Publications by authors named "Ales Cvekl"

81 Publications

Crystallin gene expression: Insights from studies of transcriptional bursting.

Exp Eye Res 2021 Apr 21;207:108564. Epub 2021 Apr 21.

Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, 10461, USA; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.

Cellular differentiation is marked by temporally and spatially regulated gene expression. The ocular lens is one of the most powerful mammalian model system since it is composed from only two cell subtypes, called lens epithelial and fiber cells. Lens epithelial cells differentiate into fiber cells through a series of spatially and temporally orchestrated processes, including massive production of crystallins, cellular elongation and the coordinated degradation of nuclei and other organelles. Studies of transcriptional and posttranscriptional gene regulatory mechanisms in lens provide a wide range of opportunities to understand global molecular mechanisms of gene expression as steady-state levels of crystallin mRNAs reach very high levels comparable to globin genes in erythrocytes. Importantly, dysregulation of crystallin gene expression results in lens structural abnormalities and cataracts. The mRNA life cycle is comprised of multiple stages, including transcription, splicing, nuclear export into cytoplasm, stabilization, localization, translation and ultimate decay. In recent years, development of modern mRNA detection methods with single molecule and single cell resolution enabled transformative studies to visualize the mRNA life cycle to generate novel insights into the sequential regulatory mechanisms of gene expression during embryogenesis. This review is focused on recent major advancements in studies of transcriptional bursting in differentiating lens fiber cells, analysis of nascent mRNA expression from bi-directional promoters, transient nuclear accumulation of specific mRNAs, condensation of chromatin prior lens fiber cell denucleation, and outlines future studies to probe the interactions of individual mRNAs with specific RNA-binding proteins (RBPs) in the cytoplasm and regulation of translation and mRNA decay.
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http://dx.doi.org/10.1016/j.exer.2021.108564DOI Listing
April 2021

Morphometric analysis of the lens in human aniridia and mouse Small eye.

Exp Eye Res 2021 02 26;203:108371. Epub 2020 Nov 26.

Department of Biological Sciences, University of Delaware, Newark, DE, USA. Electronic address:

Congenital aniridia is caused by heterozygous mutations in the PAX6 gene. In this disease, congenital iris and foveal hypoplasia is associated with juvenile onset cataract, glaucoma, and corneal keratopathy. In rodents, Pax6 mutations result in a congenital reduction in ocular size that is not typically described in human aniridia. Here, the ocular morphometry of aniridia patients is compared with the lens phenotype of Pax6 mice to reveal whether there are species differences in Pax6 regulation of lens development and homeostasis. Ultrasound biometry (UBM) revealed that eleven percent of aniridia patients exhibited mild microphthalmia while the anterior chamber depth of aniridic eyes was significantly reduced from 6 months of age onward. Although aniridic lens thickness was normal from birth, it was significantly decreased in aniridic lenses older than 30. Notably, 86% of aniridic lenses exhibited cataractous changes in this cohort. In addition, a significant proportion of aniridia patients develop lens subluxation as they age associated with reduced lens diameter as measured by anterior segment optical coherence tomography (AS-OCT). Analysis of young adult Pax6 mouse lenses by micro-computed tomography (microCT), bright field and dark field imaging revealed that they are reduced in size but did not exhibit overt cataracts at this age. Overall, this study reveals that congenital microphthalmia as assessed by axial length, or microphakia, as assessed by lens thickness, are not typical in human aniridia, although these are primary manifestations of Pax6 mutations in mice, suggesting that PAX6 regulates some aspects of lens development differently between these species.
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http://dx.doi.org/10.1016/j.exer.2020.108371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867623PMC
February 2021

Transcriptomic analysis and novel insights into lens fibre cell differentiation regulated by Gata3.

Open Biol 2019 12 18;9(12):190220. Epub 2019 Dec 18.

Departments of Ophthalmology and Visual Sciences and Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Gata3 is a DNA-binding transcription factor involved in cellular differentiation in a variety of tissues including inner ear, hair follicle, kidney, mammary gland and T-cells. In a previous study in 2009, Maeda . (, 2280-2291; doi:10.1002/dvdy.22035) found that Gata3 mutants could be rescued from midgestational lethality by the expression of a Gata3 transgene in sympathoadrenal neuroendocrine cells. The rescued embryos clearly showed multiple defects in lens fibre cell differentiation. To determine whether these defects were truly due to the loss of Gata3 expression in the lens, we generated a lens-specific Gata3 loss-of-function model. Analogous to the previous findings, our Gata3 null embryos showed abnormal regulation of cell cycle exit during lens fibre cell differentiation, marked by reduction in the expression of the cyclin-dependent kinase inhibitors Cdkn1b/p27 and Cdkn1c/p57, and the retention of nuclei accompanied by downregulation of Dnase IIβ. Comparisons of transcriptomes between control and mutated lenses by RNA-Seq revealed dysregulation of lens-specific crystallin genes and intermediate filament protein Bfsp2. Both Cdkn1b/p27 and Cdkn1c/p57 loci are occupied by Gata3, as well as Prox1 and c-Jun, in lens chromatin. Collectively, our studies suggest that Gata3 regulates lens differentiation through the direct regulation of the Cdkn1b/p27and Cdkn1c/p57 expression, and the direct/or indirect transcriptional control of Bfsp2 and Dnase IIβ.
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http://dx.doi.org/10.1098/rsob.190220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936257PMC
December 2019

A distal enhancer that directs Otx2 expression in the retinal pigment epithelium and neuroretina.

Dev Dyn 2020 02 19;249(2):209-221. Epub 2019 Nov 19.

Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, New York.

Background: Homeodomain transcription factor Otx2 is essential for embryonic development of multiple head tissues, including retinal pigment epithelium (RPE) and neuroretina. Temporospatial regulation of Otx2 expression is critical for its functions. Molecular dissection of the cis-acting enhancers will help elucidate how Otx2 expression is regulated.

Results: We comprehensively characterized distal enhancer hs1150 that was previously identified in a high throughput study. We established multiple transgenic mouse lines in which human hs1150, corresponding mouse hs1150, and two highly conserved sub-fragments in the mouse hs1150 were individually fused to a minimal hsp68 promoter to drive reporter expression. We found that hs1150 enhancer directed reporter expression in the RPE, neuroretina, and brain in a developmentally regulated manner. Human hs1150-directed reporter expression largely recapitulated Otx2 expression in the RPE, in the early neuroretina, and to a lesser degree in the early brain. Mouse hs1150, although shorter than human hs1150, exhibited similar enhancer activity, indicating functional conservation of hs1150 enhancer across species. Both of the highly conserved subfragments in mouse hs1150 enhancer directed reporter expression in the early neuroretina, indicating that the hs1150 enhancer has two functional components.

Conclusions: Our findings provide insight into the molecular mechanisms underlying the regulation of Otx2 retinal expression.
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http://dx.doi.org/10.1002/dvdy.127DOI Listing
February 2020

Rinf Regulates Pluripotency Network Genes and Tet Enzymes in Embryonic Stem Cells.

Cell Rep 2019 08;28(8):1993-2003.e5

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA; Department of Genetics, Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA. Electronic address:

The Retinoid inducible nuclear factor (Rinf), also known as CXXC5, is a nuclear protein, but its functions in the context of the chromatin are poorly defined. We find that in mouse embryonic stem cells (mESCs), Rinf binds to the chromatin and is enriched at promoters and enhancers of Tet1, Tet2, and pluripotency genes. The Rinf-bound regions show significant overlapping occupancy of pluripotency factors Nanog, Oct4, and Sox2, as well as Tet1 and Tet2. We found that Rinf forms a complex with Nanog, Oct4, Tet1, and Tet2 and facilitates their proper recruitment to regulatory regions of pluripotency and Tet genes in ESCs to positively regulate their transcription. Rinf deficiency in ESCs reduces expression of Rinf target genes, including several pluripotency factors and Tet enzymes, and causes aberrant differentiation. Together, our findings establish Rinf as a regulator of the pluripotency network genes and Tet enzymes in ESCs.
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http://dx.doi.org/10.1016/j.celrep.2019.07.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716522PMC
August 2019

Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids.

Proc Natl Acad Sci U S A 2019 05 9;116(22):10824-10833. Epub 2019 May 9.

Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY 10461;

Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were -passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium-retinal organoid-that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.
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http://dx.doi.org/10.1073/pnas.1901572116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561190PMC
May 2019

Profiling of chromatin accessibility and identification of general cis-regulatory mechanisms that control two ocular lens differentiation pathways.

Epigenetics Chromatin 2019 05 3;12(1):27. Epub 2019 May 3.

The Departments of Genetics, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.

Background: Promoters and enhancers are cis-regulatory DNA sequences that control specificity and quantity of transcription. Both are rich on clusters of cis-acting sites that interact with sequence-specific DNA-binding transcription factors (TFs). At the level of chromatin, these regions display increased nuclease sensitivity, reduced nucleosome density, including nucleosome-free regions, and specific combinations of posttranslational modifications of core histone proteins. Together, "open" and "closed" chromatins represent transcriptionally active and repressed states of individual genes, respectively. Cellular differentiation is marked by changes in local chromatin structure. Lens morphogenesis, regulated by TF Pax6, includes differentiation of epithelial precursor cells into lens fibers in parallel with differentiation of epithelial precursors into the mature lens epithelium.

Results: Using ATAC-seq, we investigated dynamics of chromatin changes during mouse lens fibers and epithelium differentiation. Tissue-specific features of these processes are demonstrated via comparative studies of embryonic stem cells, forebrain, and liver chromatins. Unbiased analysis reveals cis-regulatory logic of lens differentiation through known (e.g., AP-1, Ets, Hsf4, Maf, and Pax6 sites) and novel (e.g., CTCF, Tead, and NF1) motifs. Twenty-six DNA-binding TFs, recognizing these cis-motifs, are markedly up-regulated in differentiating lens fibers. As specific examples, our ATAC-seq data uncovered both the regulatory regions and TF binding motifs in Foxe3, Prox1, and Mip loci that are consistent with previous, though incomplete, experimental data. A cross-examination of Pax6 binding with ATAC-seq data demonstrated that Pax6 bound to both open (H3K27ac and P300-enriched) and closed chromatin domains in lens and forebrain.

Conclusions: Our study has generated the first lens chromatin accessibility maps that support a general model of stage-specific chromatin changes associated with transcriptional activities of batteries of genes required for lens fiber cell formation. Analysis of active (or open) promoters and enhancers reveals important cis-DNA motifs that establish the molecular foundation for temporally and spatially regulated gene expression in lens. Together, our data and models open new avenues for the field to conduct mechanistic studies of transcriptional control regions, reconstruction of gene regulatory networks that govern lens morphogenesis, and identification of cataract-causing mutations in noncoding sequences.
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http://dx.doi.org/10.1186/s13072-019-0272-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498704PMC
May 2019

Bidirectional Analysis of Cryba4-Crybb1 Nascent Transcription and Nuclear Accumulation of Crybb3 mRNAs in Lens Fibers.

Invest Ophthalmol Vis Sci 2019 01;60(1):234-244

Departments of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States.

Purpose: Crystallin gene expression during lens fiber cell differentiation is tightly spatially and temporally regulated. A significant fraction of mammalian genes is transcribed from adjacent promoters in opposite directions ("bidirectional" promoters). It is not known whether two proximal genes located on the same allele are simultaneously transcribed.

Methods: Mouse lens transcriptome was analyzed for paired genes whose transcriptional start sites are separated by less than 5 kbp to identify coexpressed bidirectional promoter gene pairs. To probe these transcriptional mechanisms, nascent transcription of Cryba4, Crybb1, and Crybb3 genes from gene-rich part of chromosome 5 was visualized by RNA fluorescent in situ hybridizations (RNA FISH) in individual lens fiber cell nuclei.

Results: Genome-wide lens transcriptome analysis by RNA-seq revealed that the Cryba4-Crybb1 pair has the highest Pearson correlation coefficient between their steady-state mRNA levels. Analysis of Cryba4 and Crybb1 nascent transcription revealed frequent simultaneous expression of both genes from the same allele. Nascent Crybb3 transcript visualization in "early" but not "late" differentiating lens fibers show nuclear accumulation of the spliced Crybb3 transcripts that was not affected in abnormal lens fiber cell nuclei depleted of chromatin remodeling enzyme Snf2h (Smarca5).

Conclusions: The current study shows for the first time that two highly expressed lens crystallin genes, Cryba4 and Crybb1, can be simultaneously transcribed from adjacent bidirectional promoters and do not show nuclear accumulation. In contrast, spliced Crybb3 mRNAs transiently accumulate in early lens fiber cell nuclei. The gene pairs coexpressed during lens development showed significant enrichment in human "cataract" phenotype.
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http://dx.doi.org/10.1167/iovs.18-25921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336207PMC
January 2019

Six3 and Six6 Are Jointly Required for the Maintenance of Multipotent Retinal Progenitors through Both Positive and Negative Regulation.

Cell Rep 2018 11;25(9):2510-2523.e4

Departments of Ophthalmology and Visual Sciences and Genetics, Albert Einstein College of Medicine, New York, NY 10461, USA. Electronic address:

Gene regulation of multipotent neuroretinal progenitors is partially understood. Through characterizing Six3 and Six6 double knockout retinas (DKOs), we demonstrate Six3 and Six6 are jointly required for the maintenance of multipotent neuroretinal progenitors. Phenotypes in DKOs were not found in either Six3 nulls or Six6 nulls. At the far periphery, ciliary margin (CM) markers Otx1 and Cdon together with Wnt3a and Fzd1 were ectopically upregulated, whereas neuroretinal progenitor markers Sox2, Notch1, and Otx2 were absent or reduced. At the mid periphery, multi-lineage differentiation was defective. The gene set jointly regulated by Six3 and Six6 significantly overlapped with the gene networks regulated by WNT3A, CTNNB1, POU4F2, or SOX2. Stimulation of Wnt/β-catenin signaling by either Wnt-3a or a GS3Kβ inhibitor promoted CM progenitors at the cost of neuroretinal identity at the periphery of eyecups. Therefore, Six3 and Six6 together directly or indirectly suppress Wnt/β-catenin signaling but promote retinogenic factors for the maintenance of multipotent neuroretinal progenitors.
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http://dx.doi.org/10.1016/j.celrep.2018.10.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317371PMC
November 2018

Promoter-enhancer looping and shadow enhancers of the mouse αA-crystallin locus.

Biol Open 2018 Dec 7;7(12). Epub 2018 Dec 7.

Departments Ophthalmology and Visual Sciences and Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave, Ullmann 123, Bronx, NY 10461, USA

Gene regulation by enhancers is important for precise temporal and spatial gene expression. Enhancers can drive gene expression regardless of their location, orientation or distance from the promoter. Changes in chromatin conformation and chromatin looping occur to bring the promoter and enhancers into close proximity. αA-crystallin ranks among one of the most abundantly expressed genes and proteins in the mammalian lens. The αA-crystallin locus is characterized by a 16 kb chromatin domain marked by two distal enhancers, 5' DCR1 and 3' DCR3. Here we used chromatin conformation capture (3C) analysis and transgenic approaches to analyze temporal control of the mouse αA-crystallin gene. We find that DCR1 is necessary, but not sufficient alone to drive expression at E10.5 in the mouse lens pit. Chromatin looping revealed interaction between the promoter and the region 3' to DCR1, identifying a novel enhancer region in the αA-crystallin locus. We determined that this novel enhancer region, DCR1S, recapitulates the temporal control by DCR1. Acting as shadow enhancers, DCR1 and DCR1S are able to control expression in the lens vesicle at E11.5. It remains to be elucidated however, which region of the αA-crystallin locus is responsible for expression in the lens pit at E10.5.
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http://dx.doi.org/10.1242/bio.036897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310886PMC
December 2018

Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers.

Exp Eye Res 2019 02 22;179:32-46. Epub 2018 Oct 22.

Departments Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY, 10461, USA; Genetics, Albert Einstein College of Medicine, Bronx, NY, 10461, USA. Electronic address:

Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular compositions of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development and pathology in vitro. To compare protein compositions between the lens epithelium and fibers, we employed tandem mass spectrometry (2D-LC/MS) analysis of microdissected mouse P0.5 lenses. Functional classifications of the top 525 identified proteins into gene ontology categories by molecular processes and subcellular localizations, were adapted for the lens. Expression levels of both epithelial and fiber proteomes were compared with whole lens proteome and mRNA levels using E14.5, E16.5, E18.5, and P0.5 RNA-Seq data sets. During this developmental time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. As expected, crystallins showed a high correlation between their mRNA and protein levels. Comprehensive data analysis confirmed and/or predicted roles for transcription factors (TFs), RNA-binding proteins (e.g. Carhsp1), translational apparatus including ribosomal heterogeneity and initiation factors, microtubules, cytoskeletal [e.g. non-muscle myosin IIA heavy chain (Myh9) and βB2-spectrin (Sptbn2)] and membrane proteins in lens formation and maturation. Our data highlighted many proteins with unknown functions in the lens that were preferentially enriched in epithelium or fibers, setting the stage for future studies to further dissect the roles of these proteins in fiber cell differentiation vs. epithelial cell maintenance. In conclusion, the present proteomic datasets represent the first mouse lens epithelium and fiber cell proteomes, establish comparative analyses of protein and RNA-Seq data, and characterize the major proteome remodeling required to form the mature lens fiber cells.
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http://dx.doi.org/10.1016/j.exer.2018.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360118PMC
February 2019

Identification of Novel Gata3 Distal Enhancers Active in Mouse Embryonic Lens.

Dev Dyn 2018 11 10;247(11):1186-1198. Epub 2018 Nov 10.

Departments of Ophthalmology and Visual Sciences and Genetics, Albert Einstein College of Medicine, Bronx, New York.

Background: The tissue-specific transcriptional programs during normal development require tight control by distal cis-regulatory elements, such as enhancers, with specific DNA sequences recognized by transcription factors, coactivators, and chromatin remodeling enzymes. Gata3 is a sequence-specific DNA-binding transcription factor that regulates formation of multiple tissues and organs, including inner ear, lens, mammary gland, T-cells, urogenital system, and thyroid gland. In the eye, Gata3 has a highly restricted expression domain in the posterior part of the lens vesicle; however, the underlying regulatory mechanisms are unknown.

Results: Here we describe the identification of a novel bipartite Gata3 lens-specific enhancer located ∼18 kb upstream from its transcriptional start site. We also found that a 5-kb Gata3 promoter possesses low activity in the lens. The bipartite enhancer contains arrays of AP-1, Ets-, and Smad1/5-binding sites as well as binding sites for lens-associated DNA-binding factors. Transient transfection studies of the promoter with the bipartite enhancer showed enhanced activation by BMP4 and FGF2.

Conclusions: These studies identify a novel distal enhancer of Gata3 with high activity in lens and indicate that BMP and FGF signaling can up-regulate expression of Gata3 in differentiating lens fiber cells through the identified Gata3 enhancer and promoter elements. Developmental Dynamics 247:1186-1198, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
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http://dx.doi.org/10.1002/dvdy.24677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6246825PMC
November 2018

Transcriptional burst fraction and size dynamics during lens fiber cell differentiation and detailed insights into the denucleation process.

J Biol Chem 2018 08 29;293(34):13176-13190. Epub 2018 Jun 29.

From the Departments of Genetics,

Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction ( the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell differentiation. In the ocular lens, the positions of lens fiber cells correlate precisely with their differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the β-actin and lens differentiation-specific α-, β-, and γ-crystallin genes by RNA fluorescent hybridization (FISH) in the lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular differentiation dramatically alters the burst fraction in synchronized waves across the lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration.
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http://dx.doi.org/10.1074/jbc.RA118.001927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109918PMC
August 2018

A comprehensive spatial-temporal transcriptomic analysis of differentiating nascent mouse lens epithelial and fiber cells.

Exp Eye Res 2018 10 5;175:56-72. Epub 2018 Jun 5.

Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address:

Elucidation of both the molecular composition and organization of the ocular lens is a prerequisite to understand its development, function, pathology, regenerative capacity, as well as to model lens development and disease using in vitro differentiation of pluripotent stem cells. Lens is comprised of the anterior lens epithelium and posterior lens fibers, which form the bulk of the lens. Lens fibers differentiate from lens epithelial cells through cell cycle exit-coupled differentiation that includes cellular elongation, accumulation of crystallins, cytoskeleton and membrane remodeling, and degradation of organelles within the central region of the lens. Here, we profiled spatiotemporal expression dynamics of both mRNAs and non-coding RNAs from microdissected mouse nascent lens epithelium and lens fibers at four developmental time points (embryonic [E] day 14.5, E16.5, E18.5, and P0.5) by RNA-seq. During this critical time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. Throughout this developmental window, 3544 and 3518 genes show consistently and significantly greater expression in the nascent lens epithelium and fibers, respectively. Comprehensive data analysis confirmed major roles of FGF-MAPK, Wnt/β-catenin, PI3K/AKT, TGF-β, and BMP signaling pathways and revealed significant novel contributions of mTOR, EIF2, EIF4, and p70S6K signaling in lens formation. Unbiased motif analysis within promoter regions of these genes with consistent expression changes between epithelium and fiber cells revealed an enrichment for both established (e.g. E2Fs, Etv5, Hsf4, c-Maf, MafG, MafK, N-Myc, and Pax6) transcription factors and a number of novel regulators of lens formation, such as Arntl2, Dmrta2, Stat5a, Stat5b, and Tulp3. In conclusion, the present RNA-seq data serves as a comprehensive reference resource for deciphering molecular principles of normal mammalian lens differentiation, mapping a full spectrum of signaling pathways and DNA-binding transcription factors operating in both lens compartments, and predicting novel pathways required to establish lens transparency.
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http://dx.doi.org/10.1016/j.exer.2018.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167154PMC
October 2018

BNIP3L/NIX is required for elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during eye lens organelle-free zone formation.

Exp Eye Res 2018 09 4;174:173-184. Epub 2018 Jun 4.

Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, 33431, USA. Electronic address:

The formation and life-long growth of the ocular lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To achieve their mature structure and transparent function, newly formed lens fiber cells undergo a series of cellular remodeling events including the complete elimination of cellular organelles to form the lens organelle-free zone (OFZ). To date, the mechanisms and requirements for organelle elimination by lens fiber cells remain to be fully elucidated. In previous studies, we detected the presence of mitochondria contained within autophagolysosomes throughout human and chick lenses suggesting that proteins targeting mitochondria for degradation by mitophagy could be required for the elimination of mitochondria during OFZ formation. Consistently, high-throughput RNA sequencing of microdissected embryonic chick lenses revealed that expression of a protein that targets mitochondria for elimination during erythrocyte formation, called BCL2 interacting protein 3-like (BNIP3L/NIX), peaks in the region of lens where organelle elimination occurs. To examine the potential role for BNIP3L in the elimination of mitochondria during lens fiber cell remodeling, we analyzed the expression pattern of BNIP3L in newborn mouse lenses, the effect of its deletion on organelle elimination and its co-localization with lens organelles. We demonstrate that the expression pattern of BNIP3L in the mouse lens is consistent with it playing an important role in the elimination of mitochondria during lens fiber cell organelle elimination. Importantly, we demonstrate that deletion of BNIP3L results in retention of mitochondria during lens fiber cell remodeling, and, surprisingly, that deletion of BNIP3L also results in the retention of endoplasmic reticulum and Golgi apparatus but not nuclei. Finally, we show that BNIP3L localizes to the endoplasmic reticulum and Golgi apparatus of wild-type newborn mouse lenses and is contained within mitochondria, endoplasmic reticulum and Golgi apparatus isolated from adult mouse liver. These data identify BNIP3L as a novel requirement for the elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell remodeling and they suggest a novel function for BNIP3L in the regulation of endoplasmic reticulum and Golgi apparatus populations in the lens and non-lens tissues.
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http://dx.doi.org/10.1016/j.exer.2018.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110959PMC
September 2018

Evolutionary Origins of Pax6 Control of Crystallin Genes.

Genome Biol Evol 2017 08;9(8):2075-2092

Department of Genetics, Albert Einstein College of Medicine, Bronx, New York.

The birth of novel genes, including their cell-specific transcriptional control, is a major source of evolutionary innovation. The lens-preferred proteins, crystallins (vertebrates: α- and β/γ-crystallins), provide a gateway to study eye evolution. Diversity of crystallins was thought to originate from convergent evolution through multiple, independent formation of Pax6/PaxB-binding sites within the promoters of genes able to act as crystallins. Here, we propose that αB-crystallin arose from a duplication of small heat shock protein (Hspb1-like) gene accompanied by Pax6-site and heat shock element (HSE) formation, followed by another duplication to generate the αA-crystallin gene in which HSE was converted into another Pax6-binding site. The founding β/γ-crystallin gene arose from the ancestral Hspb1-like gene promoter inserted into a Ca2+-binding protein coding region, early in the cephalochordate/tunicate lineage. Likewise, an ancestral aldehyde dehydrogenase (Aldh) gene, through multiple gene duplications, expanded into a multigene family, with specific genes expressed in invertebrate lenses (Ω-crystallin/Aldh1a9) and both vertebrate lenses (η-crystallin/Aldh1a7 and Aldh3a1) and corneas (Aldh3a1). Collectively, the present data reconstruct the evolution of diverse crystallin gene families.
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http://dx.doi.org/10.1093/gbe/evx153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737492PMC
August 2017

Signaling and Gene Regulatory Networks in Mammalian Lens Development.

Trends Genet 2017 10 31;33(10):677-702. Epub 2017 Aug 31.

Departments of Ophthalmology, Pathology and Cell Biology, Columbia University Medical Center, New York, NY 10032, USA. Electronic address:

Ocular lens development represents an advantageous system in which to study regulatory mechanisms governing cell fate decisions, extracellular signaling, cell and tissue organization, and the underlying gene regulatory networks. Spatiotemporally regulated domains of BMP, FGF, and other signaling molecules in late gastrula-early neurula stage embryos generate the border region between the neural plate and non-neural ectoderm from which multiple cell types, including lens progenitor cells, emerge and undergo initial tissue formation. Extracellular signaling and DNA-binding transcription factors govern lens and optic cup morphogenesis. Pax6, c-Maf, Hsf4, Prox1, Sox1, and a few additional factors regulate the expression of the lens structural proteins, the crystallins. Extensive crosstalk between a diverse array of signaling pathways controls the complexity and order of lens morphogenetic processes and lens transparency.
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http://dx.doi.org/10.1016/j.tig.2017.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627649PMC
October 2017

N-myc regulates growth and fiber cell differentiation in lens development.

Dev Biol 2017 09 14;429(1):105-117. Epub 2017 Jul 14.

Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Electronic address:

Myc proto-oncogenes regulate diverse cellular processes during development, but their roles during morphogenesis of specific tissues are not fully understood. We found that c-myc regulates cell proliferation in mouse lens development and previous genome-wide studies suggested functional roles for N-myc in developing lens. Here, we examined the role of N-myc in mouse lens development. Genetic inactivation of N-myc in the surface ectoderm or lens vesicle impaired eye and lens growth, while "late" inactivation in lens fibers had no effect. Unexpectedly, defective growth of N-myc-deficient lenses was not associated with alterations in lens progenitor cell proliferation or survival. Notably, N-myc-deficient lens exhibited a delay in degradation of DNA in terminally differentiating lens fiber cells. RNA-sequencing analysis of N-myc-deficient lenses identified a cohort of down-regulated genes associated with fiber cell differentiation that included DNaseIIβ. Further, an integrated analysis of differentially expressed genes in N-myc-deficient lens using normal lens expression patterns of iSyTE, N-myc-binding motif analysis and molecular interaction data from the String database led to the derivation of an N-myc-based gene regulatory network in the lens. Finally, analysis of N-myc and c-myc double-deficient lens demonstrated that these Myc genes cooperate to drive lens growth prior to lens vesicle stage. Together, these findings provide evidence for exclusive and cooperative functions of Myc transcription factors in mouse lens development and identify novel mechanisms by which N-myc regulates cell differentiation during eye morphogenesis.
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http://dx.doi.org/10.1016/j.ydbio.2017.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5586101PMC
September 2017

Six3 in a small population of progenitors at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice.

Authors:
Wei Liu Ales Cvekl

Dev Biol 2017 08 1;428(1):164-175. Epub 2017 Jun 1.

Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA; Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.

Neuroretina and retinal pigment epithelium (RPE) are differentiated from the progenitors in optic vesicles, but it is unclear when and how the two lineages are segregated. Manipulation of chick embryos reveals that the early anteroventral optic vesicle is crucial for neuroretinal development, but the molecular mechanism is unclear. Homeodomain transcription factor Six3 is required for neuroretinal specification and is dispensable for RPE formation, but the cell fates of Six3-deficient progenitors and the origins of remnant RPE are unknown. Here, we performed lineage tracing of Six3-Cre positive cells in wild-type and Six3-deficient mouse embryos. Six3-Cre positive progenies were found in a population of progenitors in the anteroventral optic pits/vesicles starting at E8.5, and were found in neuroretina, optic stalk, ventral forebrain, but not RPE, at E10.5. Six3-deletion in the small population of progenitors at E8.5 was sufficient to cause rostral expansion of Wnt8b and drastic reduction of Fgf8/MAPK signaling, ablating neuroretinal specification without affecting RPE. Lineage tracing revealed Six3-deficient progenitors at E8.5 were eventually lost and the remnant RPE was derived from Six3-Cre negative cells. Thus, Six3 in a small population of progenitors expressing Six3-Cre at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice.
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http://dx.doi.org/10.1016/j.ydbio.2017.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533277PMC
August 2017

Programmed mitophagy is essential for the glycolytic switch during cell differentiation.

EMBO J 2017 06 2;36(12):1688-1706. Epub 2017 May 2.

Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain

Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis-associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX-deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX-dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts.
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http://dx.doi.org/10.15252/embj.201695916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470043PMC
June 2017

Pax6 associates with H3K4-specific histone methyltransferases Mll1, Mll2, and Set1a and regulates H3K4 methylation at promoters and enhancers.

Epigenetics Chromatin 2016 9;9(1):37. Epub 2016 Sep 9.

Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 USA ; Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY 10461 USA.

Background: Pax6 is a key regulator of the entire cascade of ocular lens formation through specific binding to promoters and enhancers of batteries of target genes. The promoters and enhancers communicate with each other through DNA looping mediated by multiple protein-DNA and protein-protein interactions and are marked by specific combinations of histone posttranslational modifications (PTMs). Enhancers are distinguished from bulk chromatin by specific modifications of core histone H3, including H3K4me1 and H3K27ac, while promoters show increased H3K4me3 PTM. Previous studies have shown the presence of Pax6 in as much as 1/8 of lens-specific enhancers but a much smaller fraction of tissue-specific promoters. Although Pax6 is known to interact with EP300/p300 histone acetyltransferase responsible for generation of H3K27ac, a potential link between Pax6 and histone H3K4 methylation remains to be established.

Results: Here we show that Pax6 co-purifies with H3K4 methyltransferase activity in lens cell nuclear extracts. Proteomic studies show that Pax6 immunoprecipitates with Set1a, Mll1, and Mll2 enzymes, and their associated proteins, i.e., Wdr5, Rbbp5, Ash2l, and Dpy30. ChIP-seq studies using chromatin prepared from mouse lens and cultured lens cells demonstrate that Pax6-bound regions are mostly enriched with H3K4me2 and H3K4me1 in enhancers and promoters, though H3K4me3 marks only Pax6-containing promoters. The shRNA-mediated knockdown of Pax6 revealed down-regulation of a set of direct target genes, including Cap2, Farp1, Pax6, Plekha1, Prox1, Tshz2, and Zfp536. Pax6 knockdown was accompanied by reduced H3K4me1 at enhancers and H3K4me3 at promoters, with little or no changes of the H3K4me2 modifications. These changes were prominent in Plekha1, a gene regulated by Pax6 in both lens and retinal pigmented epithelium.

Conclusions: Our study supports a general model of Pax6-mediated recruitment of histone methyltransferases Mll1 and Mll2 to lens chromatin, especially at distal enhancers. Genome-wide data in lens show that Pax6 binding correlates with H3K4me2, consistent with the idea that H3K4me2 PTMs correlate with the binding of transcription factors. Importantly, partial reduction of Pax6 induces prominent changes in local H3K4me1 and H3K4me3 modification. Together, these data open the field to mechanistic studies of Pax6, Mll1, Mll2, and H3K4me1/2/3 dynamics at distal enhancers and promoters of developmentally controlled genes.
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http://dx.doi.org/10.1186/s13072-016-0087-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018195PMC
September 2016

Sip1 regulates the generation of the inner nuclear layer retinal cell lineages in mammals.

Development 2016 08 6;143(15):2829-41. Epub 2016 Jul 6.

Department of Human Molecular Genetics and Biochemistry, Faculty of Medicine and Sagol School of Neuroscience, Tel-Aviv University, Tel Aviv 69978, Israel

The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals.
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http://dx.doi.org/10.1242/dev.136101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004910PMC
August 2016

Chromatin remodeling enzyme Snf2h regulates embryonic lens differentiation and denucleation.

Development 2016 06;143(11):1937-47

Department of Ophthalmology & Visual Sciences and Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIβ, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation.
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http://dx.doi.org/10.1242/dev.135285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920164PMC
June 2016

Intercellular Adhesion-Dependent Cell Survival and ROCK-Regulated Actomyosin-Driven Forces Mediate Self-Formation of a Retinal Organoid.

Stem Cell Reports 2016 05 28;6(5):743-756. Epub 2016 Apr 28.

Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address:

In this study we dissected retinal organoid morphogenesis in human embryonic stem cell (hESC)-derived cultures and established a convenient method for isolating large quantities of retinal organoids for modeling human retinal development and disease. Epithelialized cysts were generated via floating culture of clumps of Matrigel/hESCs. Upon spontaneous attachment and spreading of the cysts, patterned retinal monolayers with tight junctions formed. Dispase-mediated detachment of the monolayers and subsequent floating culture led to self-formation of retinal organoids comprising patterned neuroretina, ciliary margin, and retinal pigment epithelium. Intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces are required for the self-organization. Our data supports a hypothesis that newly specified neuroretina progenitors form characteristic structures in equilibrium through minimization of cell surface tension. In long-term culture, the retinal organoids autonomously generated stratified retinal tissues, including photoreceptors with ultrastructure of outer segments. Our system requires minimal manual manipulation, has been validated in two lines of human pluripotent stem cells, and provides insight into optic cup invagination in vivo.
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http://dx.doi.org/10.1016/j.stemcr.2016.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939656PMC
May 2016

PAX6: 25th anniversary and more to learn.

Exp Eye Res 2017 03 25;156:10-21. Epub 2016 Apr 25.

Laboratory of Behavioral and Developmental Genetics, K.U. Leuven, VIB, 3000, Leuven, Belgium. Electronic address:

The DNA-binding transcription factor PAX6 was cloned 25 years ago by multiple teams pursuing identification of human and mouse eye disease causing genes, cloning vertebrate homologues of pattern-forming regulatory genes identified in Drosophila, or abundant eye-specific transcripts. Since its discovery in 1991, genetic, cellular, molecular and evolutionary studies on Pax6 mushroomed in the mid 1990s leading to the transformative thinking regarding the genetic program orchestrating both early and late stages of eye morphogenesis as well as the origin and evolution of diverse visual systems. Since Pax6 is also expressed outside of the eye, namely in the central nervous system and pancreas, a number of important insights into the development and function of these organs have been amassed. In most recent years, genome-wide technologies utilizing massively parallel DNA sequencing have begun to provide unbiased insights into the regulatory hierarchies of specification, determination and differentiation of ocular cells and neurogenesis in general. This review is focused on major advancements in studies on mammalian eye development driven by studies of Pax6 genes in model organisms and future challenges to harness the technology-driven opportunities to reconstruct, step-by-step, the transition from naïve ectoderm, neuroepithelium and periocular mesenchyme/neural crest cells into the three-dimensional architecture of the eye.
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http://dx.doi.org/10.1016/j.exer.2016.04.017DOI Listing
March 2017

Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling.

J Biol Chem 2016 Feb 30;291(8):3947-58. Epub 2015 Dec 30.

From the Departments of Ophthalmology and Visual Sciences and Genetics, Albert Einstein College of Medicine, Bronx, New York 10461,

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.
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http://dx.doi.org/10.1074/jbc.M115.705103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759173PMC
February 2016

Unfolded-protein response-associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract.

FASEB J 2016 Mar 20;30(3):1087-95. Epub 2015 Nov 20.

*Ministry of Education Key Laboratory of Bio-resource and Eco-environment, College of Life Science, Sichuan University, Sichuan China; Laboratory for Nutrition and Vision Research, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, USA; Department of Biological Sciences, University of Delaware, Newark, Delaware, USA; Department of Genetics and Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, New York, USA; and Department of Ophthalmology, School of Medicine, University of Missouri, Columbia, Missouri, USA

Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei.
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http://dx.doi.org/10.1096/fj.15-278036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4750420PMC
March 2016

Chromatin features, RNA polymerase II and the comparative expression of lens genes encoding crystallins, transcription factors, and autophagy mediators.

Mol Vis 2015 28;21:955-73. Epub 2015 Aug 28.

Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY ; Department of Genetics, Albert Einstein College of Medicine, Bronx, NY.

Purpose: Gene expression correlates with local chromatin structure. Our studies have mapped histone post-translational modifications, RNA polymerase II (pol II), and transcription factor Pax6 in lens chromatin. These data represent the first genome-wide insights into the relationship between lens chromatin structure and lens transcriptomes and serve as an excellent source for additional data analysis and refinement. The principal lens proteins, the crystallins, are encoded by predominantly expressed mRNAs; however, the regulatory mechanisms underlying their high expression in the lens remain poorly understood.

Methods: The formaldehyde-assisted identification of regulatory regions (FAIRE-Seq) was employed to analyze newborn lens chromatin. ChIP-seq and RNA-seq data published earlier (GSE66961) have been used to assist in FAIRE-seq data interpretation. RNA transcriptomes from murine lens epithelium, lens fibers, erythrocytes, forebrain, liver, neurons, and pancreas were compared to establish the gene expression levels of the most abundant mRNAs versus median gene expression across other differentiated cells.

Results: Normalized RNA expression data from multiple tissues show that crystallins rank among the most highly expressed genes in mammalian cells. These findings correlate with the extremely high abundance of pol II all across the crystallin loci, including crystallin genes clustered on chromosomes 1 and 5, as well as within regions of "open" chromatin, as identified by FAIRE-seq. The expression levels of mRNAs encoding DNA-binding transcription factors (e.g., Foxe3, Hsf4, Maf, Pax6, Prox1, Sox1, and Tfap2a) revealed that their transcripts form "clusters" of abundant mRNAs in either lens fibers or lens epithelium. The expression of three autophagy regulatory mRNAs, encoding Tfeb, FoxO1, and Hif1α, was found within a group of lens preferentially expressed transcription factors compared to the E12.5 forebrain.

Conclusions: This study reveals novel features of lens chromatin, including the remarkably high abundance of pol II at the crystallin loci that exhibit features of "open" chromatin. Hsf4 ranks among the most abundant fiber cell-preferred DNA-binding transcription factors. Notable transcripts, including Atf4, Ctcf, E2F4, Hey1, Hmgb1, Mycn, RXRβ, Smad4, Sp1, and Taf1 (transcription factors) and Ctsd, Gabarapl1, and Park7 (autophagy regulators) have been identified with high levels of expression in lens fibers, which suggests specific roles in lens fiber cell terminal differentiation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551281PMC
May 2016

Lens Biology and Biochemistry.

Prog Mol Biol Transl Sci 2015 4;134:169-201. Epub 2015 Jun 4.

Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, USA. Electronic address:

The primary function of the lens resides in its transparency and ability to focus light on the retina. These require both that the lens cells contain high concentrations of densely packed lens crystallins to maintain a refractive index constant over distances approximating the wavelength of the light to be transmitted, and a specific arrangement of anterior epithelial cells and arcuate fiber cells lacking organelles in the nucleus to avoid blocking transmission of light. Because cells in the lens nucleus have shed their organelles, lens crystallins have to last for the lifetime of the organism, and are specifically adapted to this function. The lens crystallins comprise two major families: the βγ-crystallins are among the most stable proteins known and the α-crystallins, which have a chaperone-like function. Other proteins and metabolic activities of the lens are primarily organized to protect the crystallins from damage over time and to maintain homeostasis of the lens cells. Membrane protein channels maintain osmotic and ionic balance across the lens, while the lens cytoskeleton provides for the specific shape of the lens cells, especially the fiber cells of the nucleus. Perhaps most importantly, a large part of the metabolic activity in the lens is directed toward maintaining a reduced state, which shelters the lens crystallins and other cellular components from damage from UV light and oxidative stress. Finally, the energy requirements of the lens are met largely by glycolysis and the pentose phosphate pathway, perhaps in response to the avascular nature of the lens. Together, all these systems cooperate to maintain lens transparency over time.
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http://dx.doi.org/10.1016/bs.pmbts.2015.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538311PMC
February 2016

Lens Development and Crystallin Gene Expression.

Prog Mol Biol Transl Sci 2015 12;134:129-67. Epub 2015 Jun 12.

Departments of Genetics and Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, New York, USA.

The eye and lens represent excellent models to understand embryonic development at cellular and molecular levels. Initial 3D formation of the eye depends on a reciprocal invagination of the lens placode/optic vesicle to form the eye primordium, i.e., the optic cup partially surrounding the lens vesicle. Subsequently, the anterior part of the lens vesicle gives rise to the lens epithelium, while the posterior cells of the lens vesicle differentiate into highly elongated lens fibers. Lens fiber differentiation involves cytoskeletal rearrangements, cellular elongation, accumulation of crystallin proteins, production of extracellular matrix for the lens capsule, and degradation of organelles. This chapter summarizes recent advances in lens development and provides insights into the regulatory mechanisms and differentiation at the level of chromatin structure and dynamics, the emerging field of noncoding RNAs, and novel strategies to fill the gaps in our understanding of lens development.
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http://dx.doi.org/10.1016/bs.pmbts.2015.05.001DOI Listing
February 2016