Gene Expression Profiles as Molecular Indicators of Dissolved Oxygen Stress in Grass Shrimp \u3ci\u3ePalaemonetes pugio\u3c/i\u3e Holthuis 1949

Occurrence and severity of hypoxia is increasing in coastal and estuarine environments, and recovery of impacted habitats and living resources is slow. Detection of early biological effects of hypoxia is needed for timely remedial action to be taken. The overall objectives of this research was to develop molecular indicators of dissolved oxygen stress to assess the biological impact of hypoxia in coastal estuaries and validate their use through a combination of laboratory and field studies. To achieve these goals, grass shrimp, Palaemonetes pugio, oxygen-sensitive and hypoxia-tolerant species abundant in estuarine systems, were exposed to hypoxia under controlled laboratory conditions, and significant changes in gene expression were identified. Grass shrimp were collected from hypoxic field sites to evaluate if these hypoxia-responsive genes can be used as indicators of dissolved oxygen (DO) stress in the aquatic environment. Hypoxia inducible factor l a (HIF-la), a key transcription factor that controls a variety of cellular and systemic homeostatic responses to hypoxic stress, was successfully cloned and characterized in crustaceans using RT-PCR and RACE. Grass shrimp HIF-la protein shows a high level of conservation with other HIF-la proteins in the bHLH, PAS, ODD, and TAD domains. Phylogenetic analysis indicates that grass shrimp and vertebrate HIFs belong to distinct clades within the HIF protein family. HIF mRNA levels were not responsive to chronic or cyclic hypoxia. Six libraries of expressed sequence tags (ESTs) were constructed by suppression subtractive hybridization (SSH) from the grass shrimp exposed to environmental stress: moderate (DO 2.5 mg/L) and severe (1.5 mg/L) hypoxia, cyclic hypoxia (1.5 —\u3e1 mg/L), contaminant-induced stress (pyrene and copper), and biological stress (molt). Gene Ontology (GO) analysis of libraries showed several genes that were present in only one library suggesting that their expression may be stressor specific. The molting process was accompanied by changes in expression of many genes not found in the hypoxia/copper/pyrene libraries. The resulting annotated transcripts were used to design and construct a cDNA microarray to measure the expression changes in response to hypoxia conditions. The microarrays were used to examine differentially expressed genes in hypoxic vs. normoxic groups at 6 (H6), 12 (H12), 24 (H24), 48 (H48), 120 (H120), and 240 (H240) hours exposure to chronic hypoxia. Cluster analysis showed two response patterns, composed of an up- (including H6, H24, and H120) and down-regulated (including H12, H48, and H240) dominated cluster. Changes in gene expression are dynamic and transient. There is no differentially expressed gene up- or down-regulated common to all six groups. Differentially expressed genes were determined in hypoxic vs. normoxic groups after 1, 2, 5 and 10 days exposure to cyclic hypoxia. Sampling on each day was conducted at two different time series, one in the morning (representing low DO, CA) and one in the afternoon (representing high DO, CP). There are distinct differences between the number and identity of specific genes that are significantly down- or up-regulated in shrimp collected at the low DO and high DO points of the cyclic DO cycle. Only a few genes are differentially expressed in grass shrimp exposed to cyclic hypoxia in the field relative to those collected from a normoxic reference site. In conclusion, grass shrimp HIF is constitutively expressed and not induced by chronic and cyclic hypoxia exposures in both laboratory and field studies. Some differentially expressed genes appear unique at certain time points during laboratory and field exposures. However, changes of significant genes are too dynamic to serve as biomarkers of hypoxia stress in grass shrimp. Gene expression changes of grass shrimp in response to cyclic hypoxia conditions are not only dependent on the duration of exposure but also on the time of day

Analysis of neuronal injury transcriptional response identifies CTCF and YY1 as co-operating factors regulating axon regeneration

Injured sensory neurons activate a transcriptional program necessary for robust axon regeneration and eventual target reinnervation. Understanding the transcriptional regulators that govern this axon regenerative response may guide therapeutic strategies to promote axon regeneration in the injured nervous system. Here, we used cultured dorsal root ganglia neurons to identify pro-regenerative transcription factors. Using RNA sequencing, we first characterized this neuronal culture and determined that embryonic day 13.5 DRG (eDRG) neurons cultured for 7 days are similar to e15.5 DRG neuron

Gill Na+, K+-ATPase as a function of size and salinity in the blue crab, Callinectes sapidus Rathbun

Some kinetic properties of gill Na+,K+-ATPase of the blue crab, Callinectes sapidus, and its relation to osmotic regulation in juvenile and adult crabs were analyzed. Results suggest the presence of some differences in transport mechanisms for juvenile and adult blue crabs to maintain hemolymph concentration in dilute media. Adult and sub-adult crabs demonstrated fairly low levels of Na+,K+-ATPase activity in both anterior and posterior gills regardless of acclimation salinity. Juvenile blue crabs had much higher levels of Na+,K+-ATPase activity in both anterior and posterior gills relative to adults. Crabs acclimated to low salinity showed a significant increase in enzyme activity, both in anterior and posterior gills. The levels of enzyme activity in the anterior gills of juveniles showed a marked increase at low salinities. Enzyme activity in the posterior gills of juvenile crabs at 150 mOsm increased (35%) over sea water. However, there is a markedly higher specific activity increase (80%) in the anterior gills of smaller, juvenile crabs at this lower salinity. Sex had no effect on the enzyme activity relative to salinity. Abrupt transfer of juvenile and adult crabs from 1000mOsm to 150mOsm resulted in a gradual change in the Na+,K+-ATPase activity that reached steady-state levels within 7-10 days after the transfer. The time course for the increase in activity was preceded by an increase in the expression of the Na+,K+-ATPase a-subunit mRNA. The Na+,K+-ATPase of the posterior gills of both juvenile and sub-adult crabs showed an increased affinity for ATP at lower salinities, while that of the posterior gills of juvenile crabs had a higher affinity for Na+ than that of sub-adults at low salinity

Sequence specificity incompletely defines the genome-wide occupancy of Myc

BACKGROUND: The Myc-Max heterodimer is a transcription factor that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by Enhancer box (E-box) DNA elements, CACGTG or variants, to which the heterodimer binds in vitro. RESULTS: By analyzing ChIP-Seq datasets, we demonstrate that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II, Pol II, transcription machinery significantly better than with E-boxes. Metagene analyses show that in promoter regions, Myc is uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 15 bp upstream of Myc. We re-evaluate the DNA binding properties of full length Myc-Max proteins. Electrophoretic mobility shift assay results demonstrate Myc-Max heterodimers display significant sequence preference, but have high affinity for any DNA. Quantification of the relative affinities of Myc-Max for all possible 8-mers using universal protein-binding microarray assays shows that sequences surrounding core 6-mers significantly affect binding. Compared to the in vitro sequence preferences, Myc-Max genomic occupancy measured by ChIP-Seq is largely, although not completely, independent of sequence specificity. CONCLUSIONS: We quantified the affinity of Myc-Max to all possible 8-mers and compared this with the sites of Myc binding across the human genome. Our results indicate that the genomic occupancy of Myc cannot be explained by its intrinsic DNA specificity and suggest that the transcription machinery and associated promoter accessibility play a predominant role in Myc recruitment. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13059-014-0482-3) contains supplementary material, which is available to authorized users

Histone demethylase PHF8 promotes epithelial to mesenchymal transition and breast tumorigenesis

Histone demethylase PHF8 is upregulated and plays oncogenic roles in various cancers; however, the mechanisms underlying its dysregulation and functions in carcinogenesis remain obscure. Here, we report the novel functions of PHF8 in EMT (epithelial to mesenchymal transition) and breast cancer development. Genome-wide gene expression analysis revealed that PHF8 overexpression induces an EMT-like process, including the upregulation of SNAI1 and ZEB1. PHF8 demethylates H3K9me1, H3K9me2 and sustains H3K4me3 to prime the transcriptional activation of SNAI1 by TGF-β signaling. We show that PHF8 is upregulated and positively correlated with MYC at protein levels in breast cancer. MYC post-transcriptionally regulates the expression of PHF8 via the repression of microRNAs. Specifically, miR-22 directly targets and inhibits PHF8 expression, and mediates the regulation of PHF8 by MYC and TGF-β signaling. This novel MYC/microRNAs/PHF8 regulatory axis thus places PHF8 as an important downstream effector of MYC. Indeed, PHF8 contributes to MYC-induced cell proliferation and the expression of EMT-related genes. We also report that PHF8 plays important roles in breast cancer cell migration and tumor growth. These oncogenic functions of PHF8 in breast cancer confer its candidacy as a promising therapeutic target for this disease