Replication-dependent histone genes are actively transcribed in differentiating and aging retinal neurons

Abstract

In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as "replication-dependent." Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons.

Keywords: AA; CE; Chr; GCL; INL; ISH; NE; ONBL; ONL; PCR; RPE; amino acid; chromosome; cytoplasmic extract; development; expression; ganglion cell layer; histones; in situ hybridization; inner nuclear layer; isotypes; nuclear extract; outer neuroblastic layer; outer nuclear layer; polymerase chain reaction; qPCR; quantitative PCR; replication-dependent; retina; retinal pigment epithelium; transcription; variant nucleosome.

Figure 1.

Expression of replication-dependent histone genes by deep sequence analysis. Shown here are the heat maps representing the FPKM values converted to log₂. (A) Expression of genes with known expression kinetics in E16 and P0 CE. Expression of H1 isotypes (B), H2a isotypes (C), H2b isotypes (D), H3 isotypes (E), and H4 isotypes (F) in E16 and P0 CE. Shown in the box in the top right corner is the key representing the expression levels ranging from 12 (blue) to 0 (yellow).

Figure 2.

Validation of fractionation/quality of cDNA. Shown here are graphs representing expression values determined by qPCR analysis, with the x-axis showing different time points at which the retinae were harvested. The y-axis is in log scale showing gene expression levels normalized to Gapdh values. Here the error bars represent the standard error of the mean. Dashed lines indicate expression levels of the genes tested in the nuclear fraction, while the solid lines represent expression in the cytoplasmic fraction. (A) Normalized expression of Xist (red) and Malat1 (black) across all time points and fractions. (B) Normalized expression of Rho (red) and Fgf15 (black) across all time points and fractions.

Figure 3.

Histone mRNA expression across retinal development and fractions as determined by qPCR analysis. Shown here are graphs representing expression values determined by qPCR analysis, with the x-axis showing different time points at which the retinae were harvested. The y-axis is in log scale showing gene expression levels normalized to Gapdh values. Here the error bars represent the standard error of the mean. Dashed lines indicate expression levels of the genes tested in the nuclear fraction, while the solid lines represent expression in the cytoplasmic fraction. (A) Normalized expression of 4 H1 histone genes, including Hist1h1a (blue), Hist1h1b (black), Hist1h1e (green), and Hist1h1c (red). (B) Normalized expression of 3 H2a histone genes, including Hist3h2a (red), Hist1h2ac (blue), and Hist1h2ab (black). (C) Normalized expression of 3 H2b histone genes, including Hist2h2bb (red), Hist3h2ba (blue), and Hist2h2be (black). (D) Normalized expression of H3 histone genes, including all H3 histone genes (red), Hist1h3e and Hist1h3f (black), and Hist2h3c2 (blue). (E) Normalized expression of H4 histone genes, including Hist1h4d (red), Hist4h4 (black), and Hist1h4a (blue). (F) Additional validation of cDNA preparation across retinal development interrogated by the expression of Nr2e3 (red), Nrl (black), and Pax6 (green).

Figure 4.

Spatiotemporal expression analysis by in situ hybridization for all H3 histone mRNA across retinal development. In situ hybridization with DIG-labeled anti-sense RNA probe detecting all H3 histone mRNA across retinal development from E14 to P14. (A) All H3 histone mRNA in situ signal in E14 section including the brain and retina. (A′) and (A″) are magnified images of the boxes around the retina in (A) and (A′), respectively, and (A″′) is a magnified image of the box around the brain in (A). (B) All H3 histone mRNA in situ signal in E16 section including the brain and retina. (B′) and (B″) are magnified images of the boxes around the retina and brain (B), respectively. (C) All H3 histone mRNA in situ signal in E18 retinal section. (C′), (C″), and (C′″) are magnified images of the boxes in the central retina in (C), the peripheral retina in (C), and the peripheral retina in (C″), respectively. (D) All H3 histone mRNA in situ signal for E18 brain section. (D′) is a magnified image of the box in (D). (E) All H3 histone mRNA in situ signal in P0 retinal section. (E′) and (E″) are magnified images of the boxes in the peripheral and central retina in (E), respectively. (F) All H3 histone mRNA in situ signal in P3 retinal section. (F′) and (F″) are magnified images of the boxes around the peripheral and central retina in (F), respectively. (G) All H3 histone mRNA in situ signal in P7 retinal section. (G′) and (G″) are magnified images of the boxes around the peripheral and central retina in (G), respectively. (H) All H3 histone mRNA in situ signal in P14 retinal section.

Figure 5.

Spatiotemporal expression analysis by in situ hybridization for Hist1h1c across retinal development. In situ hybridization with DIG-labeled anti-sense RNA probe detecting Hist1h1c across retinal development from E14 to P14. (A) Hist1h1c in situ signal in E14 section including the brain and retina. (A′), (A″), and (A’″) are magnified images of the boxes around the retina in (A), the retina in (A′), and the brain in (A), respectively. (B) Hist1h1c in situ signal in E16 section including brain and retina. (B′), (B″), and (B′″) are magnified images of the boxes around the retina in (B), the peripheral retina in (B′), and the brain in (B), respectively. (C) Hist1h1c in situ signal in E18 section including brain and retina*. (C′) and (C″) are magnified images of the boxes around the retina in (C) and the peripheral retina in (C′), respectively. (C″′) Hist1h1c in situ signal in the brain at E18. (D) Hist1h1c in situ signal in P0 retinal section. (D′) and (D″) are magnified images of the central and peripheral retina from (D), respectively. (E) Hist1h1c in situ signal in P3 retinal section. (E′) and (E″) are magnified images of the central and peripheral retina from (E), respectively. (F) Hist1h1c in situ signal in P7 retinal section. (F′) and (F″) are magnified images of the central and peripheral retina from (F), respectively. (G) Hist1h1c in situ signal in P14 retinal section.

Figure 6.

Cell type-specific expression of histone genes by single-cell microarray analysis. Shown here is a heat map (blue = high, yellow = low) reflecting expression of different histone genes within specific cells at different time points (embryonic and postnatal). GC = ganglion cell, AC = amacrine cell, BP = bipolar cell, MG = Müller glia.

Figure 7.

Expression of replication-dependent histone genes in the aging retina. Shown here is the heat map reflecting expression values (blue = high, yellow = low) of histone transcripts as observed by microarray analysis. Genes with known expression kinetics in the retina were used to contextualize the expression of the histone genes and are shown in red.

Figure 8.

Cyclin and histone transcription in rapamycin-treated retinae. (A) Validation of rapamycin treatment shown by immunoblot analysis, using rabbit α-pS6_240/244 (1:1000; Cell Signaling) antibody. Total S6 detected by rabbit α-S6 (1:1000; Cell Signaling) antibody used as the loading control. (B) Shown are normalized fold expression changes as detected by qPCR analysis in untreated (ctrl) and rapamycin-treated (rap) for Ccnd1, Ccne1, Ccne2, and Npat. Normalized fold expression of the various histone genes in ctrl and rap detected by qPCR for Hist1h1a, Hist1h1c (C); Hist1h2ab, Hist3h2a (D); Hist2h2be, Hist3h2ba (E); Hist2h3c2, all H3s (F); and Hist1h4a and Hist4h4 (G). All qPCR values for ctrl (n = 3) and rap (n = 4) were first normalized to the geometric mean of Gapdh values, followed by averaging and 2-tailed t-test, which showed no statistical significance for all values shown. (H) Gapdh, Ccnd1, Ccne1, and Ccne2 expression levels detected in the microarray analysis of the aging retina.

Figure 9.

Variant nucleosome model. Shown here is the schematic of DNA wrapped around the core octamer and connected to the linker histone. Shown here are pie charts reflecting the total mRNA contributions (qPCR data) of the different histone isotypes to the total RNA of that histone family across retinal development. (A) Relative contributions of Hist1h1a (blue), Hist1h1b (red), Hist1h1e (green), and Hist1h1c (purple) to the production of the linker histone H1 across retinal development. (B) Relative contributions of Hist1h2ab (green), Hist3h2a (blue), and Hist1h2ac (red) to the production of the core histone H2a across retinal development. (C) Relative contributions of Hist3h2ba (red), Hist2h2bb (blue), and Hist2h2be (green) to the production of the core histone H2b across retinal development.