Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior

Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. We investigate this question by examining Drosophila melanogaster fru P1 neurons that underlie reproductive behaviors in both sexes. We developed a method to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. Here, we use Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. We find substantial changes in chromatin modification profiles across development and fewer differences between males and females. Additionally, we find chromatin modifications that persist in different sets of genes from pupal to adult stages, which may point to genes important for cell fate determination in fru P1 neurons. We generated cell-type-specific RNA-seq data sets, using translating ribosome affinity purification (TRAP). We identify actively translated genes in fru P1 neurons, revealing novel stage- and sex-differences in gene expression. We also find chromatin modification enrichment patterns that are associated with gene expression. Next, we use the chromatin modification data to identify cell-type-specific super-enhancer-containing genes. We show that genes with super-enhancers in fru P1 neurons differ across development and between the sexes. We validated that a set of genes are expressed in fru P1 neurons, which were chosen based on having a super-enhancer and TRAP-enriched expression in fru P1 neurons.     

New Tools for Comparing Microscopy Images: Quantitative Analysis of Cell Types in Bacillus subtilis

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed.     

Local and global regulators linking anaerobiosis to cupA fimbrial gene expression in Pseudomonas aeruginosa

The cupA gene cluster of Pseudomonas aeruginosa encodes components and assembly factors of a putative fimbrial structure that enable this opportunistic pathogen to form biofilms on abiotic surfaces. In P. aeruginosa the control of cupA gene expression is complex, with the H-NS-like MvaT protein functioning to repress phase-variable (on/off) expression of the operon. Here we identify four positive regulators of cupA gene expression, including three unusual regulators encoded by the cgrABC genes and Anr, a global regulator of anaerobic gene expression. We show that the cupA genes are expressed in a phase-variable manner under anaerobic conditions and that the cgr genes are essential for this expression. We show further that cgr gene expression is negatively controlled by MvaT and positively controlled by Anr and anaerobiosis. Expression of the cupA genes therefore appears to involve a regulatory cascade in which anaerobiosis, signaled through Anr, stimulates expression of the cgr genes, resulting in a concomitant increase in cupA gene expression. Our findings thus provide mechanistic insight into the regulation of cupA gene expression and identify anaerobiosis as an inducer of phase-variable cupA gene expression, raising the possibility that phase-variable expression of fimbrial genes important for biofilm formation may occur in P. aeruginosa persisting in the largely anaerobic environment of the cystic fibrosis host lung.     

Conditional Survival as a Selection Strategy To Identify Plant-Inducible Genes of Pseudomonas syringae

A novel strategy termed habitat-inducible rescue of survival (HIRS) was developed to identify genes of Pseudomonas syringae that are induced during growth on bean leaves. This strategy is based on the complementation of metXW, two cotranscribed genes that are necessary for methionine biosynthesis and required for survival of P. syringae on bean leaves exposed to conditions of low humidity. We constructed a promoter trap vector, pTrap, containing a promoterless version of the wild-type P. syringae metXW genes. Only with an active promoter fused to metXW on pTrap did this plasmid restore methionine prototrophy to the P. syringae metXW mutant B7MX89 and survival of this strain on bean leaves. To test this method, a partial library of P. syringae genomic DNA was constructed in pTrap and a total of 1,400 B7MX89 pTrap clones were subjected to HIRS selection on bean leaves. This resulted in the enrichment of five clones, each with a unique RsaI restriction pattern of their DNA insert. Sequence analysis of these clones revealed those P. syringae genes for which putative plant-inducible activity could be assigned. Promoter activity experiments with a gfp reporter gene revealed that these plant-inducible gene promoters had very low levels of expression in minimal medium. Based on green fluorescent protein fluorescence levels, it appears that many P. syringae genes have relatively low expression levels and that the metXW HIRS strategy is a sensitive method to detect weakly expressed P. syringae genes that are active on plants. Furthermore, we found that protected sites on the leaf surface provided a higher level of enrichment for P. syringae expressing metXW than exposed sites. Thus, the metXW HIRS strategy should lead to the identification of P. syringae genes that are expressed primarily in these areas on the leaf.     

PLIN1 deficiency affects testicular gene expression at the meiotic stage in the first wave of spermatogenesis

PLIN1, a lipid droplet associated protein, has been implicated in playing a key role in the regulation of lipolysis and lipid storage in adipocytes. PLIN1 is found to be highly expressed in Leydig cells of testis, suggesting a potential role in steroidogenesis and spermatogenesis. In this study, we showed that PLIN1 was expressed in testis and that its mRNA levels declined significantly with development. To investigate the role of PLIN1, we take advantage of PLIN1-null mice. We found that the number of seminiferous tubules containing round spermatids was significantly increased at P21 (postnatal day 21). Furthermore, microarray analysis showed that there were 538 differentially expressed genes between PLIN1-null and wild-type mice at P21. The up-regulated genes in knockout mice were enriched in spermatogenesis by Gene Ontology classification. Among them, Prm1 and Wbp2nl are important for spermatogenesis which were confirmed by real-time PCR. Unexpectedly, the levels of serum testosterone and serum 17β-estradiol as well as steroidogenic genes are not altered in the PLIN1-null mice. Compared to the wild-type mice, no significant difference of fertility was found in the PLIN1-null mice. Therefore, these findings indicated that PLIN1 disruption leads to the increase of round spermatid-containing seminiferous tubules at the meiotic stage of the first wave of spermatogenesis through regulating spermatogenic related genes.     

Expression of cohesin and condensin genes during zebrafish development supports a non-proliferative role for cohesin

Cohesin and condensin are similar, but distinct multi-subunit protein complexes that have well-described roles in sister chromatid cohesion and chromosome condensation, respectively. Recently it has emerged that cohesin, and proteins that regulate cohesin function have additional developmental roles. To further understand the role of cohesin in development, we analyzed the expression of genes encoding cohesin and condensin subunits in developing zebrafish embryos and juvenile brain. We found that cohesin subunits are expressed in a pattern that is similar (but not quite identical) to the expression of condensin subunits. Cohesin genes smc1a, rad21, pds5b and smc3 were expressed in the forebrain ventricular zone, the tectum, the mid-hindbrain boundary, the fourth ventricle, branchial arches, the otic vesicle, the eye and faintly in the developing pectoral fins. Condensin genes smc2 and smc4 were expressed in the forebrain ventricular zone, the tectum, the mid-hindbrain boundary, the fourth ventricle, branchial arches, eye and pectoral fins. Condensin genes were additionally expressed in the hindbrain proliferative zone, an area in which cohesin genes were not detected. A comparison with pcna expression and BrdU incorporation revealed that the expression of cohesins and condensins closely overlap with zones of proliferation. Interestingly, cohesin genes were expressed in non-proliferating cells flanking rhombomere boundaries in the developing brain. In mature brain and eye, cohesin was expressed in both proliferating cells and in broad zones of post-mitotic cells. The distribution of cohesin and condensin mRNAs supports existing evidence for a non-cell cycle role for cohesin in the developing brain.     

Identification of gene expression profile of neural crest-derived cells isolated from submandibular glands of adult mice

Neural crest cells in the embryo migrate to reach target sites as neural crest-derived cells (NCDCs) where they differentiate into a variety of derivatives. Some NCDCs are maintained in an undifferentiated state throughout the life of the animal and are considered to be a useful cell source for regenerative medicine. However, no established method to obtain NCDCs sufficient for regenerative medicine from adults with high purity has been presented, since their distribution in adult tissues is not fully understood. It is critical to identify reliable markers for NCDCs in adults, as the expressions of P0 and Wnt1, the most reliable NCDC markers, are shut off in the embryonic stage. To analyze the characteristics of NCDCs in adult tissues, we utilized a double transgenic mouse strain, P0-Cre/CAG-CAT-EGFP transgenic mice (P0 mice), in which NCDCs were shown to express EGFP and we were able to recognize GFP-positive cells in those. We focused on the submandibular glands (SMGs), which are known to be derived from the neural crest. GFP-positive cells were shown to be scattered like islands in the SMGs of adult P0 mice. We surgically removed SMGs from adult mice and digested samples into single cell suspensions. GFP-positive cells separated using flow cytometry expressed a high level of Sox10, a marker of embryonic neural crest cells, suggesting successful isolation of NCDCs. To identify candidate marker genes in isolated NCDCs, we performed DNA microarray analyses and real-time PCR analysis of GFP-positive and -negative cells isolated from P0 mice, then selected genes showing differential gene expression patterns. As compared to GFP-negative cells, GFP-positive cells expressed Gpr4 and Ednrb at higher levels, whereas Pdgfra and Pdgfrb were expressed at lower levels. Furthermore, DNA microarray analysis showed that GFP-positive cells were positive for aquaporin 5, a marker for acinar cells. Together, our results indicate that NCDCs in adult SMGs have characteristic gene expression profiles specially their cell surface molecules. Cell sorting using a combination of these specific cell surface proteins would be a useful strategy for isolation of NCDCs from SMGs with high purity.