Transcription Start Sites Of Genes Research Articles

PPARG in osteocytes controls sclerostin expression, bone mass, marrow adiposity and mediates TZD-induced bone loss.

The peroxisome proliferator activated receptor gamma (PPARG) nuclear receptor regulates energy metabolism and insulin sensitivity. In this study, we present novel evidence for an essential role of PPARG in the regulation of osteocyte function, and support for the emerging concept of the conjunction between regulation of energy metabolism and bone mass. We report that PPARG is essential for sclerostin production, a recently approved target to treat osteoporosis. Our mouse model of osteocyte-specific PPARG deletion (Dmp1CrePparγflfl or γOTKO) is characterized with increased bone mass and reduced bone marrow adiposity, which is consistent with upregulation of WNT signaling and increased bone forming activity of endosteal osteoblasts. An analysis of osteocytes derived from γOTKO and control mice showed an excellent correlation between PPARG and SOST/sclerostin at the transcript and protein levels. The 8kb sequence upstream of Sost gene transcription start site possesses multiple PPARG binding elements (PPREs) with at least two of them binding PPARG with dynamics reflecting its activation with full agonist rosiglitazone and correlating with increased levels of Sost transcript and sclerostin protein expression (Pearson's r=0.991, p=0.001). Older γOTKO female mice are largely protected from TZD-induced bone loss providing proof of concept that PPARG in osteocytes can be pharmacologically targeted. These findings demonstrate that transcriptional activities of PPARG are essential for sclerostin expression in osteocytes and support consideration of targeting PPARG activities with selective modulators to treat osteoporosis.

The yeast ISW1b ATP-dependent chromatin remodeler is critical for nucleosome spacing and dinucleosome resolution

Isw1 and Chd1 are ATP-dependent nucleosome-spacing enzymes required to establish regular arrays of phased nucleosomes near transcription start sites of yeast genes. Cells lacking both Isw1 and Chd1 have extremely disrupted chromatin, with weak phasing, irregular spacing and a propensity to form close-packed dinucleosomes. The Isw1 ATPase subunit occurs in two different remodeling complexes: ISW1a (composed of Isw1 and Ioc3) and ISW1b (composed of Isw1, Ioc2 and Ioc4). The Ioc4 subunit of ISW1b binds preferentially to the H3-K36me3 mark. Here we show that ISW1b is primarily responsible for setting nucleosome spacing and resolving close-packed dinucleosomes, whereas ISW1a plays only a minor role. ISW1b and Chd1 make additive contributions to dinucleosome resolution, such that neither enzyme is capable of resolving all dinucleosomes on its own. Loss of the Set2 H3-K36 methyltransferase partly phenocopies loss of Ioc4, resulting in increased dinucleosome levels with only a weak effect on nucleosome spacing, suggesting that Set2-mediated H3-K36 trimethylation contributes to ISW1b-mediated dinucleosome separation. The H4 tail domain is required for normal nucleosome spacing but not for dinucleosome resolution. We conclude that the nucleosome spacing and dinucleosome resolving activities of ISW1b and Chd1 are critical for normal global chromatin organisation.

Distinct roles of nucleosome sliding and histone modifications in controlling the fidelity of transcription initiation

ABSTRACT Regulation of gene expression starts from the transcription initiation. Regulated transcription initiation is critical for generating correct transcripts with proper abundance. The impact of epigenetic control, such as histone modifications and chromatin remodelling, on gene regulation has been extensively investigated, but their specific role in regulating transcription initiation is far from well understood. Here we aimed to better understand the roles of genes involved in histone H3 methylations and chromatin remodelling on the regulation of transcription initiation at a genome-scale using the budding yeast as a study system. We obtained and compared maps of transcription start site (TSS) at single-nucleotide resolution by nAnT-iCAGE for a strain with depletion of MINC (Mot1-Ino80C-Nc2) by Mot1p and Ino80p anchor-away (Mot1&Ino80AA) and a strain with loss of histone methylation (set1Δset2Δdot1Δ) to their wild-type controls. Our study showed that the depletion of MINC stimulated transcription initiation from many new sites flanking the dominant TSS of genes, while the loss of histone methylation generates more TSSs in the coding region. Moreover, the depletion of MINC led to less confined boundaries of TSS clusters (TCs) and resulted in broader core promoters, and such patterns are not present in the ssdΔ mutant. Our data also exhibits that the MINC has distinctive impacts on TATA-containing and TATA-less promoters. In conclusion, our study shows that MINC is required for accurate identification of bona fide TSSs, particularly in TATA-containing promoters, and histone methylation contributes to the repression of transcription initiation in coding regions.

Intragenic DNA methylation regulates insect gene expression and reproduction through the MBD/Tip60 complex

SummaryDNA methylation is an important epigenetic modification. However, the regulations and functions of insect intragenic DNA methylation remain unknown. Here, we demonstrate that a regulatory mechanism involving intragenic DNA methylation controls ovarian and embryonic developmental processes in Bombyx mori. In B. mori, DNA methylation is found near the transcription start site (TSS) of ovarian genes. By promoter activity analysis, we observed that 5′ UTR methylation enhances gene expression. Moreover, methyl-DNA-binding domain protein 2/3 (MBD2/3) binds to the intragenic methyl-CpG fragment and recruits acetyltransferase Tip60 to promote histone H3K27 acetylation and gene expression. Additionally, genome-wide analyses showed that the peak of H3K27 acetylation appears near the TSS of methyl-modified genes, and DNA methylation is enriched in genes involved in protein synthesis in the B. mori ovary, with MBD2/3 knockdown resulting in decreased fecundity. These data uncover a mechanism of gene body methylation for regulating insect gene expression and reproduction.

Chromatin dynamics: Nucleosome occupancy and sensitivity as determinants of gene expression and cell fate.

The nucleosome, consisting of ~150bp of DNA wrapped around a core histone octamer, is a regulator of nuclear events that contributes to gene expression and cell fate. Nucleosome organization at promoters and their associated remodeling events are important regulators of access to the genome. Occupancy alone, however, is not the only nucleosomal characteristic that plays a role in genome regulation. Nucleosomes at the transcription start sites (TSSs) of genes show differential sensitivity to micrococcal nuclease (MNase) and this differential sensitivity is linked to transcription and regulatory factor binding events. Recently, lymphoblastoid cells treated with heat-killed Salmonella typhimurium were shown to exhibit increased MNase sensitivity specifically at genes implicated in immune responses. Increased sensitivity at the −1-nucleosome permitted transcription factor and RNA Pol II binding events. This system illustrates how cytoplasmic signals induce altered chromatin states to produce a specific cellular response to a stimulus. Innate immune activation is a longstanding model for inducible promoters, transcriptional activation, and differential nucleosomal sensitivity in response to immune activation and offers a model that may be largely applicable to other specific cellular responses including viral infection and cancer. Previous work has shown that early transformation events are associated with prolonged nucleosome occupancy changes that are not observed later in cancer progression. Herein, we propose a model in which we suggest that detailed studies of nucleosomal occupancy and sensitivity in response to specific stimuli will provide insight into the regulation of nuclear events in cancer and other biological processes.

The poly(ADP-ribosyl)ation of BRD4 mediated by PARP1 promoted pathological cardiac hypertrophy

The bromodomain and extraterminal (BET) family member BRD4 is pivotal in the pathogenesis of cardiac hypertrophy. BRD4 induces hypertrophic gene expression by binding to the acetylated chromatin, facilitating the phosphorylation of RNA polymerases II (Pol II) and leading to transcription elongation. The present study identified a novel post-translational modification of BRD4: poly(ADP-ribosyl)ation (PARylation), that was mediated by poly(ADP-ribose)polymerase-1 (PARP1) in cardiac hypertrophy. BRD4 silencing or BET inhibitors JQ1 and MS417 prevented cardiac hypertrophic responses induced by isoproterenol (ISO), whereas overexpression of BRD4 promoted cardiac hypertrophy, confirming the critical role of BRD4 in pathological cardiac hypertrophy. PARP1 was activated in ISO-induced cardiac hypertrophy and facilitated the development of cardiac hypertrophy. BRD4 was involved in the prohypertrophic effect of PARP1, as implied by the observations that BRD4 inhibition or silencing reversed PARP1-induced hypertrophic responses, and that BRD4 overexpression suppressed the anti-hypertrophic effect of PARP1 inhibitors. Interactions of BRD4 and PARP1 were observed by co-immunoprecipitation and immunofluorescence. PARylation of BRD4 induced by PARP1 was investigated by PARylation assays. In response to hypertrophic stimuli like ISO, PARylation level of BRD4 was elevated, along with enhanced interactions between BRD4 and PARP1. By investigating the PARylation of truncation mutants of BRD4, the C-terminal domain (CTD) was identified as the PARylation modification sites of BRD4. PARylation of BRD4 facilitated its binding to the transcription start sites (TSS) of hypertrophic genes, resulting in enhanced phosphorylation of RNA Pol II and transcription activation of hypertrophic genes. The present findings suggest that strategies targeting inhibition of PARP1-BRD4 might have therapeutic potential for pathological cardiac hypertrophy.

Bovine pyruvate carboxylase gene proximal promoter activity is regulated by saturated and unsaturated fatty acids in Madin-Darby bovine kidney cells

An increase in bovine pyruvate carboxylase (PC; EC 6.4.1.1) at calving and during feed restriction corresponds with increased circulating nonesterified fatty acids as a consequence of negative energy balance. Regulation of PC mRNA and effect of specific combinations of saturated and unsaturated fatty acid profiles has yet to be explored. Our objective was to determine the effects of chain length, degree of saturation, and copresence of saturated and unsaturated fatty acids on activity of bovine PC promoter 1 (PCP1). For these experiments, Madin-Darby bovine kidney cells were transfected with a full-length bovine PCP1 construct from -1002 to +3 bp relative to the bovine PC gene transcription start site (bovine PCP1(-1002_+3)) ligated to a Firefly luciferase reporter, or with one of a series of nested 5' serial truncations (bovine PCP1(-773_+3), bovine PCP1(-494_+3), or bovine PCP1(-222_+3)). Cells were exposed for 23 h to either individual fatty acids (C16:0, C18:0, or C18:3n-3 cis) bound to BSA or to fatty acid mixtures in ratios of 90:10, 75:25, 50:50, or 25:75, corresponding to combinations of C16:0: C18:3n-3 cis or C18:0: C18:3n-3 cis. Total fatty acid concentration was 1.00 mM. Exposure to either C16:0 or C18:3n-3 cis alone elicited a significant increase in capacity to drive bovine PCP1(-1002_+3) activity compared with 1% BSA in Dulbecco's Modified Eagle's Medium control treatment (2.29, 2.89, and 1.00 ± 0.26 fold of promoter induction for C16:0, C18:3n-3 cis, and control, respectively). Treatment with C18:3n-3 cis alone caused a greater increase in promoter activity compared with C16:0 alone, indicating a lesser response to C16:0 alone for bovine PCP1(-1002_+3). Interestingly, inclusion of C18:3n-3 cis, at any level of fatty acid ratios examined, in combination with C16:0 increased promoter activity of bovine PCP1(-773_+3) or bovine PCP1(-222_+3) compared with treatment with C16:0 alone or control. Data from the bovine PCP1 truncation and fatty acid copresence experiments reveal the potential for response elements of unsaturated fatty acids or fatty acid ligands in several bovine PCP1 promoter regions. In silico analysis of bovine PCP1 identified putative peroxisome proliferator-activated receptor α and sterol regulatory element binding protein binding sites which may be implicated in fatty acid signaling to alter bovine PCP1 activity. Pyruvate carboxylase promoter 1 activity that is mediated by unsaturated fatty acids acting through elements within -1002 and -222 bp of bovine PCPI may determine PC response during periods of negative energy balance in dairy cows.

Comparative genome-wide methylation analysis of longissimus dorsi muscles in Yorkshire and Wannanhua pigs.

DNA methylation was one of the earliest discovered epigenetic modifications in vertebrates, and is an important epigenetic mechanism involved in the expression of genes in many biological processes, including muscle growth and development. Its effects on economically important traits are evidenced in reported differences in meat quality traits between Chinese indigenous pig breeds (Wannanhua pig) and Western commercial pig breeds (Yorkshire pig), and this presents a unique model for analyzing the effects of DNA methylation on these traits. In the present study, a whole genome DNA methylation analysis was performed on the two breeds using methylated DNA immunoprecipitation. GO functional enrichment and pathway enrichment analyses identified differentially methylated genes primarily associated with fatty acid metabolism, biological processes of muscle development and signaling pathways related to muscle development and pork quality. Differentially methylated genes were verified by sodium pyrosequencing, and the results were consistent with the sequencing results. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative correlation with gene expression levels around the transcription start site of genes. In total, 41 genes were both differentially expressed and methylated; these genes were related to fat metabolism, lipid metabolism and skeletal muscle development. This study could help further explore the molecular mechanisms and phenotypic differences in pig growth and development among different breeds.

High-resolution analyses of human sperm dynamic methylome reveal thousands of novel age-related epigenetic alterations

BackgroundChildren of aged fathers are at a higher risk of developing mental disorders. Alterations in sperm DNA methylation have been implicated as a potential cause. However, age-dependent modifications of the germ cells’ epigenome remain poorly understood. Our objective was to assess the DNA methylation profile of human spermatozoa during aging.ResultsWe used a high throughput, customized methylC-capture sequencing (MCC-seq) approach to characterize the dynamic DNA methylation in spermatozoa from 94 fertile and infertile men, who were categorized as young, 48 men between 18–38 years or old 46 men between 46–71 years. We identified more than 150,000 age-related CpG sites that are significantly differentially methylated among 2.65 million CpG sites covered. We conducted machine learning using our dataset to predict the methylation age of subjects; the age prediction accuracy based on our assay provided a more accurate prediction than that using the 450 K chip approach. In addition, we found that there are more hypermethylated (62%) than hypomethylated (38%) CpG sites in sperm of aged men, corresponding to 798 of total differential methylated regions (DMRs), of which 483 are hypermethylated regions (HyperDMR), and 315 hypomethylated regions (HypoDMR). Moreover, the distribution of age-related hyper- and hypomethylated CpGs in sperm is not random; the CpG sites that were hypermethylated with advanced age were frequently located in the distal region to genes, whereas hypomethylated sites were near to gene transcription start sites (TSS). We identified a high density of age-associated CpG changes in chromosomes 4 and 16, particularly HyperDMRs with localized clusters, the chr4 DMR cluster overlaps PGC1α locus, a protein involved in metabolic aging and the chr16 DMR cluster overlaps RBFOX1 locus, a gene implicated in neurodevelopmental disease. Gene ontology analysis revealed that the most affected genes by age were associated with development, neuron projection, differentiation and recognition, and behaviour, suggesting a potential link to the higher risk of neurodevelopmental disorders in children of aged fathers.ConclusionWe identified thousands of age-related and sperm-specific epigenetic alterations. These findings provide novel insight in understanding human sperm DNA methylation dynamics during paternal aging, and the subsequently affected genes potentially related to diseases in offspring.

Computational identification of eukaryotic promoters based on cascaded deep capsule neural networks.

A promoter is a region in the DNA sequence that defines where the transcription of a gene by RNA polymerase initiates, which is typically located proximal to the transcription start site (TSS). How to correctly identify the gene TSS and the core promoter is essential for our understanding of the transcriptional regulation of genes. As a complement to conventional experimental methods, computational techniques with easy-to-use platforms as essential bioinformatics tools can be effectively applied to annotate the functions and physiological roles of promoters. In this work, we propose a deep learning-based method termed Depicter (Deep learning for predicting promoter), for identifying three specific types of promoters, i.e. promoter sequences with the TATA-box (TATA model), promoter sequences without the TATA-box (non-TATA model), and indistinguishable promoters (TATA and non-TATA model). Depicter is developed based on an up-to-date, species-specific dataset which includes Homo sapiens, Mus musculus, Drosophila melanogaster and Arabidopsis thaliana promoters. A convolutional neural network coupled with capsule layers is proposed to train and optimize the prediction model of Depicter. Extensive benchmarking and independent tests demonstrate that Depicter achieves an improved predictive performance compared with several state-of-the-art methods. The webserver of Depicter is implemented and freely accessible at https://depicter.erc.monash.edu/.

Site-Specific Phosphorylation of Histone H1.4 Is Associated with Transcription Activation.

Core histone variants, such as H2A.X and H3.3, serve specialized roles in chromatin processes that depend on the genomic distributions and amino acid sequence differences of the variant proteins. Modifications of these variants alter interactions with other chromatin components and thus the protein’s functions. These inferences add to the growing arsenal of evidence against the older generic view of those linker histones as redundant repressors. Furthermore, certain modifications of specific H1 variants can confer distinct roles. On the one hand, it has been reported that the phosphorylation of H1 results in its release from chromatin and the subsequent transcription of HIV-1 genes. On the other hand, recent evidence indicates that phosphorylated H1 may in fact be associated with active promoters. This conflict suggests that different H1 isoforms and modified versions of these variants are not redundant when together but may play distinct functional roles. Here, we provide the first genome-wide evidence that when phosphorylated, the H1.4 variant remains associated with active promoters and may even play a role in transcription activation. Using novel, highly specific antibodies, we generated the first genome-wide view of the H1.4 isoform phosphorylated at serine 187 (pS187-H1.4) in estradiol-inducible MCF7 cells. We observe that pS187-H1.4 is enriched primarily at the transcription start sites (TSSs) of genes activated by estradiol treatment and depleted from those that are repressed. We also show that pS187-H1.4 associates with ‘early estrogen response’ genes and stably interacts with RNAPII. Based on the observations presented here, we propose that phosphorylation at S187 by CDK9 represents an early event required for gene activation. This event may also be involved in the release of promoter-proximal polymerases to begin elongation by interacting directly with the polymerase or other parts of the transcription machinery. Although we focused on estrogen-responsive genes, taking into account previous evidence of H1.4′s enrichment of promoters of pluripotency genes, and its involvement with rDNA activation, we propose that H1.4 phosphorylation for gene activation may be a more global observation.

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression.

The CD69 gene encodes a C-type lectin glycoprotein with immune regulatory properties which is expressed on the cell surfaces of all activated hematopoietic cells. CD69 activation kinetics differ by developmental stage, cell linage and activating conditions, and these differences have been attributed to the participation of complex gene regulatory networks. An evolutionarily conserved regulatory element, CNS2, located 4kb upstream of the CD69 gene transcriptional start site, has been proposed as the major candidate governing the gene transcriptional activation program. To investigate the function of human CNS2, we studied the effect of its endogenous elimination via CRISPR-Cas9 on CD69 protein and mRNA expression levels in various immune cell lines. Even when the entire promoter region was maintained, CNS2-/- cells did not express CD69, thus indicating that CNS2 has promoter-like characteristics. However, like enhancers, inverted CNS2 sustained transcription, although at a diminished levels, thereby suggesting that it has dual promoter and enhancer functions. Episomal luciferase assays further suggested that both functions are combined within the CNS2 regulatory element. In addition, CNS2 directs its own bidirectional transcription into two different enhancer-derived RNAs molecules (eRNAs) which are transcribed from two independent transcriptional start sites in opposite directions. This eRNA transcription is dependent on only the enhancer sequence itself, because in the absence of the CD69 promoter, sufficient RNA polymerase II levels are maintained at CNS2 to drive eRNA expression. Here, we describe a regulatory element with overlapping promoter and enhancer functions, which is essential for CD69 gene transcriptional regulation.

DNA methylation methods: Global DNA methylation and methylomic analyses.

DNA methylation provides a pivotal layer of epigenetic regulation in eukaryotes that has significant involvement for numerous biological processes in health and disease. The function of methylation of cytosine bases in DNA was originally proposed as a "silencing" epigenetic marker and focused on promoter regions of genes for decades. Improved technologies and accumulating studies have been extending our understanding of the roles of DNA methylation to various genomic contexts including gene bodies, repeat sequences and transcriptional start sites. The demand for comprehensively describing DNA methylation patterns spawns a diversity of DNA methylation profiling technologies that target its genomic distribution. These approaches have enabled the measurement of cytosine methylation from specific loci at restricted regions to single-base-pair resolution on a genome-scale level. In this review, we discuss the different DNA methylation analysis technologies primarily based on the initial treatments of DNA samples: bisulfite conversion, endonuclease digestion and affinity enrichment, involving methodology evolution, principles, applications, and their relative merits. This review may offer referable information for the selection of various platforms for genome-wide analysis of DNA methylation.

Widespread Transcriptional Readthrough Caused by Nab2 Depletion Leads to Chimeric Transcripts with Retained Introns

Nascent RNA sequencing has revealed that pre-mRNA splicing can occur shortly after introns emerge from RNA polymerase II (RNA Pol II). Differences in co-transcriptional splicing profiles suggest regulation by cis- and/or trans-acting factors. Here, we use single-molecule intron tracking (SMIT) to identify a cohort of regulators by machine learning in budding yeast. Of these, Nab2 displays reduced co-transcriptional splicing when depleted. Unexpectedly, these splicing defects are attributable to aberrant "intrusive" transcriptional readthrough from upstream genes, as revealed by long-read sequencing. Transcripts that originate from the intron-containing gene's own transcription start site (TSS) are efficiently spliced, indicating no direct role of Nab2 in splicing per se. This work highlights the coupling between transcription, splicing, and 3' end formation in the context of gene organization along chromosomes. We conclude that Nab2 is required for proper 3' end processing, which ensures gene-specific control of co-transcriptional RNA processing.

Tissue-specific usage of transposable element-derived promoters in mouse development

BackgroundTransposable elements (TEs) are a significant component of eukaryotic genomes and play essential roles in genome evolution. Mounting evidence indicates that TEs are highly transcribed in early embryo development and contribute to distinct biological functions and tissue morphology.ResultsWe examine the epigenetic dynamics of mouse TEs during the development of five tissues: intestine, liver, lung, stomach, and kidney. We found that TEs are associated with over 20% of open chromatin regions during development. Close to half of these accessible TEs are only activated in a single tissue and a specific developmental stage. Most accessible TEs are rodent-specific. Across these five tissues, 453 accessible TEs are found to create the transcription start sites of downstream genes in mouse, including 117 protein-coding genes and 144 lincRNA genes, 93.7% of which are mouse-specific. Species-specific TE-derived transcription start sites are found to drive the expression of tissue-specific genes and change their tissue-specific expression patterns during evolution.ConclusionOur results suggest that TE insertions increase the regulatory potential of the genome, and some TEs have been domesticated to become a crucial component of gene and regulate tissue-specific expression during mouse tissue development.

Contextual Reprogramming of CAR-T Cells for Treatment of HER2+ Cancers

Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. The conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

Abstract 1256: Widespread alterations of chromatin accessibility in chronic lymphocytic leukemia (CLL)

Abstract Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in western countries that has a strong genetic basis. Using data from both chromatin immunoprecipitation and sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq), recent studies have uncovered alterations of chromatin landscape in CLL. However, the spectrum of epigenetic changes and the mechanisms of casualtyare only partially understood. In this study, we used ATAC-seq to profile chromatin accessibility across the two major CLL subtypes, IGHV-mutated (n=19) and IGHV-unmutated (n=22). DNA library of accessible chromatin regions was generated from purified CD5+/CD19+ leukemiacells. All libraries were assessed to have high data quality based on the ENCODE quality metrics. Additionally, publicly available ATAC-seq data (GSE118189 and GSE111015) from total (n=5), memory (n=6) and naïve B cells (n=6) were analyzed in parallel and used as normal reference. ATAC-seq peaks from CLL and normal B cells were merged to generate a consensus list of 107,928 high-confidence peaks that did not overlap ENCODE blacklisted regions, were present in at least two CLL or two normal B-cell samples, and had ≥1 counts-per-million in at least 10% of the samples. Raw read counts were Trimmed Mean of M-values normalized and log2 transformed. K-means clustering of differential peaks revealed an extensive reprogramming of chromatin accessibility landscape in CLL, with over 11,000 lost and nearly 9,000 gained open chromatin regions relative to normal B-cells. In particular, we identified two clusters comprising 1,401 regions that were largely open either in IGHV-mutated(571 peaks) or in IGHV-unmutated CLL (830 peaks). Open chromatin regions gained in IGHV-mutatedwere preferentially linked to genes in the B cell receptor signaling pathway and those in IGHV-mutatedto genes enriched in the interferon type I signaling pathway. These discriminatory regions are highly enriched with DNA binding motifs for transcription factors (TFs) with key roles in B cell development, such as PU.1, AP-1, c-Jun, BATF, and TCF family. In addition, 94% of the 1,401 peaks were located in distal regulatory regions, being ≥2.5 kb away from transcription start sites of genes; two-third of these distal regulatory regions overlapped H3K27ac peaks from CLL patients or immune cell types in the Roadmap reference epigenomes,indicating that enhancers are the prime sites of epigenetic differentiation between the two subtypes. Together, our analyses provide new insights into the global alterations of regulatory regions in CLL. Given the noticeable heterogeneity of chromatin landscape, further integration with histone modifications, TF binding, chromatin interaction, gene expression and genetic variation data are needed to understand the roles of epigenetic dysregulation and its interaction with genetic lesions in CLL pathogenesis. Citation Format: Huihuang Yan, Zhiquan Wang, Shulan Tian, Geffen Kleinstern, Nicholas Boddicker, Xing Li, Susan Slager, Esteban Braggio. Widespread alterations of chromatin accessibility in chronic lymphocytic leukemia (CLL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1256.

Biophysical Characterization of Interaction between E. coli Alanyl-tRNA Synethase with its Promoter DNA.

Aminoacyl-tRNA Synthetases (aaRSs) are well known for their role in the translation process. Lately investigators have discovered that this family of enzymes are also capable of executing a broad repertoire of functions that not only impact protein synthesis, but extend to a number of other activities. Till date, transcriptional regulation has so far only been described in E. coli Alanyl-tRNA synthetase and it was demonstrated that alaRS binds specifically to the palindromic DNA sequence flanking the gene's transcriptional start site and thereby regulating its own transcription. In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques. To understand the role of full length protein and oligomerization of alaRS in promoter DNA binding, two mutants were constructed, namely, N700 (a monomer, containing the N-terminal aminoacylation domain but without the C-terminal part) and G674D (previously demonstrated to form full-length monomer). Protein-DNA binding study using fluorescence spectroscopy, analytical ultracentrifugation, Isothermal Titration Calorimetry was conducted. Sedimentation equilibrium studies clearly demonstrated that monomeric variants were unable to bind promoter DNA. Isothermal Calorimetry (ITC) experiment was employed for further characterization of wild type alaRS-DNA interaction. It was observed that full length E. coli Alanyl-tRNA synthetase binds specifically with its promoter DNA and forms a dimer of dimers. On the other hand the two mutant variants were unable to bind with the DNA. In this study it was concluded that full length E. coli Alanyl-tRNA synthetase undergoes a conformational change in presence of its promoter DNA leading to formation of higher order structures. However, the exact mechanism behind this binding is currently unknown and beyond the scope of this study.

BonnMu: A Sequence-Indexed Resource of Transposon-Induced Maize Mutations for Functional Genomics Studies.

Sequence-indexed insertional libraries in maize (Zea mays) are fundamental resources for functional genetics studies. Here, we constructed a Mutator (Mu) insertional library in the B73 inbred background designated BonnMu A total of 1,152 Mu-tagged F2-families were sequenced using the Mu-seq approach. We detected 225,936 genomic Mu insertion sites and 41,086 high quality germinal Mu insertions covering 16,392 of the annotated maize genes (37% of the B73v4 genome). On average, each F2-family of the BonnMu libraries captured 37 germinal Mu insertions in genes of the Filtered Gene Set (FGS). All BonnMu insertions and phenotypic seedling photographs of Mu-tagged F2-families can be accessed via MaizeGDB.org Downstream examination of 137,410 somatic and germinal insertion sites revealed that 50% of the tagged genes have a single hotspot, targeted by Mu By comparing our BonnMu (B73) data to the UniformMu (W22) library, we identified conserved insertion hotspots between different genetic backgrounds. Finally, the vast majority of BonnMu and UniformMu transposons was inserted near the transcription start site of genes. Remarkably, 75% of all BonnMu insertions were in closer proximity to the transcription start site (distance: 542 bp) than to the start codon (distance: 704 bp), which corresponds to open chromatin, especially in the 5' region of genes. Our European sequence-indexed library of Mu insertions provides an important resource for functional genetics studies of maize.