Insights into Alu retrotransposons: mechanism of Alu transcriptome alteration in response to virus infection and novel effects on gene expression

Abstract

A large portion of the human genome is composed of repeated sequences, with Alu retrotransposons representing the most abundant repetitive elements. Alu sequences belong to the class of the Short Interspersed Nuclear Elements (SINEs) and depend on the Long Interspersed Nuclear Elements (LINEs) for their mobilization into the genome. The efficiency of Alu amplification during primate evolution suggests a positive driving force for their accumulation, bringing up to 1 million copies in the human genome. For unclear reasons, the majority of Alu sequences is repressed by tight epigenetic silencing, which is released in response to cell stresses such as virus infection and cancer progression. Adenovirus 5 (Ad5) is known to cause an increase of Alu transcription in HeLa, myelogenous leukemia and embryonic kidney cell lines, even though the virus factors that are responsible for this transcriptional enhancement have not been identified yet. Potential candidates could be represented by oncovirus proteins that induce a global remodeling of the host epigenetic landscape. For example, the Adenovirus early E1A protein interacts with the host tumor suppressor Rb, the lysine acetylase p300 and the p400 ATP-dependent chromatin remodeling complex, resulting in the induction of quiescent fibroblasts to enter the S-phase of the cell cycle. The exceptional success of Alu expansion and their retention even at the cost of a strong epigenetic silencing, which is released by virus infection, led us to investigate the molecular mechanism of Alus activation and their potential involvement in various cell processes. Firstly, genome-wide Alu profiling was performed in quiescent human primary fibroblasts infected with the Ad5 dl1500 mutant, which only expresses the oncovirus small E1A protein. A total of 1880 Polymerase III (Pol III) transcribed Alus were detected through high throughput RNA-sequencing (RNA-seq), revealing a 4-fold increase of the average Alu expression induced by small E1A. With the aim of identifying small E1A-host protein interactions that are crucial for the activation of Alu expression, the host proteins Rb, p300 and p400 were put under investigation. Alu expression profiling was performed in cells infected with small E1A mutants that are not capable to bind Rb, p300 or p400. RNA-seq and RT-qPCR data revealed that the small E1A-p400 binding mutant was the least efficient in activating Alu expression, whereas a milder effect was reported for small E1A-Rb and small E1A-p300 binding mutants. Moreover, ChIP-sequencing (ChIP-seq) analyses of dl1500 infected fibroblasts revealed an enrichment of H3K4me1 within the body of small E1A-induced Alus. Our data point toward the existence of H3K4me1 Alu loci that are recognized by p400 bound by small E1A, resulting in Alu transcription in response to virus infection. Secondly, two Alu sequences were stably overexpressed in one primary and one cancer cell line (human fibroblasts and HeLa cells, respectively) and differential gene expression in Alu-overexpressing cells was performed through RNA-seq data analysis. Among the two Alu- overexpressing fibroblast cell lines, 330 genes were detected as Differentially Expressed (DE), whereas only two genes were differentially expressed in HeLa cells. Interestingly, DE genes detected in Alu-overexpressing fibroblasts were significantly enriched in pathways belonging to cell cycle progression and mitotic entry. The promotion of cell cycle was also supported by a significantly higher percentage of cells in the S-phase compared to control samples as revealed by flow cytometry. The studies conducted in this work identify Ad5 small E1A as one of the virus factors that enhance Alu transcription, possibly through binding with the p400 complex. Interestingly, the overexpression of two Alu sequences in human fibroblasts leads to the stimulation of cell cycle progression, resulting in the same phenotype as previously observed in Ad5 infected fibroblasts. This brings us to speculate that the overexpression of Alu sequences during stress response to viral infection could be exploited by Ad5 to sustain cell proliferation.