John C. F. Hsieh

Transcriptional Activity Affects the H3K4me3 Level and Distribution in the Coding Region

ABSTRACT Histone lysine methylation and CpG DNA methylation contribute to transcriptional regulation. We have shown previously that dimethylated and trimethylated forms of histone H3 at lysine 4 (H3K4me2 and H3K4me3) are primarily depleted from CpG-methylated DNA regions by using patch-methylated stable episomes (minichromosomes) in human cells. This effect on H3K4me2 is clearly not linked to the transcriptional activity in the methylated DNA region; however, transcriptional activity may play a role in the presence of H3K4me3. Here, we present clear evidence of the impact of transcriptional activity on the overall level of H3K4me3 in the coding region and the lack of impact on H3K4me2. Our data also demonstrate the influence of transcriptional activity on the distribution of H3K4me3 and H3K4me2, but not that of total H3, in the 5′ end of the coding region relative to the 3′ end. The nature of the promoter (viral or endogenous) affects H3K4me3 much more than it affects H3K4me2, suggesting a potential fundamental difference in the recruitment of methyltransferase for H3K4 trimethylation.

Large chromosome deletions, duplications, and gene conversion events accumulate with age in normal human colon crypts

Little is known about the types and numbers of mutations that may accumulate in normal human cells with age. Such information would require obtaining enough DNA from a single cell to accurately carry out reliable analysis despite extensive amplification; and complete genomic coverage under these circumstances is difficult. We have compared colon crypts, which are putatively clonal and contain ~2000 cells each, to determine how much somatic genetic variation occurs in vivo (without ex vivo cell culturing). Using high‐density SNP microarrays, we find that chromosome deletions, duplications, and gene conversions were significantly more frequent in colons from the older individuals. These changes affected lengths ranging from 73 kb to 46 Mb. Although detection requires progeny of a single mutant stem cell to reach niche dominance over neighboring stem cells, none of the deletions appear likely to confer a selective advantage. Mutations can become fixed randomly during stem cell evolution through neutral drift in normal human crypts. The fact that chromosomal changes are detected in individual crypts with increasing age suggests that either such changes accumulate with age or single stem cell dominance increases with age, and the former is more likely. This progressive genome‐wide divergence of human somatic cells with age has implications for aging and disease in multicellular organisms.

Effects of dietary fiber on cecal short-chain fatty acid and cecal microbiota of broiler and laying-hen chicks

This experiment was conducted to evaluate the effects of feeding dietary fiber on cecal short-chain fatty acid (SCFA) concentration and cecal microbiota of broiler and laying-hen chicks. The lower fiber diet was based on corn-soybean meal (SBM) and the higher fiber diet was formulated using corn-SBM-dried distillers grains with solubles (DDGS) and wheat bran to contain 60.0 g/kg of both DDGS and wheat bran from 1 to 12 d and 80.0 g/kg of both DDGS and wheat bran from 13 to 21 d. Diets were formulated to meet or exceed NRC nutrient requirements. Broiler and laying-hen chicks were randomly assigned to the high and low fiber diets with 11 replicates of 8 chicks for each of the 4 treatments. One cecum from 3 chicks was collected from each replicate: one cecum underwent SCFA concentration analysis, one underwent bacterial DNA isolation for terminal restriction fragment length polymorphism (TRFLP), and the third cecum was used for metagenomics analyses. There were interactions between bird line and dietary fiber for acetic acid (P = 0.04) and total SCFA (P = 0.04) concentration. There was higher concentration of acetic acid (P = 0.02) and propionic acid (P < 0.01) in broiler chicks compared to laying-hen chicks. TRFLP analysis showed that cecal microbiota varied due to diet (P = 0.02) and chicken line (P = 0.03). Metagenomics analyses identified differences in the relative abundance of Helicobacter pullorum and Megamonas hypermegale and the genera Enterobacteriaceae, Campylobacter, Faecalibacterium, and Bacteroides in different treatment groups. These results provide insights into the effect of dietary fiber on SCFA concentration and modulation of cecal microbiota in broiler and laying-hen chicks.

Tumor Stroma as the Main Source of Inhibin Production in Ovarian Epithelial Tumors

PROBLEM: Elevated serum inhibin levels have been found in ovarian cancer patients; however, the source of the elevated inhibin is uncertain. Previous studies of activin in human ovarian cancer suggest that activin may promote the growth of the tumor. The aims of this study were to examine the source of elevated inhibin from ovarian epithelial tumors (OETs) and to preliminarily investigate the role of the gonadotropin–inhibin/activin relationship in the development of OET.
 METHOD OF STUDY: The protein and mRNA expression of α and βA subunits of inhibin/activin were examined by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT‐PCR) in 120 OETs, including 30 benign cystadenomas, 30 borderline tumors, and 60 carcinomas. Stromal and epithelial cells were microdissected from 23 OETs to further examine the expression of α and βA subunits by RT‐PCR. Dimeric inhibin A and activin A production were measured by using the two‐site ELISA from three OET cell lines in culture under treatment of follicle‐stimulating hormone (FSH) and luteinizing hormone (LH).
 RESULTS: βA subunit was expressed in the epithelial component of 100% of the cystadenomas, in 80% of borderline tumors, and in 75% of the carcinomas, but not in tumor stroma. Inhibin α expression was not found in the epithelium of all OETs studied, but focal inhibin α immunoreactivity was seen in the tumor stroma (mainly luteinized stromal cells) in the majority of cases. Dimeric activin A was produced by all of the three OET cell lines with a 1.5–1.9‐fold increment after FSH stimulation. However, activin A production was not augmented by LH treatment. No inhibin A was produced by the three OET cell lines with or without gonadotropin stimulation.
 CONCLUSIONS: The stroma of OET is the major source in the production of inhibin α (monomer). Dimeric inhibin A production may be the result of combined efforts of the tumor stroma (α subunit) and epithelium (βA subunit). Cellular, compartmental expression of inhibin and activin subunits may play a role in the development of OET, although the mechanism remains undefined. The unopposed activin A production stimulated by FSH in OET cell lines suggests that activin production may represent one of the cellular mechanisms of growth promotion by FSH.

Heterogeneity and randomness of DNA methylation patterns in human embryonic stem cells.

DNA methylation has been proposed to be important in many biological processes and is the subject of intense study. Traditional bisulfite genomic sequencing allows detailed high-resolution methylation pattern analysis of each molecule with haplotype information across a few hundred bases at each locus, but lacks the capacity to gather voluminous data. Although recent technological developments are aimed at assessing DNA methylation patterns in a high-throughput manner across the genome, the haplotype information cannot be accurately assembled when the sequencing reads are short or when each hybridization target only includes one or two cytosine-phosphate-guanine (CpG) sites. Whether a distinct and nonrandom DNA methylation pattern is present at a given locus is difficult to discern without the haplotype information, and the DNA methylation patterns are much less apparent because the data are often obtained only as methylation frequencies at each CpG site with some of these methods. It would facilitate the interpretation of data obtained from high-throughput bisulfite sequencing if the loci with nonrandom DNA methylation patterns could be distinguished from those that are randomly methylated. In this study, we carried out traditional genomic bisulfite sequencing using the normal diploid human embryonic stem (hES) cell lines, and utilized Hamming distance analysis to evaluate the existence of a distinct and nonrandom DNA methylation pattern at each locus studied. Our findings suggest that Hamming distance is a simple, quick, and useful tool to identify loci with nonrandom DNA methylation patterns and may be utilized to discern links between biological changes and DNA methylation patterns in the high-throughput bisulfite sequencing data sets.

Heat Stress and Lipopolysaccharide Stimulation of Chicken Macrophage-Like Cell Line Activates Expression of Distinct Sets of Genes

Acute heat stress requires immediate adjustment of the stressed individual to sudden changes of ambient temperatures. Chickens are particularly sensitive to heat stress due to development of insufficient physiological mechanisms to mitigate its effects. One of the symptoms of heat stress is endotoxemia that results from release of the lipopolysaccharide (LPS) from the guts. Heat-related cytotoxicity is mitigated by the innate immune system, which is comprised mostly of phagocytic cells such as monocytes and macrophages. The objective of this study was to analyze the molecular responses of the chicken macrophage-like HD11 cell line to combined heat stress and lipopolysaccharide treatment in vitro. The cells were heat-stressed and then allowed a temperature-recovery period, during which the gene expression was investigated. LPS was added to the cells to mimic the heat-stress-related endotoxemia. Semi high-throughput gene expression analysis was used to study a gene panel comprised of heat shock proteins, stress-related genes, signaling molecules and immune response genes. HD11 cell line responded to heat stress with increased mRNA abundance of the HSP25, HSPA2 and HSPH1 chaperones as well as DNAJA4 and DNAJB6 co-chaperones. The anti-apoptotic gene BAG3 was also highly up-regulated, providing evidence that the cells expressed pro-survival processes. The immune response of the HD11 cell line to LPS in the heat stress environment (up-regulation of CCL4, CCL5, IL1B, IL8 and iNOS) was higher than in thermoneutral conditions. However, the peak in the transcriptional regulation of the immune genes was after two hours of temperature-recovery. Therefore, we propose the potential influence of the extracellular heat shock proteins not only in mitigating effects of abiotic stress but also in triggering the higher level of the immune responses. Finally, use of correlation networks for the data analysis aided in discovering subtle differences in the gene expression (i.e. the role of the CASP3 and CASP9 genes).

Integrated host and viral transcriptome analyses reveal pathology and inflammatory response mechanisms to ALV-J injection in SPF chickens

Avian leukosis virus (ALV) is detrimental to poultry health and causes substantial economic losses from mortality and decreased performance. Because tumorigenesis is a complex mechanism, the regulatory architecture of the immune system is likely to include the added dimensions of modulation by miRNAs and long-noncoding RNA (lncRNA). To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and the associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were infected with ALV-J or maintained as non-injected controls. Spleen samples were collected at 40 days post injection (dpi), and sequenced. There were 864 genes, 7 miRNAs and 17 lncRNAs differentially expressed between infected and non-infected birds. The combined analysis of the 4 RNA expression datasets revealed that ALV infection is detected by pattern-recognition receptors (TLR9 and TLR3) leading to a type-I IFN mediated innate immune response that is modulated by IRF7 and IRF1. Co-expression network analysis of mRNA with miRNA, lncRNA and virus genes identified key elements within the complex networks utilized during ALV response. The integration of information from the host transcriptomic, epigenetic and virus response also has the potential to provide deeper insights into other host-pathogen interactions.

Host-Pathogen Protein-Protein Interaction Prediction Using an in silico Model

and Implications Newcastle Disease (ND) is caused by Newcastle Disease Virus (NDV) and is a major problem in developing countries where vaccination against NDV is not easily achievable. A step required for NDV infection is the cleavage of the NDV fusion (F) protein. Using structural information of the NDV F protein and the only known host protein binding partner, protein disulfide isomerase A3 (PDIA3), we computer modeled the interaction between the two proteins by looking at a docked structure of these two proteins. With our docked structure, we visualized one of the catalytic domains of PDIA3 being near the cleavage site of the NDV F protein. We also discovered a novel binding pocket on the NDV F protein that interacts with the second catalytic domain of PDIA3. This new insight may provide additional molecular targets for NDV vaccine development. Introduction Newcastle Disease is an avian disease with major global impact, especially in developing countries where the virulent strain is endemic. Up to 80% of chickens die within days of infection by a high virulence strain. Unfortunately, this spells trouble for poor rural families that rely on their flock for protein and income. ND is caused by NDV, and vaccines for ND are available in developed countries. Of the little that is known about the mechanism of NDV infection, the cleavage of NDV F protein is a crucial step during the virus invasion of the host cell. In 2014, chicken PDIA3 was found as the first host binding partner for NDV F protein. In this study, we used an in silico method to visualize the docking of NDV F protein to the chicken PDIA3 in order to confirm the potential activation of the NDV F protein by PDIA3.