Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain.

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6⁺/⁻ lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6⁻/⁻ lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues

RNA-binding proteins in eye development and disease: implication of conserved RNA granule components

The molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1, and Bmp4 are commonly required for their development. In contrast, our understanding of posttranscriptional regulation in eye development and disease, particularly regarding the function of RNA-binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila as well as several vertebrate models such as fish, frog, chicken, and mouse. Interestingly, of the 42 RBPs that have been investigated for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as processing bodies, stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate posttranscriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2, and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly, and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving posttranscriptional regulatory networks in eye development and disease. WIREs RNA 2016, 7:527-557. doi: 10.1002/wrna.1355 For further resources related to this article, please visit the WIREs website

Role of exosomes in malignant glioma: microRNAs and proteins in pathogenesis and diagnosis

Malignant gliomas are the most common and deadly type of central nervous system tumors. Despite some advances in treatment, the mean survival time remains only about 1.25 years. Even after surgery, radiotherapy and chemotherapy, gliomas still have a poor prognosis. Exosomes are the most common type of extracellular vesicles with a size range of 30 to 100 nm, and can act as carriers of proteins, RNAs, and other bioactive molecules. Exosomes play a key role in tumorigenesis and resistance to chemotherapy or radiation. Recent evidence has shown that exosomal microRNAs (miRNAs) can be detected in the extracellular microenvironment, and can also be transferred from cell to cell via exosome secretion and uptake. Therefore, many recent studies have focused on exosomal miRNAs as important cellular regulators in various physiological and pathological conditions. A variety of exosomal miRNAs have been implicated in the initiation and progression of gliomas, by activating and/or inhibiting different signaling pathways. Exosomal miRNAs could be used as therapeutic agents to modulate different biological processes in gliomas. Exosomal miRNAs derived from mesenchymal stem cells could also be used for glioma treatment. The present review summarizes the exosomal miRNAs that have been implicated in the pathogenesis, diagnosis and treatment of gliomas. Moreover, exosomal proteins could also be involved in glioma pathogenesis. Exosomal miRNAs and proteins could also serve as non-invasive biomarkers for prognosis and disease monitoring. Video Abstract

Insights into the biochemical and biophysical mechanisms mediating the longevity of the transparent optics of the eye lens

In the human eye, a transparent cornea and lens combine to form the “refracton” to focus images on the retina. This requires the refracton to have a high refractive index “n,” mediated largely by extracellular collagen fibrils in the corneal stroma and the highly concentrated crystallin proteins in the cytoplasm of the lens fiber cells. Transparency is a result of short-range order in the spatial arrangement of corneal collagen fibrils and lens crystallins, generated in part by post-translational modifications (PTMs). However, while corneal collagen is remodeled continuously and replaced, lens crystallins are very long-lived and are not replaced and so accumulate PTMs over a lifetime. Eventually, a tipping point is reached when protein aggregation results in increased light scatter, inevitably leading to the iconic protein condensation–based disease, age-related cataract (ARC). Cataracts account for 50% of vision impairment worldwide, affecting far more people than other well-known protein aggregation–based diseases. However, because accumulation of crystallin PTMs begins before birth and long before ARC presents, we postulate that the lens protein PTMs contribute to a “cataractogenic load” that not only increases with age but also has protective effects on optical function by stabilizing lens crystallins until a tipping point is reached. In this review, we highlight decades of experimental findings that support the potential for PTMs to be protective during normal development. We hypothesize that ARC is preventable by protecting the biochemical and biophysical properties of lens proteins needed to maintain transparency, refraction, and optical function

Biochemical and Functional characterization of the LEDGF/p75-MeCP2 Interaction in Tumor Cells

The lens epithelial derived growth factor p75 (LEDGF/p75) is a novel pro-survival and stress-inducible transcription co-activator that protects mammalian cells from various environmental stresses such as oxidative stress, heat shock, and serum starvation. This emerging cancer-related protein is highly expressed in prostate tumors and other tumor types and promotes resistance to chemotherapy in cancer cells. LEDGF/p75 is also involved in acquired immunodeficiency syndrome (AIDS) since it interacts with HIV-1 integrase to facilitate the integration and replication of the HIV virus in human cells. In addition, LEDGF/p75 has been shown to interact with MLL (mixed lineage leukemia)/menin transcription complex in leukemia cells to facilitate the transcription of cancer-associated genes and leukemic transformation. In order to understand the mechanisms by which LEDGF/p75 contributes to cancer development, we explored its interactions with other transcription factors and the influence of these interactions on its transcriptional activity. Using complementary molecular, biochemical, and cellular approaches we discovered that the amino-terminal region of LEDGF/p75 interacts with the transcription regulator and methylation associated protein MeCP2 in prostate cancer cells and other cancer cell types. We observed that both proteins regulate the expression of the heat shock protein 27 gene by transactivating its promoter region. We propose that the interaction between LEDGF/p75 and MeCP2 modulates the expression of cancer-associated genes in response to environmental stressors. These findings provide a plausible mechanism that can be targeted for the treatment of advanced prostate cancer, which is the second leading cause of cancer deaths in the United States, with a disproportional burden among African American men

Epigenetic Programming of Physiological Functions by a Prenatal Stressor and Genetic Parameters of Temperament in Cattle

This project consisted of two main objectives. Objective 1 assessed the influences of prenatal stress on 1) postnatal physiological functions and 2) the postnatal presence and prevalence of epigenetic differences, specifically degree of DNA methylation, in immune cells of calves. Objective 2 assessed the genetic parameters of temperament across an age continuum in cattle. Calves studied in Objective 1 were progeny from Brahman cows that were either transported at 60, 80, 100, 120, and 140 ± 5 d of gestation (the prenatally stressed group, PNS) or were designated as the nontransported Control group. After weaning, response to an endotoxin challenge was assessed in 16 PNS and 16 Control bull calves. In response to LPS, PNS bull calves exhibited increased rectal temperatures, IFN-γ, and TNF-α, as well as decreased serum IL-6. Additionally, a subset of bull calves (n=7 PNS; n= 7 Control) was selected from the total population for evaluation of genome-wide DNA methylation in white blood cells. There were 16,128 CpG sites, 226 CHG sites, and 391 CHH sites differentially methylated in PNS compared to Control calves. An enrichment analysis was used to associate differentially methylated sites in PNS calves with predicted alterations to biological pathways. Enrichment analysis revealed alterations to biological pathways related to functions such as immune function, HPA axis activity, and neurotransmitter signaling. Objective 2 sought to further understand the genetic components of temperament. Random regression procedures estimated genetic parameters of temperament across an age continuum in a population of commercial beef cattle. As the cattle matured over time there was an increased influence of permanent environmental effects and a decreased influence of additive genetic effects based on random regression analyses

Analysis and characterisation of the mouse Hic2 gene

5.1 Analysis and characterization of the mouse Hic2 gene Like Hic1 and γFBP (chicken), Hic2 cDNA coded five Krüppel-type C2H2 zinc finger domain. Through the sequencing and comparation with different protein sequences from the homolog proteins from different species (HIC1, Hic1, HIC2, γFBP, and Hypothetical protein), Hic2 protein shares more than 80% homology with HIC1 through the BTB/POZ and zinc finger domains, and both proteins have identical GLDLSKK/R motifs. A new part of Hic2 gene coding, exon two, and new exon one deduced full-length Hic2 protein contains 601 amino acids. Hic2 gene has two Exons (240 and 1842 bp), with one Intron (2182 bp). The location of the gene is mouse chromosome 16 (B1, UniGene Cluster Mm.103787 Mus musculus). The human gene HIC2 maps to chromosome 22q11.2, and is a homolog of the HIC1 candidate tumor suppressor gene located at 17p 13.3. (Deltour et al. 2001). Upstream from the TATA box MatInspector predicted different transcription binding sites. Between them Wilms Tumor Suppressor and p53 are most interesting transcription sites for the Hic2 gene. That’s why the Hic2 promoter activity was checked. A 1.2 kb promoter fragment of Hic2 has been characterized in a gene reporter assay system. The peak activity of the Hic2 promoter is associated with the total fragment, the next higher activity is in the fragment which included Wilms Tumor Suppressor and p53 transcription sites. The expression of the mouse Hic2 was investigated by in situ hybridisations (ISHs) of whole mount embryos and paraffin sections. Hic2 expression was detected in restricted territories of the brain, sinus centralis, olfactory bulb, canallis centralis medullae spinalis, embryonic ectoderm (neuroepithelium of neural tube), and small intestine. Because of its expression in the central nervous system, and mapping position of its human homolog HIC2, Hic2 could be involved in some syndromes. Patients with a 22q11 deletion have disrupted brain development which may involve abnormal neural crest cell migration, (Van Amelsvoort et al., 2001). It is now recognized that the 22q11.2 deletion syndrome encompasses the phenotypes previously described as DiGeorge syndrome (DGS) and velocardiofacial syndrome (Shprintzen syndrome),(Thomas and Graham 1997).Die cDNA des Hic2-Gens kodiert wie Hic1 und γFBP (Huhn) ebenfalls fünf C2H2 Zink-Finger des Krüppel-Typs. Im Vergleich der ermittelten Hic2-Sequenz mit verschiedenen Proteinsequenzen homologer Proteine aus verschiedenen Spezies (HIC1, Hic1, HIC2, γFBP, sowie ein hypothetisches Protein) zeigt Hic2 über 80% Homologie zu HIC1 im Bereich der BTB/POZ- und Zink-Finger-Domänen. Darüber hinaus besitzen beide Proteine identische GLDLSKK/R-Motive. Innerhalb dieser Arbeit wurde ein neuer Bereich des ersten und zweiten kodierenden Exons von Hic2 charakterisiert. Die kodierende Sequenz wurde vollständig bestimmt. Das vollständig abgeleitete Hic2-Protein besteht aus 601 Aminosäuren. Das Hic2-Gen enthält zwei Exons (240 bp und 1842 bp) und ein Intron von 2182 bp. Hic2 ist auf dem Maus-Chromosom 16 lokalisiert (B1, UniGene Cluster Mn. 103787 Mus musculus). Das menschliche HIC2-Gen liegt auf dem Chromosom 22q11.2. Es ist zu HIC1, einem auf 17p 13.3 lokalisierten Tumor-Suppressor Kandidaten-Gen, homolog (Deltour et al. 2001). Stromaufwärts der TATA-Box wurden durch MatInspector verschiedene Tranksriptions- Bindestellen identifiziert. Hierbei stellen der Wilms Tumor-Suppressor sowie p53 die interessantesten Transkriptionsstellen des Hic2-Gens dar. Aus diesem Grund wurde die Hic2- Promotoraktivität überprüft. Ein 1.2 kb großes Promotorfragment von Hic2 wurde in einem Gen-Reporter Nachweissystem charakterisiert. Die größte Promotoraktivität zeigte sich mit dem vollständigen, undeletierten Fragment, gefolgt von der Aktivität des Fragments zwei, welches die Wilms Tumor-Suppressor- und p53-Transkriptionsstellen enthält. Dies deutete auf eine mögliche funktionelle Bedeutung dieser Bereiche für die Hic2-Expression hin. Die Expression des Hic2-Gens der Maus wurde mit Hilfe von in situ Hybridisierungen (ISHs) auf ganzen Embryonen und Paraffin-Schnitten untersucht. Eine Hic2-Expression wurde in abgegrenzten Regionen des Gehirns, sinus centralis, Riechkolbens, canallis centralis medullae spinalis, embryonalen Ektoderms (Neuroepithelium des Neuralrohrs), und des Dünndarms detektiert. Aufgrund seiner Expression im zentralen Nervensystem sowie der durch Kartierung bestimmten Lokalisation des homologen menschlichen HIC2 könnte Hic2 an einigen durch Deletionen verursachte Syndrome beteiligt sein. Einige Befunde lieferten 94 Beweise, dass Personen mit einer 22q11-Deletion unter einer unvollständigen Gehirnentwicklung leiden, die eine abnormale Wanderung von Zellen der Neuralleiste beinhalten könnte (Van Amelsvoort et al., 2001). Mittlerweile ist bekannt, dass das 22q11.2 Deletionssyndrom mit den früher beschriebenen Phänotypen des DiGeorge-Syndroms (DGS) sowie des velokardiofaziales Syndroms (Shprintzen-Syndrom) in Verbindung steht (Thomas and Graham 1997)