Changes in relative histone abundance and heterochromatin in αA-crystallin and αB-crystallin knock-in mutant mouse lenses

Abstract

Objective: Understanding the mechanisms of cataract formation is important for age-related and hereditary cataracts caused by mutations in lens protein genes. Lens proteins of the crystallin gene families α-, β-, and γ-crystallin are the most abundant proteins in the lens. Single point mutations in crystallin genes cause autosomal dominant cataracts in multigenerational families. Our previous proteomic and RNAseq studies identified genes and proteins altered in the early stages of cataract formation in mouse models. Histones H2A, H2B, and H4 increase in abundance in αA- and αB-crystallin mutant mouse lenses and in cultured cells expressing the mutant form of αA-crystallin linked with hereditary cataracts.

Results: In this study of histones in mutant lenses, we extracted histones from adult mouse lenses from cryaa-R49C and cryab-R120G mutant knock-in mice. We characterized the histones using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)-mass spectrometric analysis and gel electrophoresis and characterized the lens nucleus morphology using electron microscopy (EM). The relative abundance of histone H3 protein decreased in lenses from cryaa-R49C mutant mice and the relative abundance of histone H2 increased in these lenses. Electron microscopy of nuclei from cryaa-R49C-homozygous mutant mouse lenses revealed a pronounced alteration in the distribution of heterochromatin.

Keywords: Cataract; Crystallin; Electron microscopy; Histones; Mass spec; Mouse lens.

Fig. 1

MALDI-TOF MS analysis of histones isolated from adult mouse lenses. WT, wild type. The histone peaks were identified based on their mass to charge (m/z) ratios. The m/z peaks on the left side of the x-axis represent doubly-charged ions for histones H4 [H4/2]²⁺, H2 [H2/2]²⁺, and H3 [H3/2]²⁺, respectively. MALDI spectra usually deliver single charged ions, but can also produce doubly-charged ions, especially for proteins

Fig. 2

MALDI-TOF MS analysis of histones isolated from cryaa-R49C-homo mouse lenses (related to Fig. 1). The insets show the spectra of the H2, H3, and H4 histones in detail

Fig. 3

Electron micrographs of mouse lens epithelial nuclei. Wild type, cryaa-R49C het, and cryaa-R49C homo lenses were analyzed. Cells in the cryaa-R49C–het lenses appeared stressed, with swollen mitochondria, vacuoles and lacunae in these lenses. Cells in the cryaa-R49C-homo lenses had heterochromatin localized to the nuclear periphery