Inferring evolutionary histories of pathway regulation from transcriptional profiling data

One of the outstanding challenges in comparative genomics is to interpret the evolutionary importance of regulatory variation between species. Rigorous molecular evolution-based methods to infer evidence for natural selection from expression data are at a premium in the field, and to date, phylogenetic approaches have not been well-suited to address the question in the small sets of taxa profiled in standard surveys of gene expression. We have developed a strategy to infer evolutionary histories from expression profiles by analyzing suites of genes of common function. In a manner conceptually similar to molecular evolution models in which the evolutionary rates of DNA sequence at multiple loci follow a gamma distribution, we modeled expression of the genes of an \emph{a priori}-defined pathway with rates drawn from an inverse gamma distribution. We then developed a fitting strategy to infer the parameters of this distribution from expression measurements, and to identify gene groups whose expression patterns were consistent with evolutionary constraint or rapid evolution in particular species. Simulations confirmed the power and accuracy of our inference method. As an experimental testbed for our approach, we generated and analyzed transcriptional profiles of four \emph{Saccharomyces} yeasts. The results revealed pathways with signatures of constrained and accelerated regulatory evolution in individual yeasts and across the phylogeny, highlighting the prevalence of pathway-level expression change during the divergence of yeast species. We anticipate that our pathway-based phylogenetic approach will be of broad utility in the search to understand the evolutionary relevance of regulatory change.Comment: 30 pages, 12 figures, 2 tables, contact authors for supplementary table

Local Regulatory Variation in Saccharomyces cerevisiae

Naturally occurring sequence variation that affects gene expression is an important source of phenotypic differences among individuals within a species. We and others have previously shown that such regulatory variation can occur both at the same locus as the gene whose expression it affects (local regulatory variation) and elsewhere in the genome at trans-acting factors. Here we present a detailed analysis of genome-wide local regulatory variation in Saccharomyces cerevisiae. We used genetic linkage analysis to show that nearly a quarter of all yeast genes contain local regulatory variation between two divergent strains. We measured allele-specific expression in a diploid hybrid of the two strains for 77 genes showing strong self-linkage and found that in 52%–78% of these genes, local regulatory variation acts directly in cis. We also experimentally confirmed one example in which local regulatory variation in the gene AMN1 acts in trans through a feedback loop. Genome-wide sequence analysis revealed that genes subject to local regulatory variation show increased polymorphism in the promoter regions, and that some but not all of this increase is due to polymorphisms in predicted transcription factor binding sites. Increased polymorphism was also found in the 3′ untranslated regions of these genes. These findings point to the importance of cis-acting variation, but also suggest that there is a diverse set of mechanisms through which local variation can affect gene expression levels

Expression Profiling of the Wheat Pathogen Zymoseptoria tritici Reveals Genomic Patterns of Transcription and Host-Specific Regulatory Programs

Host specialization by pathogens requires a repertoire of virulence factors as well as fine-tuned regulation of gene expression. The fungal wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola) is a powerful model system for the discovery of genetic elements that underlie virulence and host specialization. We transcriptionally profiled the early stages of Z. tritici infection of a compatible host (wheat) and a noncompatible host (Brachypodium distachyon). The results revealed infection regulatory programs common to both hosts and genes with striking wheat-specific expression, with many of the latter showing sequence signatures of positive selection along the Z. tritici lineage. Genes specifically regulated during infection of wheat populated two large clusters of coregulated genes that may represent candidate pathogenicity islands. On evolutionarily labile, repeat-rich accessory chromosomes (ACs), we identified hundreds of highly expressed genes with signatures of evolutionary constraint and putative biological function. Phylogenetic analyses suggested that gene duplication events on these ACs were rare and largely preceded the diversification of Zymoseptoria species. Together, our data highlight the likely relevance for fungal growth and virulence of hundreds of Z. tritici genes, deepening the annotation and functional inference of the genes of this model pathogen

Circulating miRNA Biomarkers in Early Breast Cancer Detection following Mammography

The currently accepted stepwise model of breast tumorigenesis assumes a gradual transition from normal breast epithelial cells to atypical ductal hyperplasia (ADH), to ductal carcinoma in situ (DCIS) and then to invasive ductal carcinoma (IDC). Percutaneous core needle biopsy (CNB) is the standard technique following an abnormal mammographic finding. However, CNB is less reliable in differentiating simple ADH (sADH) from ADH component coexisted with advanced lesions such as DCIS and/or IDC (cADH). Therefore, to identify and validate novel reliable molecular biomarkers is essential in order to improve the efficiency of therapeutic recommendations, as well as to minimize anxiety and unnecessary procedures. miRNAs function as tumor suppressors or oncogenes and play a critical role in cancer initiation and progression by regulating their target genes. Unlike messenger RNAs (mRNAs), which could be easily degraded, miRNAs are found to be stable not only in body fluid, but also in Formalin-Fixed, Paraffin-Embedded (FFPE) tissues. The stability of miRNAs in FFPE and blood samples suggests that they may be the ideal biomarkers for the early diagnosis and prognosis of cancer, including breast cancer. The goal of this research is to use FFPE and blood samples from the two different groups of patients, analyze the candidate miRNAs to differentiating simple sADH from cADH. In our published studies, we identified a series of miRNAs that are differentially expressed during stepwise transition of breast carcinogenesis, including miR-671-5p. In this study, we showed that the expression of miR-671-5p and miR-638 decreases in ADH, DCIS, and IDC compared with the matched adjacent normal tissues. In addition, we examined the candidate miRNA expression in two groups of ADH blood samples: 28 sADHs and 32 cADHs by qRT-PCR. We found that miR-671-5p expression was decreased in cADHs, but not in sADHs, compared with their matched normal controls. Our recent publication demonstrated that miR-671-5p functions as a tumor suppressor miRNA during breast cancer progression by regulating FOXM1 expression. Using NanoString technology, we found another miRNA, miR-545-3p to be significantly overexpressed in cADHs compared with sADH. miR-545-3p is related to Snai2, which is a member of Snail family transcription factor, encoding a transcription repressor involving in epithelial-mesenchymal transitions (EMT). Our data suggest that miRNAs, such as miR-671-5p and miR-545-3p may be potential circulating biomarkers for early breast cancer detection following mammography and CNB

Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis.

Chronic itch remains a highly prevalent disorder with limited treatment options. Most chronic itch diseases are thought to be driven by both the nervous and immune systems, but the fundamental molecular and cellular interactions that trigger the development of itch and the acute-to-chronic itch transition remain unknown. Here, we show that skin-infiltrating neutrophils are key initiators of itch in atopic dermatitis, the most prevalent chronic itch disorder. Neutrophil depletion significantly attenuated itch-evoked scratching in a mouse model of atopic dermatitis. Neutrophils were also required for several key hallmarks of chronic itch, including skin hyperinnervation, enhanced expression of itch signaling molecules, and upregulation of inflammatory cytokines, activity-induced genes, and markers of neuropathic itch. Finally, we demonstrate that neutrophils are required for induction of CXCL10, a ligand of the CXCR3 receptor that promotes itch via activation of sensory neurons, and we find that that CXCR3 antagonism attenuates chronic itch

Structural Variation among Wild and Industrial Strains of Penicillium chrysogenum

Strain selection and strain improvement are the first, and arguably most important, steps in the industrial production of biological compounds by microorganisms. While traditional methods of mutagenesis and selection have been effective in improving production of compounds at a commercial scale, the genetic changes underpinning the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54–1255). We developed an assembly-free method of identifying chromosomal rearrangements and validated the in silico predictions with a PCR-based assay and Sanger sequencing. Despite many rounds of mutagen treatment and artificial selection, WIS 54–1255 differs from its wild progenitor at only one of the identified rearrangements. We suggest that natural variants predisposed for high penicillin production were instrumental in the success of WIS 54–1255 as an industrial strain. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be high penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known high penicillin producers may be viable candidates for further improvement efforts

miR-638 mediated regulation of BRCA1 affects DNA repair and sensitivity to UV and cisplatin in triple negative breast cancer

Introduction Triple-negative breast cancer (TNBC) represents 15 to 20% of all types of breast cancer; however, it accounts for a large number of metastatic cases and deaths, and there is still no effective treatment. The deregulation of microRNAs (miRNAs) in breast cancer has been widely reported. We previously identified that miR-638 was one of the most deregulated miRNAs in breast cancer progression. Bioinformatics analysis revealed that miR-638 directly targets BRCA1. The aim of this study was to investigate the role of miR-638 in breast cancer prognosis and treatment. Methods Formalin-fixed, paraffin-embedded (FFPE) breast cancer samples were microdissected into normal epithelial and invasive ductal carcinoma (IDC) cells, and total RNA was isolated. Several breast cancer cell lines were used for the functional analysis. miR-638 target genes were identified by TARGETSCAN-VERT 6.2 and miRanda. The expression of miR-638 and its target genes was analyzed by real-time qRT-PCR and Western blotting. Dual-luciferase reporter assay was employed to confirm the specificity of miR-638 target genes. The biological function of miR-638 was analyzed by MTT chemosensitivity, matrigel invasion and host cell reactivation assays. Results The expression of miR-638 was decreased in IDC tissue samples compared to their adjacent normal controls. The decreased miR-638 expression was more prevalent in non-TNBC compared with TNBC cases. miR-638 expression was significantly downregulated in breast cancer cell lines compared to the immortalized MCF-10A epithelial cells. BRCA1 was predicted as one of the direct targets of miR-638, which was subsequently confirmed by dual-luciferase reporter assay. Forced expression of miR-638 resulted in a significantly reduced proliferation rate as well as decreased invasive ability in TNBC cells. Furthermore, miR-638 overexpression increased sensitivity to DNA-damaging agents, ultraviolet (UV) and cisplatin, but not to 5-fluorouracil (5-FU) and epirubicin exposure in TNBC cells. Host cell reactivation assays showed that miR-638 reduced DNA repair capability in post UV/cisplatin-exposed TNBC cells. The reduced proliferation, invasive ability, and DNA repair capabilities are associated with downregulated BRCA1 expression. Conclusions Our findings suggest that miR-638 plays an important role in TNBC progression via BRCA1deregulation. Therefore, miR-638 might serve as a potential prognostic biomarker and therapeutic target for breast cancer

miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer.

MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management