The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-β regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-β, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.

Transforming growth factor β (TGFβ) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFβ receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFβ signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFβ signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2ΔOC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFβ signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2ΔOC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFβ signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.