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

Abstract

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.

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

“…The differentiation state-specific expression patterns of these genes suggests that specific regulatory mechanisms operate to govern their expression levels during lens cell differentiation. Consistently, previous studies have identified multiple transcription factors [ 25 , 28 , 29 , 41 , 44 – 46 ] required for their differentiation state-specific expression patterns. Recently, epigenetic programming was shown to play an important role in the regulation of gene expression during lens fiber cell differentiation since ATAC (assay for transposase-accessible chromatin) sequencing of undifferentiated lens epithelial and differentiated fiber cells identified genome-wide changes in chromatin accessibility associated with the expression of multiple genes during lens differentiation [ 32 , 47 ].…”

“…7 A, Additional file 13 : Table S9A). Many of the enriched binding motifs are from transcription factors with well-established roles in regulation of lens gene expression, lens development, and differentiation (Additional file 13 : Table S9) [ 44 , 64 , 67 – 89 ]. Consistently, previous studies showed HIF1 to be a major regulator of lens gene expression [ 44 ], RBPJ is involved in early lens development [ 64 , 87 ], and both c-Myc and n-Myc have established roles in lens cell proliferation and differentiation [ 70 , 86 ].…”

“…Many of the enriched binding motifs are from transcription factors with well-established roles in regulation of lens gene expression, lens development, and differentiation (Additional file 13 : Table S9) [ 44 , 64 , 67 – 89 ]. Consistently, previous studies showed HIF1 to be a major regulator of lens gene expression [ 44 ], RBPJ is involved in early lens development [ 64 , 87 ], and both c-Myc and n-Myc have established roles in lens cell proliferation and differentiation [ 70 , 86 ]. Conversely, regions of DNA that exhibited decreased methylation in fiber cells and are contained within putative promoters of genes preferentially expressed in fiber cells contained, among other binding sequences, the consensus site for transcription factor NFAT5, SOX2, and the MAF family of transcription factors (Fig.…”

“…Many of the highly expressed transcription factors identified in Fig. 7 and Additional file 13 : Table S9 (total FPKM > 12) such as HIF1A [ 44 , 92 ], HEY1 [ 88 ], STAT3 [ 69 ], SOX2 [ 82 ], and MAFB [ 76 , 77 , 79 , 80 ] have known functions in lens development and differentiation (Additional file 13 : Table S9). In contrast, the transcription factors with low expression levels (total FPKM < 5) such as MAFF, Arid5a, TEAD4, and HIC2 (Additional file 13 : Table S9) do not yet have any known functions in the regulation of lens structure, homeostasis, or gene expression.…”

“…Many of the identified genes exhibiting altered mCG DNA methylation changes in association with corresponding changes in chromatin accessibility and differentiation state-specific gene expression have established requirements for specialized lens functions including lens cell structure, homeostasis and cell signaling (Additional file 15 : Table S11) [ 41 , 44 , 56 , 62 – 64 , 75 , 82 , 92 , 94 – 119 , 119 – 184 ]. For instance, the lens crystallins, CRYBB2, CRYBB1, CRYBA4, CRYGN, CRYBB3, and CRYBA1 are all critical for lens fiber cell transparency.…”