Identification of interleukin genes in Pogona vitticeps using a de novo transcriptome assembly from RNA-seq data.

Interleukins are a group of cytokines with complex immunomodulatory functions that are important for regulating immunity in vertebrate species. Reptiles and mammals last shared a common ancestor more than 350 million years ago, so it is not surprising that low sequence identity has prevented divergent interleukin genes from being identified in the central bearded dragon lizard, Pogona vitticeps, in its genome assembly. To determine the complete nucleotide sequences of key interleukin genes, we constructed full-length transcripts, using the Trinity platform, from short paired-end read RNA sequences from stimulated spleen cells. De novo transcript reconstruction and analysis allowed us to identify interleukin genes that are missing from the published P. vitticeps assembly. Identification of key cytokines in P. vitticeps will provide insight into the essential molecular mechanisms and evolution of interleukin gene families and allow for characterization of the immune response in a lizard for comparison with mammals.

HES6 enhances the motility of alveolar rhabdomyosarcoma cells.

HES6, a member of the hairy-enhancer-of-split family of transcription factors, plays multiple roles in myogenesis. It is a direct target of the myogenic transcription factor MyoD and has been shown to regulate the formation of the myotome in development, myoblast cell cycle exit and the organization of the actin cytoskeleton during terminal differentiation. Here we investigate the expression and function of HES6 in rhabdomyosarcoma, a soft tissue tumor which expresses myogenic genes but fails to differentiate into muscle. We show that HES6 is expressed at high levels in the subset of alveolar rhabdomyosarcomas expressing PAX/FOXO1 fusion genes (ARMSp). Knockdown of HES6 mRNA in the ARMSp cell line RH30 reduces proliferation and cell motility. This phenotype is rescued by expression of mouse Hes6 which is insensitive to HES6 siRNA. Furthermore, expression microarray analysis indicates that the HES6 knockdown is associated with a decrease in the levels of Transgelin, (TAGLN), a regulator of the actin cytoskeleton. Knockdown of TAGLN decreases cell motility, whilst TAGLN overexpression rescues the motility defect resulting from HES6 knockdown. These findings indicate HES6 contributes to the pathogenesis of ARMSp by enhancing both proliferation and cell motility.

Multi-level 3D genome organization deteriorates during breast cancer progression.

Breast cancer entails intricate alterations in genome organization and expression. However, how three-dimensional (3D) chromatin structure changes in the progression from a normal to a breast cancer malignant state remains unknown. To address this, we conducted an analysis combining Hi-C data with lamina-associated domains (LADs), epigenomic marks, and gene expression in an in vitro model of breast cancer progression. Our results reveal that while the fundamental properties of topologically associating domains (TADs) remain largely stable, significant changes occur in the organization of compartments and subcompartments. These changes are closely correlated with alterations in the expression of oncogenic genes. We also observe a restructuring of TAD-TAD interactions, coinciding with a loss of spatial compartmentalization and radial positioning of the 3D genome. Notably, we identify a previously unrecognized interchromosomal insertion event, wherein a locus on chromosome 8 housing the MYC oncogene is inserted into a highly active subcompartment on chromosome 10. This insertion leads to the formation of de novo enhancer contacts and activation of the oncogene, illustrating how structural variants can interact with the 3D genome to drive oncogenic states. In summary, our findings provide evidence for the degradation of genome organization at multiple scales during breast cancer progression revealing novel relationships between genome 3D structure and oncogenic processes.

Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts.

Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells.

Chromatin accessibility of a promoter is fundamental in regulating transcriptional activity. The histone variant H2A.Z has been shown to contribute to this regulation, but its role has remained poorly understood. Here, we prepare high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for all human Pol II promoters in epithelial, mesenchymal and isogenic cancer cell lines. We find that, in contrast to the prevailing model, many different types of active and inactive promoter structures are observed that differ in their nucleosome organization and sensitivity to MNase digestion. Key aspects of an active chromatin structure include positioned H2A.Z MNase resistant nucleosomes upstream or downstream of the TSS, and a MNase sensitive nucleosome at the TSS. Furthermore, the loss of H2A.Z leads to a dramatic increase in the accessibility of transcription factor binding sites. Collectively, these results suggest that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter.

The Histone Variant H2A.Z Is a Master Regulator of the Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a profound example of cell plasticity that is crucial for embryonic development and cancer. Although it has long been suspected that chromatin-based mechanisms play a role in this process, no master regulator that can specifically regulate EMT has been identified to date. Here, we show that H2A.Z can coordinate EMT by serving as either an activator or repressor of epithelial or mesenchymal gene expression, respectively. Following induction of EMT by TGF-ß, we observed an unexpected loss of H2A.Z across both downregulated epithelial and upregulated mesenchymal promoters. Strikingly, the repression of epithelial gene expression was associated with reduction of H2A.Z upstream of the transcription start site (TSS), while the activation of mesenchymal gene expression was dependent on removal of H2A.Z downstream of the TSS. Therefore, the ability of H2A.Z to regulate EMT is dependent on its position, either upstream or downstream of the TSS.

Direct interaction of Ikaros and Foxp1 modulates expression of the G protein-coupled receptor G2A in B-lymphocytes and acute lymphoblastic leukemia.

Ikaros and Foxp1 are transcription factors that play key roles in normal lymphopoiesis and lymphoid malignancies. We describe a novel physical and functional interaction between the proteins, which requires the central zinc finger domain of Ikaros. The Ikaros-Foxp1 interaction is abolished by deletion of this region, which corresponds to the IK6 isoform that is commonly associated with high-risk acute lymphoblastic leukemia (ALL). We also identify the Gpr132 gene, which encodes the orphan G protein-coupled receptor G2A, as a novel target for Foxp1. Increased expression of Foxp1 enhanced Gpr132 transcription and caused cell cycle changes, including G2 arrest. Co-expression of wild-type Ikaros, but not IK6, displaced Foxp1 binding from the Gpr132 gene, reversed the increase in Gpr132 expression and inhibited G2 arrest. Analysis of primary ALL samples revealed a significant increase in GPR132 expression in IKZF1-deleted BCR-ABL negative patients, suggesting that levels of wild-type Ikaros may influence the regulation of G2A in B-ALL. Our results reveal a novel effect of Ikaros haploinsufficiency on Foxp1 functioning, and identify G2A as a potential modulator of the cell cycle in Ikaros-deleted B-ALL.

Exercise-Induced Tendon and Bone Injury in Recreational Runners: A Test-Retest Reliability Study.

BACKGROUND: Long-distance runners are prone to injuries including Achilles tendinopathy and medial tibial stress syndrome. We have developed an Internet comprehensive self-report questionnaire examining the medical history, injury history, and running habits of adult recreational runners. OBJECTIVE: The objective of the study was to evaluate two alternative forms of test-retest reliability of a comprehensive self-report Internet questionnaire retrospectively examining the medical history, injury history, and running habits among a sample of adult recreational runners. This will contribute to the broad aims of a wider study investigating genetics and running injury. METHODS: Invitations to complete an Internet questionnaire were sent by email to a convenience pilot population (test group 1). Inclusion criteria required participants to be a recreational runner age 18 or over, who ran over 15 km per week on a consistent basis. The survey questions addressed regular running habits and any injuries (including signs, symptoms, and diagnosis) of the lower limbs that resulted in discontinuation of running for a period of 2 consecutive weeks or more, within the last 2 years. Questions also addressed general health, age, sex, height, weight, and ethnic background. Participants were then asked to repeat the survey using the Internet platform again after 10-14 days. Following analysis of test group 1, we soft-launched the survey to a larger population (test group 2), through a local running club of 900 members via email platform. The same inclusion criteria applied, however, participants were asked to complete a repeat of the survey by telephone interview after 7-10 days. Selected key questions, important to clarify inclusion or exclusion from the wider genetics study, were selected to evaluate test-retest reliability. Reliability was quantified using the kappa coefficient for categorical data. RESULTS: In response to the invitation, 28 participants accessed the survey from test group 1, 23 completed the Internet survey on the first occasion, and 20 completed the Internet retest within 10-21 days. Test-retest reliability scored moderate to almost perfect (kappa=.41 to .99) for 19/19 of the key questions analyzed. Following the invitation, 122 participants accessed the survey from test group 2, 101 completed the Internet survey on the first occasion, and 50 were randomly selected and contacted by email inviting them to repeat the survey by telephone interview. There were 33 participants that consented to the telephone interview and 30 completed the questionnaire within 7-10 days. Test-retest reliability scored moderate to almost perfect for 18/19 (kappa=.41 to .99) and slight for 1/19 of the key questions analyzed.

Immunofluorescent staining reveals hypermethylation of microchromosomes in the central bearded dragon, Pogona vitticeps.

BACKGROUND: Studies of model organisms have demonstrated that DNA cytosine methylation and histone modifications are key regulators of gene expression in biological processes. Comparatively little is known about the presence and distribution of epigenetic marks in non-model amniotes such as non-avian reptiles whose genomes are typically packaged into chromosomes of distinct size classes. Studies of chicken karyotypes have associated the gene-richness and high GC content of microchromosomes with a distinct epigenetic landscape. To determine whether this is likely to be a common feature of amniote microchromosomes, we have analysed the distribution of epigenetic marks using immunofluorescence on metaphase chromosomes of the central bearded dragon (Pogona vitticeps). This study is the first to study the distribution of epigenetic marks on non-avian reptile chromosomes. RESULTS: We observed an enrichment of DNA cytosine methylation, active modifications H3K4me2 and H3K4me3, as well as the repressive mark H3K27me3 in telomeric regions on macro and microchromosomes. Microchromosomes were hypermethylated compared to macrochromosomes, as they are in chicken. However, differences between macro- and microchromosomes for histone modifications associated with actively transcribed or repressed DNA were either less distinct or not detectable. CONCLUSIONS: Hypermethylation of microchromosomes compared to macrochromosomes is a shared feature between P. vitticeps and avian species. The lack of the clear distinction between macro- and microchromosome staining patterns for active and repressive histone modifications makes it difficult to determine at this stage whether microchrosome hypermethylation is correlated with greater gene density as it is in aves, or associated with the greater GC content of P. vitticeps microchromosomes compared to macrochromosomes.

Epigenetic priming of a pre-B cell-specific enhancer through binding of Sox2 and Foxd3 at the ESC stage.

Modifications to the core histones are thought to contribute to ESC pluripotency by priming tissue-specific promoters and enhancers for later activation. However, it is unclear how these marks are targeted in ESCs and maintained during differentiation. Here, we show that the ESC factor Sox2 targets H3K4 methylation to monovalent and bivalent domains. In ESCs, Sox2 contributes to the formation of a monovalent mark at an enhancer in the pro/pre-B cell-specific lambda5-VpreB1 locus. Binding of Foxd3 suppresses intergenic transcription of the enhancer and surrounding sequences. In pro-B cells, enhancer activity is dependent on the Sox and Fox binding sites, and the enhancer is bound by Sox4, which is required for efficient expression of lambda5. Our results lead us to propose a factor relay model whereby ESC factors establish active epigenetic marks at tissue specific elements before being replaced by cell type-specific factors as cells differentiate.

Notch signaling regulates the differentiation of post-mitotic intestinal epithelial cells.

The intestinal epithelium comprises differentiated cells of four lineages maintained by precursor cells. As the Notch pathway controls the fate of proliferating cells in many systems, we investigated the effect of conditional expression of an activated Notch mutant in intestinal epithelium. An increase in the number of goblet cells occurs within 8 h of induction, due to an effect of Notch on post-mitotic cells, not on precursors. This observation broadens the role of Notch into controlling postmitotic differentiation and indicates that the composition of the epithelium is not solely determined by progenitor cells.