Orientador: Patricia Pintor dos Reis / Resumo: Introdução: O adenocarcinoma é o subtipo histológico mais comum de câncer de pulmão e leva à óbito milhões de pacientes a cada ano, mundialmente. Biomarcadores com utilidade clínica potencial têm sido identificados; entre estes, os RNAs não codificadores e os pseudogenes apresentam um potente papel na regulação de genes-alvo e genes parentais regulados por mRNAs respectivamente, os quais estão associados a vias moleculares de tumorigênese. Objetivos: Identificar alterações na expressão de pseudogenes em adenocarcinoma pulmonar, utilizando dados de transcritoma (RNA-Seq). Material e Métodos: Este estudo incluiu 27 tumores de adenocarcinoma pulmonar e 10 tecidos pulmonares histologicamente normais, adjacentes ao tumor, dos mesmos pacientes. Dados de RNA-Seq foram gerados na plataforma Illumina HiScan SQ e utilizados para a aplicação de uma estratégia de análise a fim de identificar sequências de pseudogenes com expressão anormal em tumores. Os pseudogenes com expressão significativamente alterada (p<0,05) foram validados no banco de dados The Cancer Genome Atlas (TCGA) e utilizados para identificação funcional, in silico, utilizando métodos computacionais incluindo o IID (Integrated Interactions Database), TOPPGENES (functional enrichment analysis), mirDip (microRNA Data Integration Portal) e NAViGaTOR (Network Analysis, Visualization, & GraphingTORonto). Resultados e Discussão: Foram identificados 60 pseudogenes desregulados em adenocarcinoma pulmonar, sendo que 34 destes fora... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Background: Lung adenocarcinoma is the most common histological subtype of lung cancer and is associated with high rates of patient death (>1 million), every year. Clinically useful biomarkers have been identified; among these, non-coding RNAs and pseudogenes have a potent role in the regulation of miRNA target genes and parental genes, respectively, which are associated with tumorigenesis pathways. Objectives: To identify alterations in pseudogene expression in lung adenocarcinoma using transcriptome data (RNA-Seq). Material and Methods: This study included 27 lung adenocarcinoma and 10 histologically normal tissues, adjacent to the tumors, from the same patients. RNA-Seq data were generated on the Illumina HiScan SQ platform and utilized for application of a data analysis strategy (pipeline) in order to identify pseudogene sequences with abnormal expression in tumor compare to normal tissues. Pseudogenes with significantly altered expression (p<0,05) were validated using external dataset The Cancer Genome Atlas (TCGA) and subsequently used for in silico functional analysis, using computational tools including IID (Integrated Interactions Database), TOPPGENES (functional enrichment analysis), mirDip (microRNA Data Integration Portal) and NAViGaTOR (Network Analysis, Visualization, & Graphing TORonto). Results and Discussion: A total of 60 deregulated pseudogenes were identified in pulmonary adenocarcinoma, 34 of which were validated in the TCGA database. Some pseudogenes sho... (Complete abstract click electronic access below) / Doutor

Submitted by Maykon Nascimento (maykon.albani@hotmail.com) on 2016-03-22T19:26:21Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao Leidy Johana Madronero.pdf: 2812224 bytes, checksum: d1702433cce9cfe12ed08f4146e98588 (MD5) / Approved for entry into archive by Patricia Barros (patricia.barros@ufes.br) on 2016-03-23T15:36:18Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao Leidy Johana Madronero.pdf: 2812224 bytes, checksum: d1702433cce9cfe12ed08f4146e98588 (MD5) / Made available in DSpace on 2016-03-23T15:36:18Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao Leidy Johana Madronero.pdf: 2812224 bytes, checksum: d1702433cce9cfe12ed08f4146e98588 (MD5) / CAPES / O mamoeiro (Carica papaya L.) é uma das fruteiras mais cultivadas nas regiões tropicais e subtropicais do mundo. O Brasil faz parte do grupo dos países que mais produzem e exportam mamão no mundo. O Espírito Santo e a Bahia são responsáveis por mais de 70% da área brasileira produtora deste fruto. Porém, doenças causadas por microrganismos infecciosos afetam de modo considerável sua produção. Entre as principais doenças, destaca-se a meleira do mamoeiro, causada pelo Papaya meleira virus (PMeV), que ainda não possui uma cultivar resistente. Interessantemente os sintomas somente são desencadeados após a frutificação. Os mecanismos moleculares envolvidos no desenvolvimento dos sintomas e na resposta de defesa da planta ao PMeV ainda não foram esclarecidos. Para entender os pontos chaves desta interação, que permitam o desenvolvimento de metodologias de melhoramento genético, um estudo transcriptômico foi abordado. A tecnologia RNA-seq foi usada para o sequenciamento do transcriptoma a partir de plantas com 3, 6 e 8 meses de idade após plantio, inoculadas e não inoculadas com o PMeV. Os genes diferencialmente expressos nos 3 tempos e nas duas condições foram preditos e analisados. Estas análises revelaram um padrão de expressão geral dos genes envolvidos nesta interação. Foram encontrados 21 genes com o perfil de expressão alterado nas plantas inoculadas exclusivamente nos seis meses de idade. Destes, 8 genes envolvidos em processos de respostas de defesa e morte celular, resposta ao estresse e resposta ao estímulo biótico e abiótico foram reprimidos; enquanto os demais (13 genes), envolvidos principalmente em processos metabólicos primários, biogêneses, diferenciação e ciclo celular, comunicação e crescimento celular, bem como processos envolvidos em reprodução, e desenvolvimento da floração, foram superexpressos. Estes resultados sugerem que, aos seis meses de idade, a planta é obrigada a alterar seu programa de expressão gênica, direcionando a resposta para os processos próprios do desenvolvimento, requeridos nesse estádio fisiológico, que primam sob a resposta ao estresse, fato que finalmente leva ao desenvolvimento dos sintomas. / Papaya is one of the fruit crops most cultivated in tropical and subtropical regions. Brazil is a major producer and exporter of papaya in the world. The largest area in Brazil, about 70%, for producing papaya is located in Espiritu Santo and Bahia. However this production is affected by infectious diseases caused by pathogens. The sticky disease caused by Papaya meleira virus (PMeV) is one of the most sever diseases. Not resistance has been reported for sticky disease and interestingly their symptoms only are triggered at the ripening. The molecular mechanisms involved in both the symptoms’ development and in the papaya defense response are still unclear. To understand the key point in this pathosystem leading to purpose crops genetic improvement methodologies we conducted a transcriptomics study. Rna-seq technology was used to sequencing the transcriptome from PMeV inoculated and no inoculated plants with 3, 6 and 8 months old. The differentially expressed genes in the both conditions and in the three times were found. Using different graphics analysis we show the global gene expression patterns in this interaction. We found 21 genes exhibit an altered profile at six month just in the inoculated condition. 8 genes related with defense response like cellular death and stress responses and biotic and abiotic stimulus were down regulated whereas 13 genes involved with primary metabolic process, biogenesis, cell differentiation, cell cycle, cell communication, cell grown, well as in reproduction and flower development were up regulated. This results suggest that in the six month the plant is forced to change their gene expression program routed to response for the physiological processes involved just at this period and should this is being favored over the stress response leading to the symptoms development.

Background: Meat tenderness is the consumer's most preferred sensory attribute. This trait is affected by a number of factors, including genotype, age, animal sex, and pre-and post-slaughter management. In view of the high percentage of Zebu genes in the Brazilian cattle population, mainly Nellore cattle, the improvement of meat tenderness is important since the increasing proportion of Zebu genes in the population reduces meat tenderness. However, the measurement of this trait is difficult once it can only be made after animal slaughtering. New technologies such as RNA-Seq have been used to increase our understanding of the genetic processes regulating quantitative traits phenotypes. The objective of this study was to identify differentially expressed genes related to meat tenderness, in Nellore cattle in order to elucidate the genetic factors associated with meat quality. Samples were collected 24 h postmortem and the meat was not aged. Results: We found 40 differentially expressed genes related to meat tenderness, 17 with known functions. Fourteen genes were up-regulated and 3 were down-regulated in the tender meat group. Genes related to ubiquitin metabolism, transport of molecules such as calcium and oxygen, acid-base balance, collagen production, actin, myosin, and fat were identified. The PCP4L1 (Purkinje cell protein 4 like 1) and BoLA-DQB (major histocompatibility complex, class II, DQ beta) genes were validated by qRT-PCR. The results showed relative expression values similar to those obtained by RNA-Seq, with the same direction of expression (i.e., the two techniques revealed higher expression of PCP4L1 in tender meat samples and of BoLA-DQB in tough meat samples). Conclusions: This study revealed the differential expression of genes and functions in Nellore cattle muscle tissue, which may contain potential biomarkers involved in meat tenderness.

Gene transcription rates are influenced by proteins, known as Transcription Factors (TFs), that interact with DNA. The locations of TFs on the genome directly influence gene expression and the functional characteristics of a cell. TF binding locations can be estimated for entire genomes using high throughput chromatin immunoprecipitation sequencing (ChIP-Seq). While the analysis of ChIP-Seq binding locations is standardized for a single experiment, complications arise when data sets, taken from different labs and experimental conditions, are combined. In this thesis, I present my method for the simultaneous comparison of multiple ChIP-Seq data sets. My method of comparing multiple ChIP-Seq data sets extends the analysis of a single data set through the addition of two stages, a combination stage, and an extraction stage. Typically, one of two approaches are used to combine information from multiple datasets. Either estimated binding sites are extracted from each dataset and then combined (e.g. by various intersections or unions) or the "raw" genomic signals are analyzed by clustering or dimensionality reduction methods. Both approaches have strengths, but also substantial drawbacks. The method presented here relies both on estimating the binding sites and comparing the “raw” genomic signals between data sets. Once the binding locations have been found, the first step in the combination stage is to define an alternate feature space (AFS). The AFS is the union of all binding locations determined for all data sets. The AFS represents a subset of the genome that is likely to have TF binding in any condition where the protein is active. Once the AFS is defined, the read density is determined from the “raw” genomic signal of each of the data sets. The density is determined for all locations in the AFS resulting in a unified density matrix (UDM). The UDM is the final product of the combination stage of the analysis. After the data sets are homogenized into the UDM, the extraction stage is applied to the matrix. The extraction stage consists of applying machine learning techniques and other methods used to analyze the “raw” genomic signal, to help elucidate underlying similarities and differences between the data sets. I applied this method to the binding locations of the TF TAL1 across 22 ChIP-Seq data sets from the hematopoietic and endothelial lineages. Once the UDM had been generated and normalized, using quantile normalization, hierarchical clustering and principle component analysis (PCA) were applied. Clusters, formed by hematopoietic stem cells (HSCs), Erythroid, and T-cell acute lymphoblastic leukemia (T-ALL), were found using hierarchical clustering. The principle components (PCs) of the UDM provided weights for each peak. Using those weights I could separate groups of cellular conditions including T-ALL, Erythroid, HSC, and Endothelial Colony Forming Cells (ECFCs.) The weights also provided a quantitative measure of importance for each peak in the AFS based on how much weight they provided towards the group of interest. Functional analysis techniques, including de novo motif search and Gene Ontology, were applied to the peak partitions defined using the PCs. Motifs that were enriched in the T-ALL TAL1 partition, and not the Erythroid, were annotated and found to be similar to those that had previously been published, including Runx1 motif and a preference for the CC Ebox (CACCTG). In addition to finding the CC Ebox in T-ALL, I also show that it does not form a composite motif with GATA, indicating an alternative mechanism for the binding of TAL1 in T-ALL. This thesis establishes that heterogeneous collections of ChIP-Seq datasets, from multiple labs and experimental conditions, can be meaningfully combined, and provides an algorithmic template for doing so.

Herbicide resistance is increasing in incidence among weed populations and poses a threat to food security. In Sunflower County, MS, a population of junglerice was identified with resistance to four herbicides, fenoxaprop-P-ethyl, imazamox, quinclorac, and propanil, each representing a different mechanism of action. The target site of fenoxaprop-P-ethyl, acetyl coenzyme A carboxylase (ACCase), was investigated. The ACCase contained none of the known resistance-conferring point mutations and an enzyme assay revealed no difference in response to increasing levels of fenoxaprop-P-ethyl between the resistant biotype and a sensitive biotype, indicating that the ACCase enzyme in the resistant biotype was sensitive to the herbicide. Whole-plant dose response assays in the presence and absence of cytochrome P450 and glutathione-S-transferase (GST) inhibitors did not increase efficacy of fenoxaprop-P-ethyl in the resistant biotype. However, when malathion, a cytochrome P450 inhibitor, was applied with imazamox or quinclorac, a reduction in resistance was observed in the resistant biotype, suggesting that a cytochrome P450 was important to the resistance mechanism for these two herbicides. RNA was isolated from the resistant and sensitive biotypes before and one hour after imazamox treatment for RNA-seq analysis. The reads from all samples were pooled to assemble the first E. colona leaf transcriptome. Differential gene expression analysis comparing untreated and treated samples for both biotypes revealed that several stress response genes were upregulated following herbicide exposure. A time course examining six of these genes showed that expression peaked between 4 and 12 hours and then dropped to untreated levels by 48 hours. Comparison of untreated resistant and sensitive plants revealed that a kinase and GST were significantly upregulated in the resistant biotype and an F-box protein was significantly downregulated. SNP analysis of cytochrome P450 sequences identified several nonsynonymous point mutations of interest including two transcripts that had premature stop codons in the sensitive but not the resistant biotype. These transcripts and their products should be the subject of future studies to determine if and how they are involved in resistance.

In recent years, the RNA-sequencing (RNA-seq) method, which measures the transcriptome by counting short sequencing reads obtained by high-throughput sequencing, is replacing the microarray technology as the major platform in gene expression studies. The large amount of discrete data in RNA-seq experiments calls for effective analysis methods. In this dissertation, a new method to detect differentially expressed genes based on quasi-likelihood theory is developed in experiments with a completely randomized design with two experimental conditions. The proposed method estimates the variance function empirically and consequently it has similar sensitivities and FDRs across distributions with different variance functions. In a simulation study, the method is shown to have similar sensitivities and FDRs across the data with three different types of variance functions compared with some other popular methods. This method is applied to a real dataset with two experimental conditions along with some competing methods. The new method is then extended to more complex designs such as an experiment with multiple experimental conditions, an experiment with block design and an experiment with factorial design. The same advantages for the new method have been found in simulation studies. This method and some competing methods are applied to three real datasets with complex designs. The new method is also applied to analyze reads per kilobase per million mapped reads (RPKM) data. In the simulation, the method is compared with the Linear Models for Microarray Data (LIMMA) originally developed for microarray analysis (Smyth, 2004) and the question of normalization is also examined. It is shown that the new method and the LIMMA method have similar performance. Further normalization is required for the proper analysis of the RPKM data and the best such normalization is the scaling method. Analyzing raw count data properly has better performance than analyzing the RPKM data. Different normalization and statistical methods are applied to a real dataset with varied gene length across samples. / Thesis / Doctor of Philosophy (PhD)

Vision is a crucial aspect of life for humans and animals. Impaired vision can lead to reduced quality of life along with other complications. Cataracts are a leading cause of impaired vision and blindness worldwide. Cataracts develop as a process of aging, although several environmental and lifestyle factors increase the risk of this disease. The toxic metal cadmium (Cd) has been associated with cataract formation and other ocular diseases such as macular degeneration. Cadmium exposure experiments were conducted to investigate potential pathways or mechanisms by which Cd may contribute to cataract formation and ocular disease. Zebra fish larvae (72, 96, and 120 hours post fertilization), adult zebra sh (6-month male, 10-month male, and 10-month female) and the B3 human lens epithelial (HLE) cell line were acutely exposed to varying concentrations of Cd. Transcriptomic changes relative to control (0 μM Cd) were determined using microarray analysis for zebra sh larvae and RNA sequencing (RNA-Seq) for adult zebra sh and HLE cells. Gene Ontology (GO) enrichment analysis for the zebra sh larvae exposure (50 μM Cd for 4 or 8 hours) enriched the "retina development in camera-type eye" term, and genes involved in enrichment (dnmt1, ccna2, fen1, mcm3 and slbp) were down-regulated. Gene set enrichment analysis (GSEA) for the 10-month male zebra sh exposure (50 μM Cd for 4 hours) enriched the "embryonic eye morphogenesis" gene set and signi ficant genes involved in enrichment (tcf7l1a, pitx2, fzd8a, sfrp5, lmx1bb, mfap2, six3b, lum, phactr4b, and foxc1a) were down-regulated. GSEA for the 10-month female zebra sh (50 μM Cd for 4 hours) enriched the "photoreceptor cell differentiation" gene set and signi cant genes involved in enrichment (odc1, thrb, and ush2a) were up-regulated. GO enrichment analysis for up-regulated genes in the HLE cell exposure (10 μM Cd for 4 hours) enriched the terms "eye development" (22 genes) and "lens development in camera-type eye" (CITED2, SKIL, CRYAB, SLC7A11, TGFB2, EPHA2, BCAR3, WNT5B, and BMP4). These results show cadmium is capable of altering transcription of eye-related genes in both zebra sh and human models, which may contribute to the formation of ocular disease. Many of these genes are involved in lens and retina development, yet they are also associated with diseases in these eye structures. Future studies could assess the consequences of altered transcription of these genes which could help elucidate the mechanisms of these changes and the overall effect of cadmium exposure on ocular disease. Ultimately, our study characterized the regulation of eye-related genes in response to Cd exposure and provided valuable knowledge laying the foundation for identi fication of the molecular mechanisms contributing to ocular diseases. / Thesis / Master of Science (MSc) / The eye is a sphere-like organ which is important for visualizing your surroundings. It is composed of many different structures such as the cornea, lens and retina. Many eye diseases have been characterized by abnormalities in eye structures; for example, a cataract occurs when the lens becomes cloudy and unable to focus light while macular degeneration is defined by progressive deterioration of the retinal macula region. While these diseases can occur through the natural aging process, certain environmental factors can increase risk. Exposure to cadmium, a toxic heavy metal which causes negative effects in animals, has shown to be associated with eye disease like cataracts and macular degeneration. In order to expand on this knowledge, we exposed both zebrafish and human lens cells to cadmium. By utilizing different experimental methods such as microarray analysis and RNA sequencing, eye-related genes which were affected by cadmium were revealed. Identifying the relationship between eye diseases, cadmium and gene expression will help identify the mechanism by which cadmium contributes to eye disease formation.

Droplet-based microfluidics is a powerful tool permitting massive-scale single-cell analysis in pico-/nano-liter water-in-oil droplets. It has been integrated into various library preparation techniques to accomplish high-throughput scRNA-seq, scDNA-seq, scATAC-seq, scChIP-seq, as well as scMulti-omics-seq. These advanced technologies have been providing unique and novel insights into both normal differentiation and disease development at single-cell level. In this thesis, we develop four new droplet-based tools for single-cell omics profiling. First, the developed Drop-BS is the first droplet-based platform to construct single-cell bisulfite sequencing libraries for DNA methylome profiling and allows production of BS library of 2,000-10,000 single cells within 2 d. We applied the technology to separately profile mixed cell lines, mouse brain tissues, and human brain tissues to reveal cell type heterogeneity. Second, the new Drop-ChIP platform only requires two steps of droplet generation to achieve multiple steps of reactions in droplets such as single-cell lysis, chromatin fragmentation, ChIP, and barcoding. Third, we aim to establish a droplet-based platform to accomplish high-throughput full-length RNA-seq (Drop-full-seq), which both current tube-based and droplet-based methods cannot realize. Last, we constructed an in-house droplet-based tool to assist single-cell ATAC-seq library preparation (Drop-ATAC), which provided a low-cost and facile protocol to conduct scATAC-seq in laboratories without the expensive instrument. / Doctor of Philosophy / Microfluidics is a collection of techniques to manipulate fluids in the micrometer scale. One of microfluidic techniques is called "droplet-based microfluidics". It can manipulate (i.e., generate, merge, sort, split, etc) pico-/nano-liter of water-in-oil droplets. First, since the water phase is separated by the continuous oil phase, these droplets are discrete and individual reactors. Second, droplet-based microfluidics can achieve highly parallel manipulation of thousands to millions of droplets. These two advantages make droplet-based microfluidics an ideal tool to perform single-cell assays. Over the past 10 years, various droplet-based platforms have been developed to study single-cell transcriptome, genome, epigenome, as well as multi-ome. To expand droplet-based tools for single-cell analysis, we aim to develop four novel platforms in this thesis. First, Drop-BS, by integrating droplet generation and droplet fusion techniques, can achieve high-throughput single-cell bisulfite sequencing library preparation. It can generate 10,000 single-cell BS libraries within 2 days which is difficult to achieve for conventional library preparation in tubes/microwells. Second, we developed a novel and facile Drop-ChIP platform to prepare single-cell ChIP-seq library. It is easy to operate since it only requires two steps of droplet generation. It also generates higher quality of data compared to previous work. In addition, we are working on the development and characterization of the other two droplet-based tools to achieve full-length single-cell RNA-seq and single-cell ATAC-seq.

Transcriptional regulation is a complicated process controlled by numerous factors such as transcription factors (TFs), chromatin remodeling enzymes, nucleosomes, post-transcriptional machineries, and cis-acting DNA sequence. I explored the complex transcriptional regulation in eukaryotes through three distinct studies to comprehensively understand the functional genomics at various steps. Although a variety of high throughput approaches have been developed to understand this complex system on a genome wide scale with high resolution, a lack of accurate and comprehensive annotation transcription start sites (TSS) and polyadenylation sites (PAS) has hindered precise analyses even in Saccharomyces cerevisiae, one of the simplest eukaryotes. We developed Simultaneous Mapping Of RNA Ends by sequencing (SMORE-seq) and identified the strongest TSS and PAS of over 90% of yeast genes with single nucleotide resolution. Owing to the high accuracy of TSS identified by SMORE-seq, we detected possibly mis-annotated 150 genes that have a TSS downstream of the annotated start codon. Furthermore, SMORE-seq showed that 5’-capped non-coding RNAs were highly transcribed divergently from TATA-less promoters in wild-type cells under normal conditions. Mapping of DNA-protein interactions is essential to understanding the role of TFs in transcriptional regulation. ChIP-seq is the most widely used method for this purpose. However, careful attention has not been given to technical bias reflected in final target calling due to many experimental steps of ChIP-seq including fixation and shearing of chromatin, immunoprecipitation, sequencing library construction, and computational analysis. While analyzing large-scale ChIP-seq data, we observed that unrelated proteins appeared to bind to the gene bodies of highly transcribed genes across datasets. Control experiments including input, IgG ChIP in untagged cells, and the Golgi factor Mnn10 ChIP also showed the strong binding at the same loci, indicating that the signals were obviously derived from bias that is devoid of biological meaning. In addition, the appearance of nucleosomal periodicity in ChIP-seq data for proteins localizing to gene bodies is another bias that can be mistaken for false interactions with nucleosomes. We alleviated these biases by correcting data with proper negative controls, but the biases could not be completely removed. Therefore, caution is warranted in interpreting the results from ChIP-seq. Nucleosome positioning is another critical mechanism of transcriptional regulation. Global mapping of nucleosome occupancy in S. cerevisiae strains deleted for chromatin remodeling complexes has elucidated the role of these complexes on a genome wide scale. In this study, loss of chromodomain helicase DNA binding protein 1 (Chd1) resulted in severe disorganization of nucleosome positioning. Despite the difficulties of performing ChIP-seq for chromatin remodeling complexes due to their transient and dynamic localization on chromatin, we successfully mapped the genome-wide occupancy of Chd1 and quantitatively showed that Chd1 co-localizes with early transcription elongation factors, but not late transcription elongation factors. Interestingly, Chd1 occupancy was independent of the methylation levels at H3K36, indicating the necessity of a new working model describing Chd1 localization.

Transcription factors activated by exogenous or endogenous stimuli alter gene expression with major effects on chromatin accessibility and the epigenome. This thesis investigates that impact of environmental chemical and hormonal exposure on liver chromatin accessibility in a mouse liver model. Exposure to the constitutive androstane receptor (CAR)-specific agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) induces localized changes in chromatin accessibility at several thousand DNase hypersensitive sites (DHS). Activating histone marks, associated with enhancers and promoters, were induced by TCPOBOP and were highly enriched at opening DHS. Opening DHS were highly enriched for CAR binding sites and nuclear receptor direct repeat-4 motifs. These DHS were also enriched for the CAR heterodimeric partner RXRA, binding by CEBPA and CEBPB, and motifs for other liver-specific factors. Thus, TCPOBOP alters the enhancer landscape through changes in histone marks and by mechanisms linked to induced CAR binding. In other studies, the impact of pituitary growth hormone (GH) secretion patterns on chromatin accessibility changes associated with sex-biased liver gene expression was examined. In adult male liver, the transcription factor STAT5 is directly activated by each successive plasma GH pulse. In female liver, STAT5 is persistently activated by the near-continuous stimulation by plasma GH. A majority of the ~4,000 GH-regulated, sex-biased DHS have chromatin marks characteristic of enhancers and were enriched for proximity to sex-biased gene promoters. Chromatin accessibility is thus a key feature of sex-differential gene expression. Two major classes of male-biased DHS were identified: dynamic male-biased DHS, almost all bound by STAT5, which undergo repeated cycles of chromatin opening and closing induced by each GH pulse; and static male-biased DHS, whose accessibility is unaffected GH/STAT5 pulses and whose sex bias results from these chromatin sites being more closed in female liver. Sites with STAT5 binding showed greater chromatin opening, many of which also contain the STAT5 motif. Finally, the effect of a single GH pulse on hypophysectomized male mouse liver was investigated to identify DHS responsive to the male, pulsatile-GH, secretion pattern. These studies demonstrate that widespread epigenetic changes associated with target gene expression are induced by xenobiotics and hormones regulating liver gene expression. / 2022-01-31T00:00:00Z