to nuclear pores. This association is shown to have modulating effects on gene expression