(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



The LHX2-OTX2 transcriptional regulatory module controls retinal pigmented epithelium differentiation and underlies genetic risk for age-related macular degeneration [1]

['Mazal Cohen-Gulkar', 'Department Of Human Molecular Genetics', 'Biochemistry', 'Sackler Faculty Of Medicine', 'Sagol School Of Neurosciences', 'Tel Aviv University', 'Tel Aviv', 'Ahuvit David', 'Naama Messika-Gold', 'Mai Eshel']

Date: 2023-01

Tissue - specific transcription factors (TFs) control the transcriptome through an association with noncoding regulatory regions (cistromes). Identifying the combination of TFs that dictate specific cell fate, their specific cistromes and examining their involvement in complex human traits remain a major challenge. Here, we focus on the retinal pigmented epithelium (RPE), an essential lineage for retinal development and function and the primary tissue affected in age-related macular degeneration (AMD), a leading cause of blindness. By combining mechanistic findings in stem-cell-derived human RPE, in vivo functional studies in mice and global transcriptomic and proteomic analyses, we revealed that the key developmental TFs LHX2 and OTX2 function together in transcriptional module containing LDB1 and SWI/SNF (BAF) to regulate the RPE transcriptome. Importantly, the intersection between the identified LHX2-OTX2 cistrome with published expression quantitative trait loci, ATAC-seq data from human RPE, and AMD genome-wide association study (GWAS) data, followed by functional validation using a reporter assay, revealed a causal genetic variant that affects AMD risk by altering TRPM1 expression in the RPE through modulation of LHX2 transcriptional activity on its promoter. Taken together, the reported cistrome of LHX2 and OTX2, the identified downstream genes and interacting co-factors reveal the RPE transcription module and uncover a causal regulatory risk single-nucleotide polymorphism (SNP) in the multifactorial common blinding disease AMD.

Funding: RA-P laboratory is supported by grants from the Israel Science Foundation (1128/20), Binational Science Foundation (2013016) and European Union COST program under COST Action CA-18116, ANIRIDIA-NET supported (in part) by grant no. 317652 from the Chief Scientist Office of the Ministry of Health, Israel and the Cancer Biology Research Center, Tel Aviv University. RA-P and RE are supported by the Israel Ministry of Science (3-17557). MC’s PhD scholarship is supported by the Claire and Amedee Maratier Institute for the Study of Blindness and Visual Disorders, Sackler Faculty of Medicine, Tel Aviv University, Israel. R.E. is a Faculty Fellow of the Edmond J. Safra Center for Bioinformatics at Tel Aviv University. ME was supported in part by a fellowship from the Edmond J. Safra Center for Bioinformatics at Tel Aviv University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The RNA-seq and ChIP-seq datasets generated in this study are available from the GEO, under accession number GSE178166. All other relevant data are within the paper and its Supporting Information files.

Copyright: © 2023 Cohen-Gulkar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Another important early eye determinant is the Lim HD TF Lhx2, the homolog of the fly Apterous (ap) gene. Lhx2 is required for optic vesicle formation [ 20 ], a function that has been attributed to both its regulation of the PE and retinal TFs, Mitf and Vsx2, and its role in the patterning of the neural primordium by restricting the fate of midline structures [ 21 – 24 ]. In the developing, retina Lhx2, together with its obligatory co-factors the LIM-domain-binding proteins LDB1/2 (dLDB/Chip in the fly), function in regulating of retinal progenitor proliferation and competence [ 25 – 28 ]. The role of Lhx2 in RPE differentiation in the specified optic cup progenitors has not yet been so far directly addressed. Nevertheless, LHX2’s importance in human RPE differentiation has been implicated by its identification among 9 RPE core genes, which include OTX2 and MITF, which bear the potential to induce, when mis-expressed, the direct transdifferentiation of human fibroblasts into human RPE-like cells [ 29 ]. Currently, however, it is not known how these factors function together in RPE differentiation, including the composition of the transcriptional complexes, the associated cis-regulatory sites, the involvement of epigenetic remodelers, and the relevance to retinal disease mechanisms. Here, we studied Lhx2/LHX2 in developing mouse and human RPE generated from stem cells (hES-RPE). Through functional and genomic analyses, we reveal that LHX2 functions upstream and together with OTX2 on shared genomic regions (cistrome) in regulation of target genes. Through this feed-forward regulatory module LHX2-OTX2 control the RPE transcriptional program. Proteomic analyses further exposed co-factors of LHX2 and OTX2 that likely mediate the tissue-specific gene regulation, including LDB1 and the SWI/SNF chromatin remodeling complex. Finally, intersecting the map of the LHX2-OTX2 bound cistrome with published genomic data on AMD revealed a causal noncoding risk-SNP that acts by altering TRPM1 expression in the RPE through the modulation of LHX2 binding to its promoter. The study exemplifies how delineation of tissue-specific transcriptional regulators, their cistromes, and downstream gene-regulatory networks can provide insights into a complex disease’s pathology.

Several TFs have been reported to play a role in RPE differentiation and maintenance based on phenotypic analyses of mutant mice. Among these, Otx2 the paired-type homeodomain (HD) TF, is essential for patterning of anterior neural plate and subsequent formation of forebrain, midbrain, and early eye primordia, and is also required later in development for RPE differentiation [ 8 – 12 ]. Otx2 is also maintained in the adult RPE, where it is required for the expression of multiple RPE genes [ 13 – 16 ]. Activity of Otx2 in RPE is attributed to its regulation of and interaction with the microphthalmia-associated TF (Mitf), a basic domain helix–loop–helix leucine zipper (bHLH-LZ) TF that is required for the specification and differentiation of melanocytes and ocular pigmented cells [ 15 – 19 ].

The RPE originates from the bipotential neuroectoderm of the optic vesicle. Following specification and morphogenesis, the neuroectoderm cells turn into the bilayer optic cup, populated by an outer layer of pigmented epithelium (PE) progenitors and an inner layer of retinal progenitor cells. The RPE gradually acquires its cell-specific phenotype during embryogenesis and postnatal stages, which include the formation of a selective retina–blood barrier, protection from light-induced oxidative toxicity through the pigment granules, expression of the visual-cycle genes required for the recycling of retina cells, and renewal of the outer segment by circadian phagocytosis of the shed discs (reviewed in [ 5 – 7 ]).

The development of AMD involves interactions between several tissue types, with primary roles attributed to cells of the retinal pigmented epithelium (RPE), a monolayered barrier of polarized, pigmented epithelia located between the choriocapillaris and the photoreceptors, which is vital for the development, health, survival, and function of the retinal photoreceptors and the choroid [ 3 , 4 ]. In AMD, RPE dysfunction results in progressive accumulation of deposits, termed drusen, on the basal membrane of the RPE, which ultimately leads to hypoxia and choroidal neovascularization and/or progressive loss of RPE and photoreceptor cells. The acquisition and maintenance of tissue-specific gene expression, including that of the RPE, is accomplished through the combined activity of DNA-binding transcription factors (TFs) and epigenetic factors that together restrict the transcriptional activity to approximately 2% of the genome that carries enhancer function. Mapping tissue-specific cis-regulatory elements (CREs) of the RPE and the TFs responsible for these regulatory elements’ selection is fundamental for understanding the mechanisms governing acquisition and maintenance of cell-specific transcriptional programs and for resolving the contribution of the CREs to complex diseases, such as AMD.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual impairment in older people, accounting for approximately 50% of legal blindness in western countries. Susceptibility to AMD depends on a combination of genetic components and environmental factors [ 1 , 2 ]. Several genome-wide association studies (GWASs) have been applied to AMD, resulting in the identification of approximately 50 loci that are significantly associated with increased risk for this disease. These AMD-associated loci contain over 1,000 candidate risk single-nucleotide polymorphisms (SNPs). For most of these AMD loci, the causal variants, their mode of action, and the affected target genes are unknown. This is primarily due to our limited understanding of the functional significance of noncoding genomic variants. Deciphering the functional significance of genomic variations in complex diseases, such as AMD, therefore requires identification and study of the regulatory regions that control the differentiation and maintenance of the lineages involved in disease pathology.

Results

Identification and characterization of LHX2-bound enhancers and target genes in human RPE The genes whose expression in hES-RPE or Lhx2-PE-cKO mouse RPE is altered following LHX2 KD may be direct targets of LHX2 or indirect targets affected by developmental regulators, which are themselves direct targets of LHX2. To identify the direct targets of LHX2 and delineate its gene-regulatory network in the RPE, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) using an LHX2 antibody, to systematically map the genomic regions bound by endogenous LHX2 in d14 hES-RPE cells. This ChIP-seq analysis identified 1,278 peaks, which were mapped to 984 genes based on their distance to the nearest transcription start site (TSS) (S4 Table). As expected, de novo motif analysis using Homer [40] showed that the detected peaks were highly enriched for the known LHX2-binding motif (84% of the LHX2 peaks contained an LHX2-binding motif, p = 1 × 10−174; Fig 2J). Notably, the second top-scoring motif was the OTX2-binding motif, detected in 73% of the LHX2 peaks (p = 1 × 10−76; Fig 2J, see below), indicating potential functional cooperation between these 2 TFs in regulation of the RPE transcriptome. We next integrated the LHX2-KD RNA-seq and LHX2 ChIP-seq datasets by linking each peak to its nearest gene’s TSS. Of the 984 genes mapped to the LHX2 peaks, 741 were detected in the differentiated hES-RPE cells based on the RNA-seq. Notably, as a group, the expression level of these 741 putative LHX2 target genes was significantly down-regulated upon LHX2 KD (Wilcoxon’s test p = 2.5 × 10−85; Fig 2K). The putative direct targets of LHX2 that were significantly down-regulated upon LHX2 KD in differentiated hES-RPE cells included genes associated with important functional categories for RPE fate, morphology, and physiology, including cell adhesion, ECM molecules, and ion channels (cadherin: CDH1, PCDH7; collagens: COL8A1, COL4A3; integrins: ITGB8; laminins: LAMA2 and ion channels TRPM1/3), and key transcriptional regulators (MITF, OTX2, SOX5, SOX8; S2A Table). These results strongly indicate that LHX2 is a direct regulator of multiple RPE genes that are required for different aspects of cell fate, morphology, and function.

The LHX2 and OTX2 protein co-factors in hES-RPE The numerous genomic regions bound by both LHX2 and OTX2 and reduced expression of the genes associated with these regions (based on proximity) suggest that these factors function together on CREs to regulate gene expression in the RPE. To identify factors interacting with LHX2 and OTX2, we immuno-purified LHX2 or OTX2 from nuclear extracts prepared from hES-RPE (d14) and identified significantly associated polypeptides compared to IgG control using mass spectrometry (IP-MS). In both immunoprecipitates, we detected strong enrichment of the respective proteins corresponding to the antibodies used for the immuno-purification, supporting the specificity of the antibodies (Fig 5A). Moreover, among the proteins interacting with OTX2 was MITF, which was previously reported to interact with OTX2 using pull-down assays in tissue cultures [16,18]. However, we did not detect LHX2 peptides in the OTX2 immunoprecipitate or vice versa. This could be due to low stability of the protein complex and/or to the indirect nature of the interaction between these proteins which might depend on additional co-factors. Interestingly, in both immunoprecipitates, we detected LDB1/2 as well as SSBP2 (single-strand-binding protein 2)—a known co-factor of LDB1 complexes [44,45], detected in the 3 replicates of LHX2 and in 2 of the 3 OTX2 immuno-purified samples (Fig 5A, S9 Table). We further validated the interaction between OTX2 and LDB1 by co-immunoprecipitation of Flag-LDB1 from 293T cells overexpressing OTX2 or LDB1 alone or together, or in combination with LHX2, followed by western blot analysis with OTX2 antibodies (Fig 5B). The detection of OTX2 in the Flag-LDB1 immunoprecipitate, with or without LHX2, further supports a direct interaction of human LDB1/2 with OTX2 in hES-RPE. From these results, we infer that LDB1 is directly associated with OTX2 in hES-RPE and that this interaction does not require, and is not inhibited by, LHX2. These findings suggest that LDB1, which is known to interact with LHX2, may mediate the interaction between OTX2 and LHX2, at least on some of the co-bound CREs in the hES-RPE. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. The LHX2 and OTX2 co-factors in human RPE. (A) Table summarizing the statistical analysis (t test, two-tailed, paired) and the Log2 fold change in proteins bound by LHX2, OTX2, and BRG1 compared to proteins bound by nonspecific IgG based on immunoprecipitation followed by mass spectrometry (IP-MS) analyses. (B) 293T cells overexpressing OTX2, LDB1, LHX2 in the indicated combinations (Input), were subjected to IP with Flag antibodies followed by western blot analyses and immunolabeling with OTX2 antibody, the arrow points to OTX2. S1 Raw image.). (C) Illustration (using Biorander) of the identified co-factors and key downstream targets in hES-RPE. IP, immunoprecipitation; Mitf, microphthalmia-associated TF; RPE, retinal pigmented epithelium; TF, transcription factor; TSS, transcription start site. https://doi.org/10.1371/journal.pbio.3001924.g005 In addition to association with LDB1, we detected several SWI/SNF complex subunits in the OTX2 immunoprecipitates, including BRG1/BRM (SMARCA4/2), BAF155/170 (SMARCC1/2), BAF57 (SMARCE1), BAF60a, 60b (SMARCD1/2), and ARID1A/B. Reciprocally, we detected OTX2 in the BRG1 pulldown, which further supports the physical association between BRG1 and OTX2 (Fig 5B, S9 Table). Overall, the results from the IP-MS proteomic analyses suggest that the interaction between LHX2 and OTX2 is mediated through LDB1/2 (Fig 5C). The detection of direct interactions of OTX2 with the BAF complexes suggests a chromatin-remodeling activity for OTX2 and its bound co-factors, allowing robust transcriptional activation of multiple tissue-specific genes (scheme of proposed transcriptional module and co-factors, Fig 5C).

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001924

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.