Toshiro Iwagawa

Roles of histone H3K27 trimethylase Ezh2 in retinal proliferation and differentiation.

The histone modification H3K27me3 regulates transcription negatively, and Jmjd3 and Ezh2 demethylate and methylate H3K27me3 and H3K27, respectively. We demonstrated previously that Jmjd3 plays pivotal roles in the differentiation of subsets of bipolar (BP) cells by regulating H3K27me3 levels at the Bhlhb4 and Vsx1 loci, both of which are transcription factors essential for the maturation of BP cell subsets. In this study, we examined the role of Ezh2 in retinal development using retina‐specific Ezh2 conditional knockout mice (Ezh2‐CKO). The eyes of the Ezh2‐CKO mice were microphthalemic, and the proliferation of retinal cells was diminished postnatally in Ezh2‐CKO. Differentiation of all examined retinal subsets was observed with higher proportion of BP cell subsets, which was determined by immunostaining using specific retinal markers. The onsets of Müller glia and rod photoreceptor differentiation were accelerated. The expression of Bhlhb4 was increased in postnatal retinas, which was accompanied by the loss of H3K27me3 modifications at these genetic loci. Decreased expression of proneural genes in postnatal stage was observed. As reported previously in other Ezh2‐KO tissues, increased expression of Arf/Ink4a was observed in the Ezh2‐CKO retinas. The ectopic expression of Arf or Ink4a in the retina suppressed proliferation and increased apoptosis. In addition, earlier onset of Müller glia differentiation was observed in Ink4a‐expressing cells. These results support an important role for histone H3K27me3 modification in regulating the proliferation and maturation of certain subsets of interneurons in the retina.

Histone demethylase Jmjd3 is required for the development of subsets of retinal bipolar cells

Significance The role of repressive gene regulation through histone modifications is known in various biological processes. Histone H3 trimethyl Lys27 (H3K27me3) represses gene expression, and our goal was to understand how this methylation regulates cell differentiation in the vertebrate retina. In this work, we focused on the role of demethylase (Jmjd3) of H3K27me3 in retinal development. Spatiotemporal expression patterns of Jmjd3 during retinal development were shown. Suppression of Jmjd3 expression during retinal development resulted in the failure of differentiation of retinal cell subsets. Lowered expression of genes essential for differentiation of the subsets by loss of expression of Jmjd3 was observed. Therefore, we propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal cells. Di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) is an important gene repression mechanism. H3K27me2/3-specific demethylase, Jmjd3, was expressed in the inner nuclear layer during late retinal development. In contrast, H3K27 methyltransferase, Ezh2, was highly expressed in the embryonic retina but its expression decreased rapidly after birth. Jmjd3 loss of function in the developing retina resulted in failed differentiation of PKC-positive bipolar cell subsets (rod-ON-BP) and reduced transcription factor Bhlhb4 expression, which is critical for the differentiation of rod-ON-BP cells. Overexpression of Bhlhb4, but not of other BP cell-related genes, such as transcription factors Neurod and Chx10, in Jmjd3-knockdown retina rescued loss of PKC-positive BP cells. Populations of other retinal cell subsets were not significantly affected. In addition, proliferation activity and apoptotic cell number during retinal development were not affected by the loss of Jmjd3. Levels of histone H3 trimethyl Lys27 (H3K27me3) in the Bhlhb4 locus were lower in Islet-1–positive BP cells and amacrine cells than in the Islet-1–negative cell fraction. The Islet-1–negative cell fraction consisted mainly of photoreceptors, suggestive of lineage-specific demethylation of H3K27me3 in the Bhlhb4 locus. We propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal cells.

Selection of RNA aptamers against mouse embryonic stem cells

Embryonic stem cells (ESCs) are capable of unlimited self-renewal and differentiation into multiple cell types. Recent large-scale analyses have identified various cell surface molecules on ESCs. Some of them are considered to be beneficial markers for characterization of cellular phenotypes and/or play an essential role for regulating the differentiation state. Thus, it is desired to efficiently produce affinity reagents specific to these molecules. In this study, to develop such reagents for mouse ESCs (mESCs), we selected RNA aptamers against intact, live mESCs using several selection strategies. The initial selection provided us with several anti-mESC aptamers of distinct sequences, which unexpectedly react with the same molecule on mESCs. Then, to isolate aptamers against different surface markers on mESCs, one of the selected aptamers was used as a competitor in the subsequent selections. In addition, one of the selections further employed negative selection against differentiated mouse cells. Consequently, we successfully isolated three classes of anti-mESC aptamers that do not compete with one another. The isolated aptamers were shown to distinguish mESCs from differentiated mouse cell lines and trace the differentiation process of mESCs. These aptamers could prove useful for developing molecular probes and manipulation tools for mESCs.

Expression of Sox4 and Sox11 is regulated by multiple mechanisms during retinal development

Sox11 and Sox4 play critical roles in retinal development, during which they display specific and unique expression patterns. The expression of Sox11 and Sox4 is temporally sequential, albeit spatially overlapping in some retinal subtypes. Gain‐of‐function and loss‐of‐function analyses suggested that Notch signaling suppresses Sox4 expression in the early developing retina but not during the later period of development. The levels of histone H3‐acetylation and H3‐lysine 4 tri‐methylation at the Sox11 locus declined during development, as did the levels of Sox11. A similar but less marked change was seen for Sox4. For both genes, histone H3‐lysine 27 methylation was low during development and increased markedly in the adult.

Transition of differential histone H3 methylation in photoreceptors and other retinal cells during retinal differentiation

To analyze cell lineage-specific transitions in global transcriptional and epigenetic changes during retinogenesis, we purified retinal cells from normal mice during postnatal development into two fractions, namely, photoreceptors and other retinal cells, based on Cd73 expression, and performed RNA sequencing and ChIP sequencing of H3K27me3 and H3K4me3. Genes expressed in the photoreceptor lineage were marked with H3K4me3 in the Cd73-positive cell fraction; however, the level of H3K27me3 was very low in both Cd73-positive and -negative populations. H3K27me3 may be involved in spatio-temporal onset of a subset of bipolar-related genes. Subsets of genes expressed in amacrine and retinal ganglion cells, which are early-born retinal cell types, were suggested to be maintained in a silent state by H3K27me3 during late-stage retinogenesis. In the outer nuclear layer, upregulation of Rho and rod-related genes were observed in Ezh2-ablated retina, suggesting a role for H3K27me3 in the maintenance of proper expression levels. Taken together, our data on the transition of lineage-specific molecular signatures during development suggest that histone methylation is involved in retinal differentiation and maintenance through cell lineage-specific mechanisms.

Analysis of Müller glia specific genes and their histone modification using Hes1-promoter driven EGFP expressing mouse

Retinal neurons and Müller glia are generated from a common population of multipotent retinal progenitor cells. We purposed to identify Müller glia-specific molecular signatures during retinal development. Using transgenic mice carrying the Hes1 promoter (pHes1) followed by EGFP, we purified EGFP-positive Müller glia and other EGFP-negative retinal cells from developing retinas and subjected them to RNA sequencing analysis. Gene expression pattern of EGFP-positive cell was similar to genes expressed in retinal progenitors, and they were downregulated in other cell lineages. Then, we examined the modification profiles of H3K27me3 and H3K4me3 by referring to chromatin immunoprecipitation-sequencing data of rods and other cells. Clustering of the H3K4me3 and H3K27me3 values followed by ontology analysis revealed a high incidence of transcription factors including Hes1 in clusters with high H3K27me3 levels. Hes1 expression level decreased dramatically, and the H3K27me3 level at the Hes1-locus was upregulated strongly during retinal development. Furthermore, the Hes1 expression level was upregulated in an Ezh2-knockout retina. These results suggest that downregulation of Müller glia-related genes in other lineage rather than upregulation of them in Müller glia contributed Müller-specific molecular features, and a role for modified H3K27me3 in suppressing Müller glia-related genes in other retinal cell lineages to avoid unfavorable expression.

Enhancer/Promoter Activities of the Long/Middle Wavelength-Sensitive Opsins of Vertebrates Mediated by Thyroid Hormone Receptor β2 and COUP-TFII

Cone photopigments (opsins) are crucial elements of, and the first detection module in, color vision. Individual opsins have different wavelength sensitivity patterns, and the temporal and spatial expression patterns of opsins are unique and stringently regulated. Long and middle wavelength-sensitive (L/M) opsins are of the same phylogenetic type. Although the roles of thyroid hormone/TRß2 and COUP-TFs in the transcriptional regulation of L/M opsins have been explored, the detailed mechanisms, including the target sequence in the enhancer of L/M opsins, have not been revealed. We aimed to reveal molecular mechanisms of L/M opsins in vertebrates. Using several human red opsin enhancer/promoter-luciferase reporter constructs, we found that TRß2 increased luciferase activities through the 5′-UTR and intron 3–4 region, whereas the presence of T3 affected only the intron 3–4 region-dependent luciferase activity. Furthermore, COUP-TFII suppressed intron 3–4 region-dependent luciferase activities. However, luciferase expression driven by the mouse M opsin intron 3–4 region was only slightly increased by TRß2, and rather enhanced by COUP-TFII. To determine whether these differential responses reflect differences between primates and rodents, we examined the enhancer/promoter region of the red opsin of the common marmoset. Interestingly, while TRß2 increased 5′-UTR- or intron 3–4 region-driven luciferase expression, as observed for the human red opsin, expression of the latter luciferase was not suppressed by COUP-TFII. In fact, immunostaining of common marmoset retinal sections revealed expression of COUP-TFII and red opsin in the cone cells.

Roles of Nmnat1 in the survival of retinal progenitors through the regulation of pro-apoptotic gene expression via histone acetylation

Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous dystrophy of the retina and mutations in the nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) gene is one of causal factors of LCA. NMNAT1 is a nuclear enzyme essential for nicotinamide adenine dinucleotide (NAD) biosynthesis pathways, but the mechanisms underlying the LCA pathology and whether NMNAT1 has a role in normal retinal development remain unclear. Thus, we examined the roles of Nmnat1 in retinal development via short hairpin (sh)-RNA-mediated downregulation. Retinal explants expressing sh-Nmnat1 showed large numbers of apoptotic retinal progenitor cells in the inner half of the neuroblastic layer. Decreased intracellular NAD content was observed and the addition of NAD to the culture medium attenuated sh-Nmnat1-induced apoptosis. Of the nuclear Sirtuin (Sirt) family, the expression of sh-Sirt1 and sh-Sirt6 resulted in a phenotype similar to that of sh-Nmnat1. Sirt proteins are histone deacetylases and the expression of sh-Nmnat1 increased the levels of acetylated histones H3 and H4 in the retina. Expression of sh-Nmnat1 resulted in significantly increased expression of Noxa and Fas, two pro-apoptotic genes. Acetylation of the genomic 5′-untranslated regions of Noxa and Fas loci was upregulated by sh-Nmnat1 expression. The co-expression of sh-Fas with sh-Nmnat1 reduced the number of apoptotic cells induced by sh-Nmnat1 expression alone. Taken together, our data suggested that the increased expression of Noxa and Fas explains, at least in part, the phenotype associated with sh-Nmnat1 in the retina. Taken together, these findings demonstrate the importance of the NAD biosynthesis pathway in normal development of the retina.

Author Correction: Analysis of Müller glia specific genes and their histone modification using Hes1-promoter driven EGFP expressing mouse

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Molecular mechanisms of H3K27me3 and H3K4me3 in retinal development

The retina consists of six types of neuron and Müller glia, and they are individually derived from common retinal progenitors in a chronologically defined order. Therefore, the signaling environment and competency of retinal progenitors change during retinal development, and the retina serves as an excellent model system to analyze molecular events during development. Much attention has been given to the identification of transcription factors and epigenetic mechanisms. The dynamic changing of the histone modification levels of retina-specific genes has been observed, and the modification patterns of H3K4me3 and H3K27me3 are regulated in a retinal cell type-specific manner. Therefore, it appears that the dynamism of histone modification in the developing retina is regulated both chronologically and in a cell type-specific manner in a particular gene category. Loss- and gain-of-function analyses of enzymes involved in the methylation and demethylation of H3K4 and K27 in the retina have indicated their critical roles in proliferation, differentiation, and determinations of the timing for differentiation. We summarize recent findings related to the roles of H3K4me3 and H3K27me3 in retinal development to discuss how the retinal system provides intriguing data on and contributes to concepts regarding the roles of histone modification in the chronological regulation of tissue development.