Cortical Thickness Related to Compensatory Viewing Strategies in Patients With Macular Degeneration

Retinal diseases like age-related macular degeneration (AMD) or hereditary juvenile macular dystrophies (JMD) lead to a loss of central vision. Many patients compensate for this loss with a pseudo fovea in the intact peripheral retina, the so-called “preferred retinal locus” (PRL). How extensive eccentric viewing associated with central vision loss (CVL) affects brain structures responsible for visual perception and visually guided eye movements remains unknown. CVL results in a reduction of cortical gray matter in the “lesion projection zone” (LPZ) in early visual cortex, but the thickness of primary visual cortex appears to be largely preserved for eccentric-field representations. Here we explore how eccentric viewing strategies are related to cortical thickness (CT) measures in early visual cortex and in brain areas involved in the control of eye movements (frontal eye fields, FEF, supplementary eye fields, SEF, and premotor eye fields, PEF). We determined the projection zones (regions of interest, ROIs) of the PRL and of an equally peripheral area in the opposite hemifield (OppPRL) in early visual cortex (V1 and V2) in 32 patients with MD and 32 age-matched controls (19–84 years) by functional magnetic resonance imaging. Subsequently, we calculated the CT in these ROIs and compared it between PRL and OppPRL as well as between groups. Additionally, we examined the CT of FEF, SEF, and PEF and correlated it with behavioral measures like reading speed and eccentric fixation stability at the PRL. We found a significant difference between PRL and OppPRL projection zones in V1 with increased CT at the PRL, that was more pronounced in the patients, but also visible in the controls. Although the mean CT of the eye fields did not differ significantly between patients and controls, we found a trend to a positive correlation between CT in the right FEF and SEF and fixation stability in the whole patient group and between CT in the right PEF and reading speed in the JMD subgroup. The results indicate a possible association between the compensatory strategies used by patients with CVL and structural brain properties in early visual cortex and cortical eye fields

Enhancer decommissioning by Snail1-induced competitive displacement of TCF7L2 and down-regulation of transcriptional activators results in EPHB2 silencing

Transcriptional silencing is a major cause for the inactivation of tumor suppressor genes, however, the underlying mechanisms are only poorly understood. The EPHB2 gene encodes a receptor tyrosine kinase that controls epithelial cell migration and allocation in intestinal crypts. Through its ability to restrict cell spreading, EPHB2 functions as a tumor suppressor in colorectal cancer whose expression is frequently lost as tumors progress to the carcinoma stage. Previously we reported that EPHB2 expression depends on a transcriptional enhancer whose activity is diminished in EPHB2 non-expressing cells. Here we investigated the mechanisms that lead to EPHB2 enhancer inactivation. We show that expression of EPHB2 and SNAIL1 - an inducer of epithelial-mesenchymal transition (EMT) - is anti-correlated in colorectal cancer cell lines and tumors. In a cellular model of Snail1-induced EMT, we observe that features of active chromatin at the EPHB2 enhancer are diminished upon expression of murine Snail1. We identify the transcription factors FOXA1, MYB, CDX2 and TCF7L2 as EPHB2 enhancer factors and demonstrate that Snail1 indirectly inactivates the EPHB2 enhancer by downregulation of FOXA1 and MYB. In addition, Snail1 induces the expression of Lymphoid enhancer factor 1 (LEF1) which competitively displaces TCF7L2 from the EPHB2 enhancer. In contrast to TCF7L2, however, LEF1 appears to repress the EPHB2 enhancer. Our findings underscore the importance of transcriptional enhancers for gene regulation under physiological and pathological conditions and show that SNAIL1 employs a combinatorial mechanism to inactivate the EPHB2 enhancer based on activator deprivation and competitive displacement of transcription factors

Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells.

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases

Distinct molecular response patterns of activating STAT3 mutations associate with penetrance of lymphoproliferation and autoimmunity

Germline STAT3 gain-of-function (GOF) mutations have been linked to poly-autoimmunity and lymphoproliferation with variable expressivity and incomplete penetrance. Here we studied the impact of 17 different STAT3 GOF mutations on the canonical STAT3 signaling pathway and correlated the molecular results with clinical manifestations. The mutations clustered in three groups. Group 1 mutants showed altered STAT3 phosphorylation kinetics and strong basal transcriptional activity. They were associated with the highest penetrance of lymphoproliferation and autoimmunity. Group 2 mutants showed a strongly inducible transcriptional reporter activity and were clinically less penetrant. Group 3 mutants were mostly located in the DNA binding domain and showed the strongest DNA binding affinity despite a poor transcriptional reporter response. Thus, the GOF effect of STAT3 mutations is determined by a heterogeneous response pattern at the molecular level. The correlation of response pattern and clinical penetrance indicates a significant contribution of mutation-determined effects on disease manifestations

fMRI with Central Vision Loss: Effects of Fixation Locus and Stimulus Type

Purpose In patients with central visual field scotomata, a large part of visual cortex is not adequately stimulated. Patients often use a new eccentric fixation area on intact peripheral retina (preferred retinal locusPRL) that functions as a pseudo-fovea. We used functional magnetic resonance imaging (fMRI) to examine whether stimulating this pseudo-fovea leads to increased activation or altered activation patterns in visual cortex in comparison to stimulating a comparable peripheral area in the opposite hemifield (OppPRL). Methods Nineteen patients with binocular central scotomata caused by hereditary retinal dystrophies and an age-matched control group were tested. The center of the visual field, PRL, and OppPRL were stimulated with flickering checkerboard stimuli and object pictures during fMRI measurement. Results Results show that stimulation with pictures of everyday objects led to overall larger BOLD (blood oxygen level dependent) responses in visual cortex compared to that evoked by stimulation with flickering checkerboards. Patients showed this enhancement as early as in V1. When the PRL was directly stimulated with object pictures, the central representation area in early visual cortex was coactivated in the patients but not in the controls. In higher visual areas beyond retinotopic cortex, BOLD responses to stimulation of the PRL with object pictures were significantly enhanced in comparison to stimulation of the OppPRL area. Highly stable eccentric fixation with the PRL was associated with a higher BOLD signal in visual cortex in patients, and this effect was most pronounced in the conditions with object picture stimulation. Conclusions The observed results suggest that naturalistic images are more likely to trigger top-down processes that regulate activation in early visual cortex in patients with central vision loss

SNAIL1-mediated downregulation of FOXA proteins facilitates the inactivation of transcriptional enhancer elements at key epithelial genes in colorectal cancer cells

<div><p>Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of <i>SNAIL1</i> and <i>FOXA</i> genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the <i>CDH1</i>, <i>CDX2</i> and <i>EPHB3</i> genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing <i>in situ</i> impaired enhancer activity and completely abolished the active chromatin state of the <i>EPHB3</i> enhancer. Conversely, expression of FOXA factors in cells with inactive <i>CDX2</i> and <i>EPHB3</i> enhancers led to chromatin opening and <i>de novo</i> deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.</p></div

Acetylation of Human TCF4 (TCF7L2) Proteins Attenuates Inhibition by the HBP1 Repressor and Induces a Conformational Change in the TCF4::DNA Complex

<div><p>The members of the TCF/LEF family of DNA-binding proteins are components of diverse gene regulatory networks. As nuclear effectors of Wnt/β-catenin signaling they act as assembly platforms for multimeric transcription complexes that either repress or activate gene expression. Previously, it was shown that several aspects of TCF/LEF protein function are regulated by post-translational modification. The association of TCF/LEF family members with acetyltransferases and deacetylases prompted us to investigate whether vertebrate TCF/LEF proteins are subject to acetylation. Through co-expression with p300 and CBP and subsequent analyses using mass spectrometry and immunodetection with anti-acetyl-lysine antibodies we show that TCF4 can be acetylated at lysine K₁₅₀ by CBP. K₁₅₀ acetylation is restricted to TCF4E splice variants and requires the simultaneous presence of β-catenin and the unique TCF4E C-terminus. To examine the functional consequences of K₁₅₀ acetylation we substituted K₁₅₀ with amino acids representing the non-acetylated and acetylated states. Reporter gene assays based on Wnt/β-catenin-responsive promoter regions did not indicate a general role of K₁₅₀ acetylation in transactivation by TCF4E. However, in the presence of CBP, non-acetylatable TCF4E with a K₁₅₀R substitution was more susceptible to inhibition by the HBP-1 repressor protein compared to wild-type TCF4E. Acetylation of K₁₅₀ using a bacterial expression system or amino acid substitutions at K₁₅₀ alter the electrophoretic properties of TCF4E::DNA complexes. This result suggests that K₁₅₀ acetylation leads to a conformational change that may also represent the mechanism whereby acetylated TCF4E acquires resistance against HBP1. In summary, TCF4 not only recruits acetyltransferases but is also a substrate for these enzymes. The fact that acetylation affects only a subset of TCF4 splice variants and is mediated preferentially by CBP suggests that the conditional acetylation of TCF4E is a novel regulatory mechanism that diversifies the transcriptional output of Wnt/β-catenin signaling in response to changing intracellular signaling milieus.</p></div

Mutation of the FOX binding sites at the <i>EPHB3</i> enhancer leads to chromatin compaction at the enhancer and promoter and abolishes <i>EPHB3</i> expression.

<p>(A) ChIP analysis to monitor FOXA1 occupancy at the <i>EPHB3</i> locus in LS174T cell clones genome-edited with the CRISPR/Cas9 system. Data are given as percent input. Shown is the mean and SEM; n≥3. Statistical significance was calculated relative to the LS174T wild-type cell pool. (B) FAIRE analyses to test chromatin compaction at the <i>EPHB3</i> enhancer and promoter in LS174T cell clones genome-edited with the CRISPR/Cas9 system. Data are given as relative (rel.) FAIRE enrichment; n = 3. Statistical significance was calculated relative to the LS174T wild-type cell pool. (C) ChIP analyses to monitor presence of the histone modifications H3K27ac and H3K4me1 at the <i>EPHB3</i> locus in LS174T cell clones genome-edited with the CRISPR/Cas9 system. Data are given as percent input normalized (norm.) to H3 to compensate for variations in nucleosome density; n≥3. Statistical significance was calculated relative to the LS174T wild-type cell pool. (D) qRT-PCR (upper) and Western Blot (lower) analyses to assess EPHB3 expression in LS174T cell clones with mutations in transcription factor binding sites at the <i>EPHB3</i> enhancer. Data are shown as mean and SEM; n = 3; rel. expr.: relative expression. M_W = molecular weight in kDa. α-TUBULIN (α-TUB) immunodetection served as loading control.</p

<i>CDH1</i>, <i>CDX2</i> and <i>EPHB3</i> enhancer activity is dependent on FOXA binding.

<p>(A) (Top) Scheme of the ECR at +7.8 kb at the <i>CDH1</i> locus with the described tandem GRHL binding sites [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007109#pgen.1007109.ref042" target="_blank">42</a>] and two potential FOX binding motifs. (Bottom) EMSA to test for <i>in vitro</i> binding of FOXA1 and FOXA3 to the <i>CDH1</i> +7.8 kb enhancer. Asterisks: non-specific bands. mut: mutated, P: probe. (B) (Top) Scheme of the <i>CDX2</i> +10.0 kb enhancer with the described RFX1, FOXA and nuclear hormone receptor-II (NHR-II) transcription factor binding sites [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007109#pgen.1007109.ref047" target="_blank">47</a>]. (Bottom) EMSA to test for <i>in vitro</i> binding of FOXA1 and FOXA3 to the <i>CDX2</i> +10.0 kb enhancer. (C) (Top) Scheme of the <i>EPHB3</i> −2.3 kb enhancer with its known transcription factor binding sites. TBE: TCF/LEF-binding element. (Bottom) EMSA to test for <i>in vitro</i> binding of FOXA1 and FOXA3 to the <i>EPHB3</i> −2.3 kb enhancer. (D, E, F) Luciferase reporter assay in LS174T cells with constructs covering the <i>CDH1</i> (D), the <i>CDX2</i> (E), and the <i>EPHB3</i> (F) enhancer. Mutations of the respective FOX binding motifs are indicated by red crosses. Shown is the mean and SEM; n≥3. RLA: relative luciferase activity. Statistical significance was calculated relative to the wild-type luciferase reporter constructs.</p

LS174T cell clones with absent/low FOXA expression show a mesenchymal morphology and upregulation of mesenchymal marker proteins.

<p>(A) Phase contrast images of parental LS174T cells and cell clones subjected to CRISPR/Cas9-mediated genome editing of the FOXA1 locus. Scale bar: 100 μm. (B) Western Blot analyses to assess expression of FOXA1, FOXA2, FOXA3, E-CADHERIN (E-CAD), CLAUDIN3 (CLDN3), CADHERIN11 (CAD11), LEF1 and SNAIL2/SLUG in parental LS174T cells and cell clones subjected to CRISPR/Cas9-mediated genome editing of the FOXA1 locus. RNA polymerase II (RNAPII) and GSK3β immunodetections served as loading controls. Analytes which were detected on separate membranes are grouped together including their corresponding loading controls. M_W = molecular weight in kDa.</p