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Precise insertion of fluorescent tags by CRISPR-Cas9-mediated homologous recombination (HR) in mammalian genes is a powerful tool allowing to study gene function and protein gene products. Here, we present a protocol for efficient HR-mediated targeted insertion of fluorescent markers in the genome of hard-to-transfect erythroid cell lines MEL (mouse erythroleukemic) and MEDEP (mouse ES cell-derived erythroid progenitor line). We describe steps for plasmid construction, electroporation, amplification, and verification of genome editing. We then detail procedures for isolating positive clones and validating knockin clones. For complete details on the use and execution of this protocol, please refer to Deleuze et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in (KI), remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines. We demonstrate robust fluorescent protein KI efficiency in low expressed transcription factor (TF) genes (e.g., Myb or Zeb1 ). We further show the ability to target two independent loci in individual cells, exemplified by MYB-GFP and NuMA-Cherry double KI, allowing multicolor labeling of regulatory factors at physiological endogenous levels. Our KI tagging approach allowed us to perform genome-wide TF analysis at increased signal-to-noise ratios, and highlighted previously unidentified MYB target genes and pathways. Overall, we establish a versatile CRISPR-Cas9-based platform, offering attractive opportunities for the dissection of the erythroid differentiation process., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Genome-wide analysis of transcription factors and epigenomic features is instrumental to shed light on DNA-templated regulatory processes such as transcription, cellular differentiation or to monitor cellular responses to environmental cues. Two decades of technological developments have led to a rich set of approaches progressively pushing the limits of epigenetic profiling towards single cells. More recently, disruptive technologies using innovative biochemistry came into play. Assays such as CUT&RUN, CUT&Tag and variations thereof show considerable potential to survey multiple TFs or histone modifications in parallel from a single experiment and in native conditions. These are in the path to become the dominant assays for genome-wide analysis of TFs and chromatin modifications in bulk, single-cell, and spatial genomic applications. The principles together with pros and cons are discussed., (Copyright © 2023. Published by Elsevier Inc.)

Studies of the regulatory networks and signals controlling erythropoiesis have brought important insights in several research fields of biology and have been a rich source of discoveries with far-reaching implications beyond erythroid cells biology. The aim of this review is to highlight key recent discoveries and show how studies of erythroid cells bring forward novel concepts and refine current models related to genome and 3D chromatin organization, signaling and disease, with broad interest in life sciences.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors., (© 2021 by The American Society of Hematology.)

GATA1 is an essential transcriptional regulator of myeloid hematopoietic differentiation towards red blood cells. During erythroid differentiation, GATA1 forms different complexes with other transcription factors such as LDB1, TAL1, E2A and LMO2 ("the LDB1 complex") or with FOG1. The functions of GATA1 complexes have been studied extensively in definitive erythroid differentiation; however, the temporal and spatial formation of these complexes during erythroid development is unknown. We applied proximity ligation assay (PLA) to detect, localize and quantify individual interactions during embryonic stem cell differentiation and in mouse fetal liver (FL) tissue. We show that GATA1/LDB1 interactions appear before the proerythroblast stage and increase in a subset of the CD71 + /TER119 - cells to activate the terminal erythroid differentiation program in 12.5 day FL. Using Ldb1 and Gata1 knockdown FL cells, we studied the functional contribution of the GATA1/LDB1 complex during differentiation. This shows that the active LDB1 complex appears quite late at the proerythroblast stage of differentiation and confirms the power of PLA in studying the dynamic interaction of proteins in cell differentiation at the single cell level. We provide dynamic insight into the temporal and spatial formation of the GATA1 and LDB1 transcription factor complexes during hematopoietic development and differentiation., (Copyright© 2020 Ferrata Storti Foundation.)

Purpose of Review: β-hemoglobinopathies, such as β-Thalassemias (β-Thal) and sickle cell disease (SCD) are among the most common inherited genetic disorders in humans worldwide. These disorders are characterized by a quantitative (β-Thal) or qualitative (SCD) defects in adult hemoglobin production, leading to anemia, ineffective erythropoiesis and severe secondary complications. Reactivation of the fetal globin genes (γ-globin), making-up fetal hemoglobin (HbF), which are normally silenced in adults, represents a major strategy to ameliorate anemia and disease severity., Recent Findings: Following the identification of the first 'switching factors' for the reactivation of fetal globin gene expression more than 10 years ago, a multitude of novel leads have recently been uncovered., Summary: Recent findings provided invaluable functional insights into the genetic and molecular networks controlling globin genes expression, revealing that complex repression systems evolved in erythroid cells to maintain HbF silencing in adults. This review summarizes these unique and exciting discoveries of the regulatory factors controlling the globin switch. New insights and novel leads for therapeutic strategies based on the pharmacological induction of HbF are discussed. This represents a major breakthrough for rational drug design in the treatment of β-Thal and SCD.

The intravitreous injection of therapeutic proteins that neutralize vascular endothelial growth factor (VEGF) family members is efficient to reduce macular edema associated with wet age-related macular degeneration (AMD), retinal vein occlusion (RVO) and diabetic retinopathy (DR). It has revolutionized the visual prognosis of patients with macular edema. The antiedematous effect is dependent on an intravitreous dose of drug, which varies between patients and requires frequent and repeated injections to maintain its effects. At the time when optimizing the duration of anti-VEGF effects is a major challenge, understanding how anti-VEGF reduces macular edema is crucial. We discuss herein how anti-VEGF exerts antiedematous effects and raise the hypothesis that mechanisms, unrelated to VEGF neutralization, might have been underestimated., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Choroidal neovascularization (CNV) is a major cause of visual impairment in patients suffering from wet age-related macular degeneration (AMD), particularly when refractory to intraocular anti-VEGF injections. Here we report that treatment with the oral mineralocorticoid receptor (MR) antagonist spironolactone reduces signs of CNV in patients refractory to anti-VEGF treatment. In animal models of wet AMD, pharmacological inhibition of the MR pathway or endothelial-specific deletion of MR inhibits CNV through VEGF-independent mechanisms, in part through upregulation of the extracellular matrix protein decorin. Intravitreal injections of spironolactone-loaded microspheres and systemic delivery lead to similar reductions in CNV. Together, our work suggests MR inhibition as a novel therapeutic option for wet AMD patients unresponsive to anti-VEGF drugs.

Gene expression in mammals is precisely regulated by the combination of promoters and gene-distal regulatory regions, known as enhancers. Several studies have suggested that some promoters might have enhancer functions. However, the extent of this type of promoters and whether they actually function to regulate the expression of distal genes have remained elusive. Here, by exploiting a high-throughput enhancer reporter assay, we unravel a set of mammalian promoters displaying enhancer activity. These promoters have distinct genomic and epigenomic features and frequently interact with other gene promoters. Extensive CRISPR-Cas9 genomic manipulation demonstrated the involvement of these promoters in the cis regulation of expression of distal genes in their natural loci. Our results have important implications for the understanding of complex gene regulation in normal development and disease.

Cellular differentiation requires precisely regulated tissue-specific and developmental stage-specific gene expression patterns. Numerous studies have highlighted the predictive power of enhancers on lineage-specific gene expression programs and have started to unravel their mechanisms of action. We review here the dynamics of the enhancer landscape during hematopoietic differentiation and how enhancers function in the context of the 3D organization of the genome. We further discuss the involvement of aberrant enhancer activity in human diseases and emerging strategies aiming at controlling enhancer activity and chromosome topology for therapeutic purposes., (© 2016 Federation of European Biochemical Societies.)

Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress., (Copyright © 2015 Elsevier Inc. All rights reserved.)

How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2-IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation.

We have identified and characterized a spontaneous Brown Norway from Janvier rat strain (BN-J) presenting a progressive retinal degeneration associated with early retinal telangiectasia, neuronal alterations, and loss of retinal Müller glial cells resembling human macular telangiectasia type 2 (MacTel 2), which is a retinal disease of unknown cause. Genetic analyses showed that the BN-J phenotype results from an autosomal recessive indel novel mutation in the Crb1 gene, causing dislocalization of the protein from the retinal Müller glia (RMG)/photoreceptor cell junction. The transcriptomic analyses of primary RMG cultures allowed identification of the dysregulated pathways in BN-J rats compared with wild-type BN rats. Among those pathways, TGF-β and Kit Receptor Signaling, MAPK Cascade, Growth Factors and Inflammatory Pathways, G-Protein Signaling Pathways, Regulation of Actin Cytoskeleton, and Cardiovascular Signaling were found. Potential molecular targets linking RMG/photoreceptor interaction with the development of retinal telangiectasia are identified. This model can help us to better understand the physiopathologic mechanisms of MacTel 2 and other retinal diseases associated with telangiectasia., (Copyright © 2015 the authors 0270-6474/15/356093-14$15.00/0.)

An intimate relationship exists between nuclear architecture and gene activity. Unraveling the fine-scale three-dimensional structure of the genome and its impact on gene regulation is a major goal of current epigenetic research, one with direct implications for understanding the molecular mechanisms underlying human phenotypic variation and disease susceptibility. In this context, the novel revolutionary genome editing technologies and emerging new ways to manipulate genome folding offer new promises for the treatment of human disorders., (© 2015 by The American Society of Hematology.)

As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

Gene correction at the site of the mutation in the chromosome is the absolute way to really cure a genetic disease. The oligonucleotide (ODN)-mediated gene repair technology uses an ODN perfectly complementary to the genomic sequence except for a mismatch at the base that is mutated. The endogenous repair machinery of the targeted cell then mediates substitution of the desired base in the gene, resulting in a completely normal sequence. Theoretically, it avoids potential gene silencing or random integration associated with common viral gene augmentation approaches and allows an intact regulation of expression of the therapeutic protein. The eye is a particularly attractive target for gene repair because of its unique features (small organ, easily accessible, low diffusion into systemic circulation). Moreover therapeutic effects on visual impairment could be obtained with modest levels of repair. This chapter describes in details the optimized method to target active ODNs to the nuclei of photoreceptors in neonatal mouse using (1) an electric current application at the eye surface (saline transpalpebral iontophoresis), (2) combined with an intravitreous injection of ODNs, as well as the experimental methods for (3) the dissection of adult neural retinas, (4) their immuno-labelling, and (5) flat-mounting for direct observation of photoreceptor survival, a relevant criteria of treatment outcomes for retinal degeneration.

The first site exhibiting hematopoietic activity in mammalian development is the yolk-sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in blast colony-forming cells to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk-sac hematopoiesis and vascular development in Ldb1(-/-) mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome-wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct-target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signaling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells.

RUNX1 is known to be an essential transcription factor for generating hematopoietic stem cells (HSC), but much less is known about its role in the downstream process of hematopoietic differentiation. RUNX1 has been shown to be part of a large transcription factor complex, together with LDB1, GATA1, TAL1, and ETO2 (N. Meier et al., Development 133:4913-4923, 2006) in erythroid cells. We used a tagging strategy to show that RUNX1 interacts with two novel protein partners, LSD1 and MYEF2, in erythroid cells. MYEF2 is bound in undifferentiated cells and is lost upon differentiation, whereas LSD1 is bound in differentiated cells. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and microarray expression analysis were used to show that RUNX1 binds approximately 9,000 target sites in erythroid cells and is primarily active in the undifferentiated state. Functional analysis shows that a subset of the target genes is suppressed by RUNX1 via the newly identified partner MYEF2. Knockdown of Myef2 expression in developing zebrafish results in a reduced number of HSC.

The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping.

Gfi-1B is a transcriptional repressor essential for the regulation of erythropoiesis and megakaryopoiesis. Here we identify Gfi-1B p32, a Gfi-1B isoform, as essential for erythroid differentiation. Gfi-1B p32 is generated by alternative splicing and lacks the two first zinc finger domains of the protein. Selective knock down of Gfi-1B p32 compromises erythroid differentiation, whereas its ectopic expression induces erythropoiesis in the absence of erythropoietin. Gfi-1B p32 isoform binds to Gfi-1B target gene promoters and associates with the LSD1-CoREST repressor complex more efficiently than the major Gfi-1B p37 isoform. Furthermore, we show that Gfi-1B includes a KSKK motif in its SNAG domain, which recruits the repressor complex only when dimethylated on lysine 8. Mutation of lysine 8 prevents Gfi-1B p32-induced erythroid development. Our results thus highlight a key role for the alternatively spliced Gfi-1B p32 isoform in erythroid development.

Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (e.g., RPS19) or large (e.g., RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

Time-lapse confocal microscopy of mouse embryo slices was developed to access and image the living aorta. In this paper, we explain how to label all hematopoietic and endothelial cells inside the intact mouse aorta with fluorescent directly labeled antibodies. Then we describe the technique to cut nonfixed labeled embryos into thick slices that are further imaged by time-lapse confocal imaging. This approach allows direct observation of the dynamic cell behavior in the living aorta, which was previously inaccessible because of its location deep inside the opaque mouse embryo. In particular, this approach is sensitive enough to allow the experimenter to witness the transition from endothelial cells into hematopoietic stem/progenitor cells in the aorta, the first site of hematopoietic stem cell generation during development. The protocol can be applied to observe other embryonic sites throughout mouse development. A complete experiment requires ∼2 d of practical work.

Transcription factors (TFs) play a central role in the development of multicellular organisms. The sequential actions of critical TFs direct cells to adopt defined differentiation pathways leading to functional, fully differentiated tissues. Here, we describe a generic experimental pipeline that integrates biochemistry, genetics and next generation sequencing with bioinformatics to characterize TF complexes composition, function and target genes at a genome-wide scale. We show an application of this experimental pipeline which aims to unravel the molecular events taking place during hematopoietic cell differentiation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Haematopoietic stem cells (HSCs), responsible for blood production in the adult mouse, are first detected in the dorsal aorta starting at embryonic day 10.5 (E10.5). Immunohistological analysis of fixed embryo sections has revealed the presence of haematopoietic cell clusters attached to the aortic endothelium where HSCs might localize. The origin of HSCs has long been controversial and several candidates of the direct HSC precursors have been proposed (for review see ref. 7), including a specialized endothelial cell population with a haemogenic potential. Such cells have been described both in vitro in the embryonic stem cell (ESC) culture system and retrospectively in vivo by endothelial lineage tracing and conditional deletion experiments. Whether the transition from haemogenic endothelium to HSC actually occurs in the mouse embryonic aorta is still unclear and requires direct and real-time in vivo observation. To address this issue we used time-lapse confocal imaging and a new dissection procedure to visualize the deeply located aorta. Here we show the dynamic de novo emergence of phenotypically defined HSCs (Sca1(+), c-kit(+), CD41(+)) directly from ventral aortic haemogenic endothelial cells.

One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex-in particular, the binding of the negative regulators Eto2 and Mtgr1-are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences.

Purpose: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo., Methods: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site., Results: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33., Conclusions: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

Along with viral vectors, non-viral strategies have been developed in order to efficiently deliver nucleic acids to ocular cells. During the last decade, we have observed that the outcome of these non-viral delivery systems depends on the genetic material used, the targeted tissue or cells, the expected effect duration, and the routes of administration. Assessment of efficiency has been evaluated in normal eyes or in animal models of ocular diseases. The chemical and physical methods that have been adapted for the delivery of nucleic acids to ocular tissues are highlighted and discussed in this review. Also, the results obtained with different non-viral strategies from their initial conception to their present development are summarized. At the present, selective targeting of ocular tissues and cells can be achieved using the most yielding route of administration to the eye in combination with an appropriate drug delivery technique.

Purpose: To study the combination of oligodeoxynucleotides (ODNs) intravitreous injection and saline transpalpebral iontophoresis on the delivery of ODNs to photoreceptors in the newborn rd1/rd1 mice., Methods: Cathodal or anodal transpalpebral iontophoresis (1.43 mA/cm(2) for 5 min) was applied to eyes of postnatal day 7 (PN7) rd1/rd1 mice immediately before the intravitreous injection of ODNs. The effect of cathodal iontophoresis after ODNs injection was also evaluated. The influence of current intensity (0.5, 1.5, and 2.5 mA) was assayed with cathodal iontophoresis performed prior to ODNs injection. The duration of current-induced facilitation of ODNs delivery to photoreceptors was evaluated for 6 h following iontophoresis. One group of control eyes received cathodal iontophoresis prior to the intravitreous injection of phosphate buffered saline (PBS) or hexachlorofluorescein (Hex). The second control group received ODN or Hex intravitreous injection without iontophoresis. The penetration of fluorescent ODNs in the outer nuclear layer (ONL) was quantified by image analysis of the ONL fluorescence intensity on cryosection microphotographs. Integrity of ODN was assessed using acrylamide gel migration after its extraction from the retina of treated mice. The integrity of retinal structure, 1 and 24 h after iontophoresis, was analyzed using light and electron microscopy., Results: Transpalpebral anodal or cathodal saline iontophoresis enhanced the penetration of ODNs in all retinal layers. Cathodal iontophoresis was more efficient than anodal iontophoresis in enhancing the tissue penetration of the injected ODN. Photoreceptor delivery of ODN was significantly higher when cathodal saline transpalpebral iontophoresis was applied prior than after the injection. The extent of enhanced tissue penetration decreased in parallel to the increased interval between iontophoresis application and the intravitreous injection. Current of 1.5 mA was safe and optimal for the delivery of ODNs to the ONL. One hour after iontophoresis followed by injection, ODN extracted from the retina of treated eyes remained intact. Histology and electron microscopy observations demonstrated that iontophoresis using the optimal parameters did not induce any permanent tissue alterations or structure damage., Conclusions: Saline transpalpebral iontophoresis facilitates the penetration of injected ODNs in photoreceptors for at least 3 h. This method may be considered for photoreceptor targeted gene therapy.

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.

Purpose: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres., Methods: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections., Results: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres., Conclusions: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.