Multiomic analysis implicates FOXO4 in genetic regulation of chick lens fiber cell differentiation.

A classic model for identification of novel differentiation mechanisms and pathways is the eye lens that consists of a monolayer of quiescent epithelial cells that are the progenitors of a core of mature fully differentiated fiber cells. The differentiation of lens epithelial cells into fiber cells follows a coordinated program involving cell cycle exit, expression of key structural proteins and the hallmark elimination of organelles to achieve transparency. Although multiple mechanisms and pathways have been identified to play key roles in lens differentiation, the entirety of mechanisms governing lens differentiation remain to be discovered. A previous study established that specific chromatin accessibility changes were directly associated with the expression of essential lens fiber cell genes, suggesting that the activity of transcription factors needed for expression of these genes could be regulated through binding access to the identified chromatin regions. Sequence analysis of the identified chromatin accessible regions revealed enhanced representation of the binding sequence for the transcription factor FOXO4 suggesting a direct role for FOXO4 in expression of these genes. FOXO4 is known to regulate a variety of cellular processes including cellular response to metabolic and oxidative stress, cell cycle withdrawal, and homeostasis, suggesting a previously unidentified role for FOXO4 in the regulation of lens cell differentiation. To further evaluate the role of FOXO4 we employed a multiomics approach to analyze the relationship between genome-wide FOXO4 binding, the differentiation-specific expression of key genes, and chromatin accessibility. To better identify active promoters and enhancers we also examined histone modification through analysis of H3K27ac. Specific methods included CUT&RUN (FOXO4 binding and H3K27ac modification), RNA-seq (differentiation state specific gene expression), and ATAC-seq (chromatin accessibility). CUT&RUN identified 20,966 FOXO4 binding sites and 33,921 H3K27ac marked regions across the lens fiber cell genome. RNA-seq identified 956 genes with significantly greater expression levels in fiber cells compared to epithelial cells (log2FC > 0.7, q < 0.05) and 2548 genes with significantly lower expression levels (log2FC < -0.7, q < 0.05). Integrated analysis identified 1727 differentiation-state specific genes that were nearest neighbors to at least one FOXO4 binding site, including genes encoding lens gap junctions (GJA1, GJA3), lens structural proteins (BFSP1, CRYBB1, ASL1), and genes required for lens transparency (HSF4, NRCAM). Multiomics analysis comparing the identified FOXO4 binding sites in published ATAC-seq data revealed that chromatin accessibility was associated with FOXO4-dependent gene expression during lens differentiation. The results provide evidence for an important requirement for FOXO4 in the regulated expression of key genes required for lens differentiation and link epigenetic regulation of chromatin accessibility and H3K27ac histone modification with the function of FOXO4 in controlling lens gene expression during lens fiber cell differentiation.

Multiomics Analysis Reveals Novel Genetic Determinants for Lens Differentiation, Structure, and Transparency.

Recent advances in next-generation sequencing and data analysis have provided new gateways for identification of novel genome-wide genetic determinants governing tissue development and disease. These advances have revolutionized our understanding of cellular differentiation, homeostasis, and specialized function in multiple tissues. Bioinformatic and functional analysis of these genetic determinants and the pathways they regulate have provided a novel basis for the design of functional experiments to answer a wide range of long-sought biological questions. A well-characterized model for the application of these emerging technologies is the development and differentiation of the ocular lens and how individual pathways regulate lens morphogenesis, gene expression, transparency, and refraction. Recent applications of next-generation sequencing analysis on well-characterized chicken and mouse lens differentiation models using a variety of omics techniques including RNA-seq, ATAC-seq, whole-genome bisulfite sequencing (WGBS), chip-seq, and CUT&RUN have revealed a wide range of essential biological pathways and chromatin features governing lens structure and function. Multiomics integration of these data has established new gene functions and cellular processes essential for lens formation, homeostasis, and transparency including the identification of novel transcription control pathways, autophagy remodeling pathways, and signal transduction pathways, among others. This review summarizes recent omics technologies applied to the lens, methods for integrating multiomics data, and how these recent technologies have advanced our understanding ocular biology and function. The approach and analysis are relevant to identifying the features and functional requirements of more complex tissues and disease states.

Patterns of Crystallin Gene Expression in Differentiation State Specific Regions of the Embryonic Chicken Lens.

Purpose: Transition from lens epithelial cells to lens fiber cell is accompanied by numerous changes in gene expression critical for lens transparency. We identify expression patterns of highly prevalent genes including ubiquitous and enzyme crystallins in the embryonic day 13 chicken lens. Methods: Embryonic day 13 chicken lenses were dissected into central epithelial cell (EC), equatorial epithelial cell (EQ), cortical fiber cell (FP), and nuclear fiber cell (FC) compartments. Total RNA was prepared, subjected to high-throughput unidirectional mRNA sequencing, analyzed, mapped to the chicken genome, and functionally grouped. Results: A total of 77,097 gene-specific transcripts covering 17,450 genes were expressed, of which 10,345 differed between two or more lens subregions. Ubiquitous crystallin gene expression increased from EC to EQ and was similar in FP and FC. Highly expressed crystallin genes fell into three coordinately expressed groups with R2 ≥ 0.93: CRYAA, CRYBB2, CRYAB, and CRYBA2; CRYBB1, CRYBA4, CRYGN, ASL1, and ASL; and CRYBB3 and CRYBA1. The highly expressed transcription factors YBX1, YBX3, PNRC1, and BASP1 were coordinately expressed with the second group of crystallins (r2 > 0.88). Conclusions: Although it is well known that lens crystallin gene expression changes during the epithelial to fiber cell transition, these data identify for the first time three distinct patterns of expression for specific subsets of crystallin genes, each highly correlated with expression of specific transcription factors. The results provide a quantitative basis for designing functional experiments pinpointing the mechanisms governing the landscape of crystallin expression during fiber cell differentiation to attain lens transparency.

Changes in DNA methylation hallmark alterations in chromatin accessibility and gene expression for eye lens differentiation.

BACKGROUND: Methylation at cytosines (mCG) is a well-known regulator of gene expression, but its requirements for cellular differentiation have yet to be fully elucidated. A well-studied cellular differentiation model system is the eye lens, consisting of a single anterior layer of epithelial cells that migrate laterally and differentiate into a core of fiber cells. Here, we explore the genome-wide relationships between mCG methylation, chromatin accessibility and gene expression during differentiation of eye lens epithelial cells into fiber cells. RESULTS: Whole genome bisulfite sequencing identified 7621 genomic loci exhibiting significant differences in mCG levels between lens epithelial and fiber cells. Changes in mCG levels were inversely correlated with the differentiation state-specific expression of 1285 genes preferentially expressed in either lens fiber or lens epithelial cells (Pearson correlation r = - 0.37, p < 1 × 10-42). mCG levels were inversely correlated with chromatin accessibility determined by assay for transposase-accessible sequencing (ATAC-seq) (Pearson correlation r = - 0.86, p < 1 × 10-300). Many of the genes exhibiting altered regions of DNA methylation, chromatin accessibility and gene expression levels in fiber cells relative to epithelial cells are associated with lens fiber cell structure, homeostasis and transparency. These include lens crystallins (CRYBA4, CRYBB1, CRYGN, CRYBB2), lens beaded filament proteins (BFSP1, BFSP2), transcription factors (HSF4, SOX2, HIF1A), and Notch signaling pathway members (NOTCH1, NOTCH2, HEY1, HES5). Analysis of regions exhibiting cell-type specific alterations in DNA methylation revealed an overrepresentation of consensus sequences of multiple transcription factors known to play key roles in lens cell differentiation including HIF1A, SOX2, and the MAF family of transcription factors. CONCLUSIONS: Collectively, these results link DNA methylation with control of chromatin accessibility and gene expression changes required for eye lens differentiation. The results also point to a role for DNA methylation in the regulation of transcription factors previously identified to be important for lens cell differentiation.

Mechanisms of organelle elimination for lens development and differentiation.

A hallmark feature of lens development and differentiation is the complete elimination of organelles from the center of the eye lens. A long unanswered question in lens biology is what are the mechanisms that control the elimination of organelles during the terminal remodeling program to form mature lens fiber cells? Recent advances have expanded our understanding of these mechanisms including newly discovered signaling pathways, proteasomal regulators, autophagy proteins, transcription factors and the hypoxic environment of the lens itself. These recent discoveries suggest that distinct mechanisms coordinate the elimination of the nucleus, mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell differentiation. Since regulation of organelle number and distribution is also a feature of the terminal remodeling programs of more complex cell-types and tissues, these advances are likely to impact a wide-variety of fields.

A functional map of genomic HIF1α-DNA complexes in the eye lens revealed through multiomics analysis.

BACKGROUND: During eye lens development the embryonic vasculature regresses leaving the lens without a direct oxygen source. Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 (HIF1), in the regulation of genes required for lens fiber cell differentiation, structure and transparency. Here, we employed a multiomics approach combining CUT&RUN, RNA-seq and ATACseq analysis to establish the genomic complement of lens HIF1α binding sites, genes activated or repressed by HIF1α and the chromatin states of HIF1α-regulated genes. RESULTS: CUT&RUN analysis revealed 8375 HIF1α-DNA binding complexes in the chick lens genome. One thousand one hundred ninety HIF1α-DNA binding complexes were significantly clustered within chromatin accessible regions (χ2 test p < 1 × 10- 55) identified by ATACseq. Formation of the identified HIF1α-DNA complexes paralleled the activation or repression of 526 genes, 116 of which contained HIF1α binding sites within 10kB of the transcription start sites. Some of the identified HIF1α genes have previously established lens functions while others have novel functions never before examined in the lens. GO and pathway analysis of these genes implicate HIF1α in the control of a wide-variety of cellular pathways potentially critical for lens fiber cell formation, structure and function including glycolysis, cell cycle regulation, chromatin remodeling, Notch and Wnt signaling, differentiation, development, and transparency. CONCLUSIONS: These data establish the first functional map of genomic HIF1α-DNA complexes in the eye lens. They identify HIF1α as an important regulator of a wide-variety of genes previously shown to be critical for lens formation and function and they reveal a requirement for HIF1α in the regulation of a wide-variety of genes not yet examined for lens function. They support a requirement for HIF1α in lens fiber cell formation, structure and function and they provide a basis for understanding the potential roles and requirements for HIF1α in the development, structure and function of more complex tissues.

Hypoxia regulates the degradation of non-nuclear organelles during lens differentiation through activation of HIF1a.

Formation of the eye lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To attain their mature structure and transparent function, nascent lens fiber cells must complete a precise cellular remodeling program hallmarked by the complete elimination of organelles to form the core lens organelle-free zone (OFZ). Lacking a blood supply, the lens resides in a hypoxic environment that results in a decreasing oxygen concentration from the lens surface to the lens core. This oxygen gradient results in a hypoxic microenvironment in the region of the lens where immature lens fiber cells initiate loss of organelles to form the core OFZ. These features of the lens suggest a potential role for low lens oxygen levels in the regulation of organelle degradation and other events critical for mature lens fiber cell formation. Hypoxia activates the master regulator of the hypoxic response, hypoxia-inducible factor 1a (HIF1a) that regulates hypoxia-responsive genes. To identify a potential role for hypoxia and HIF1a in the elimination of organelles during lens fiber cell maturation, we tested the requirement for hypoxia in the degradation of non-nuclear organelles in ex vivo cultured embryonic chick lenses by monitoring the degradation of mitochondria (MT), Golgi apparatus (GA) and endoplasmic reticulum (ER) under conditions of low (1% O2) and high (21% O2) oxygen. We also examined the requirement for HIF1a activation for elimination of these organelles under the same conditions using a specific HIF1a activator (DMOG) and a specific HIF1a inhibitor (chetomin) and examined the requirements for hypoxia and HIF1a for regulating transcription of BNIP3L that we previously showed to be required for elimination of non-nuclear lens organelles. We used ChIP-qPCR to confirm direct binding of HIF1a to the 5' untranslated region of the BNIP3L gene. Finally, we examined the effects of expressing an oxygen insensitive mutant form of HIF1a (P402A/P565A) and BNIP3L on non-nuclear organelle degradation. Our data demonstrate that hypoxia and HIF1a are required for the degradation of non-nuclear organelles during lens fiber cell formation and that they regulate this process by governing BNIP3L transcription. Our results also provide evidence that hypoxia and HIF1a are essential for achieving mature lens structure.

Lens differentiation is characterized by stage-specific changes in chromatin accessibility correlating with differentiation state-specific gene expression.

Changes in chromatin accessibility regulate the expression of multiple genes by controlling transcription factor access to key gene regulatory sequences. Here, we sought to establish a potential function for altered chromatin accessibility in control of key gene expression events during lens cell differentiation by establishing genome-wide chromatin accessibility maps specific for four distinct stages of lens cell differentiation and correlating specific changes in chromatin accessibility with genome-wide changes in gene expression. ATAC sequencing was employed to generate chromatin accessibility profiles that were correlated with the expression profiles of over 10,000 lens genes obtained by high-throughput RNA sequencing at the same stages of lens cell differentiation. Approximately 90,000 regions of the lens genome exhibited distinct changes in chromatin accessibility at one or more stages of lens differentiation. Over 1000 genes exhibited high Pearson correlation coefficients (r â€‹> â€‹0.7) between altered expression levels at specific stages of lens cell differentiation and changes in chromatin accessibility in potential promoter (-7.5kbp/+2.5kbp of the transcriptional start site) and/or other potential cis-regulatory regions ( ±10 â€‹kb of the gene body). Analysis of these regions identified consensus binding sequences for multiple transcription factors including members of the TEAD, FOX, and NFAT families of transcription factors as well as HIF1a, RBPJ and IRF1. Functional mapping of genes with high correlations between altered chromatin accessibility and differentiation state-specific gene expression changes identified multiple families of proteins whose expression could be regulated through changes in chromatin accessibility including those governing lens structure (BFSP1,BFSP2), gene expression (Pax-6, Sox 2), translation (TDRD7), cell-cell communication (GJA1), autophagy (FYCO1), signal transduction (SMAD3, EPHA2), and lens transparency (CRYBB1, CRYBA4). These data provide a novel relationship between altered chromatin accessibility and lens differentiation and they identify a wide-variety of lens genes and functions that could be regulated through altered chromatin accessibility. The data also point to a large number of potential DNA regulatory sequences and transcription factors whose functional analysis is likely to provide insight into novel regulatory mechanisms governing the lens differentiation program.