ΔSCOPE: A new method to quantify 3D biological structures and identify differences in zebrafish forebrain development.

Research in the life sciences has traditionally relied on the analysis of clear morphological phenotypes, which are often revealed using increasingly powerful microscopy techniques analyzed as maximum intensity projections (MIPs). However, as biology turns towards the analysis of more subtle phenotypes, MIPs and qualitative approaches are failing to adequately describe these phenotypes. To address these limitations and quantitatively analyze the three-dimensional (3D) spatial relationships of biological structures, we developed the computational method and program called ΔSCOPE (Changes in Spatial Cylindrical Coordinate Orientation using PCA Examination). Our approach uses the fluorescent signal distribution within a 3D data set and reorients the fluorescent signal to a relative biological reference structure. This approach enables quantification and statistical analysis of spatial relationships and signal density in 3D multichannel signals that are positioned around a well-defined structure contained in a reference channel. We validated the application of ΔSCOPE by analyzing normal axon and glial cell guidance in the zebrafish forebrain and by quantifying the commissural phenotypes associated with abnormal Slit guidance cue expression in the forebrain. Despite commissural phenotypes which display disruptions to the reference structure, ΔSCOPE was able to detect subtle, previously uncharacterized changes in zebrafish forebrain midline crossing axons and glia. This method has been developed as a user-friendly, open source program. We propose that ΔSCOPE is an innovative approach to advancing the state of image quantification in the field of high resolution microscopy, and that the techniques presented here are of broad applications to the life science field.

Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition.

Transcriptional regulation is modulated in part by chromatin-remodeling enzymes that control gene accessibility by altering chromatin compaction or nucleosome positioning. Brahma-related gene 1 (Brg1), a catalytic subunit of the mammalian SWI/SNF chromatin-remodeling enzymes, is required for both myoblast proliferation and differentiation, and the control of Brg1 phosphorylation by calcineurin, PKCß1, and p38 regulates the transition to differentiation. However, we hypothesized that Brg1 activity might be regulated by additional kinases. Here, we report that Brg1 is also a target of casein kinase 2 (CK2), a serine/threonine kinase, in proliferating myoblasts. We found that CK2 interacts with Brg1, and mutation of putative phosphorylation sites to non-phosphorylatable (Ser to Ala, SA) or phosphomimetic residues (Ser to Glu, SE) reduced Brg1 phosphorylation by CK2. Although BRG1-deleted myoblasts that ectopically express the SA-Brg1 mutant proliferated similarly to the parental cells or cells ectopically expressing wild-type (WT) Brg1, ectopic expression of the SE-Brg1 mutant reduced proliferation and increased cell death, similar to observations from cells lacking Brg1. Moreover, pharmacological inhibition of CK2 increased myoblast proliferation. Furthermore, the Pax7 promoter, which controls expression of a key transcription factor required for myoblast proliferation, was in an inaccessible chromatin state in the SE-Brg1 mutant, suggesting that hyperphosphorylated Brg1 cannot remodel chromatin. WT-, SA-, and SE-Brg1 exhibited distinct differences in interacting with and affecting expression of the SWI/SNF subunits Baf155 and Baf170 and displayed differential sub-nuclear localization. Our results indicate that CK2-mediated phosphorylation of Brg1 regulates myoblast proliferation and provides insight into one mechanism by which composition of the mammalian SWI/SNF enzyme complex is regulated.

Src tyrosine kinase phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer.

Src tyrosine kinase has long been implicated in colon cancer but much remains to be learned about its substrates. The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) has just recently been implicated in colon cancer but its role is poorly defined. Here we show that c-Src phosphorylates human HNF4α on three tyrosines in an interdependent and isoform-specific fashion. The initial phosphorylation site is a Tyr residue (Y14) present in the N-terminal A/B domain of P1- but not P2-driven HNF4α. Phospho-Y14 interacts with the Src SH2 domain, leading to the phosphorylation of two additional tyrosines in the ligand binding domain (LBD) in P1-HNF4α. Phosphomimetic mutants in the LBD decrease P1-HNF4α protein stability, nuclear localization and transactivation function. Immunohistochemical analysis of approximately 450 human colon cancer specimens (Stage III) reveals that P1-HNF4α is either lost or localized in the cytoplasm in approximately 80% of tumors, and that staining for active Src correlates with those events in a subset of samples. Finally, three SNPs in the human HNF4α protein, two of which are in the HNF4α F domain that interacts with the Src SH3 domain, increase phosphorylation by Src and decrease HNF4α protein stability and function, suggesting that individuals with those variants may be more susceptible to Src-mediated effects. This newly identified interaction between Src kinase and HNF4α has important implications for colon and other cancers.

Nuclear receptor HNF4α binding sequences are widespread in Alu repeats.

BACKGROUND: Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Recent studies, however, suggest that they may contain transcription factor binding sites and hence possibly play a role in regulating gene expression. RESULTS: Here, we show that binding sites for a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors, hepatocyte nuclear factor 4alpha (HNF4α, NR2A1), are highly prevalent in Alu repeats. We employ high throughput protein binding microarrays (PBMs) to show that HNF4α binds > 66 unique sequences in Alu repeats that are present in ~1.2 million locations in the human genome. We use chromatin immunoprecipitation (ChIP) to demonstrate that HNF4α binds Alu elements in the promoters of target genes (ABCC3, APOA4, APOM, ATPIF1, CANX, FEMT1A, GSTM4, IL32, IP6K2, PRLR, PRODH2, SOCS2, TTR) and luciferase assays to show that at least some of those Alu elements can modulate HNF4α-mediated transactivation in vivo (APOM, PRODH2, TTR, APOA4). HNF4α-Alu elements are enriched in promoters of genes involved in RNA processing and a sizeable fraction are in regions of accessible chromatin. Comparative genomics analysis suggests that there may have been a gain in HNF4α binding sites in Alu elements during evolution and that non Alu repeats, such as Tiggers, also contain HNF4α sites. CONCLUSIONS: Our findings suggest that HNF4α, in addition to regulating gene expression via high affinity binding sites, may also modulate transcription via low affinity sites in Alu repeats.

Characterizing the diverse cells that associate with the developing commissures of the zebrafish forebrain.

During embryonic development of bilaterally symmetrical organisms, neurons send axons across the midline at specific points to connect the two halves of the nervous system with a commissure. Little is known about the cells at the midline that facilitate this tightly regulated process. We exploit the conserved process of vertebrate embryonic development in the zebrafish model system to elucidate the identity of cells at the midline that may facilitate postoptic (POC) and anterior commissure (AC) development. We have discovered that three different gfap+ astroglial cell morphologies persist in contact with pathfinding axons throughout commissure formation. Similarly, olig2+ progenitor cells occupy delineated portions of the postoptic and anterior commissures where they act as multipotent, neural progenitors. Moreover, we conclude that both gfap+ and olig2+ progenitor cells give rise to neuronal populations in both the telencephalon and diencephalon; however, these varied cell populations showed significant developmental timing differences between the telencephalon and diencephalon. Lastly, we also showed that fli1a+ mesenchymal cells migrate along the presumptive commissure regions before and during midline axon crossing. Furthermore, following commissure maturation, specific blood vessels formed at the midline of the POC and immediately ventral and parallel to the AC. This comprehensive account of the cellular populations that correlate with the timing and position of commissural axon pathfinding has supported the conceptual modeling and identification of the early forebrain architecture that may be necessary for proper commissure development.

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation.

Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer.

The transcription factor encyclopedia.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.