Using TALENs to Knockout H2A.Lap1 Function in Mice

Abstract

The Tremethick laboratory in 2012 discovered a new mouse histone variant, which was designated H2A.Lap1 (Lack of an acidic patch), which, in the adult, is uniquely expressed in the testis and the brain. It was proposed that in the testis, H2A.Lap1 is involved in a new mechanism of gene regulation whereby H2A.Lap1 co-ordinately activates the expression of many genes by being targeted to and directly opening the chromatin region encompassing their transcription start site (TSS). More recently, this laboratory also identified H2A.Lap1 as a component of the intron-exon boundary of active genes expressed in the testis and the brain. These observations suggested a role in transcription and splicing but all the experiments to date have been correlative in nature. Therefore, the major aim of this thesis was to generate an in vivo mouse knockout model in order to directly test the importance of H2A.Lap1. However, the generation of a H2A.Lap1 knockout (KO) mouse was potentially a technically challenging feat because the H2A.Lap1 protein is expressed from three different genes (all located on the X chromosome). In this thesis, we report a simplified TALEN approach that achieved this goal whereby we used one pair of TALENs to simultaneously disrupt the three gene copies of H2A.Lap1. To our knowledge, this has not been done before. Importantly, bioinformatics analyses and wet-lab validation on exome sequencing data of our KO mice did not reveal any TALEN-induced off-target mutations, proving the reliability of this genome editing approach in generating a H2A.Lap1-specific KO model. Since H2A.Lap1 is predominantly expressed in the testis, we first explored whether are H2A.Lap1 KO mice are fertile. Our results showed that the loss of H2A.Lap1 caused a mild subfertility phenotype that manifested itself in smaller litter sizes when the hemizygous KO males (H2A.Lap1 -/Y) were mated with wild type females (H2A.Lap1 +/+). Given that these hemizygous KO mice are still able to reproduce despite the absence of H2A.Lap1 suggested that H2A.Lap1 is important but not essential for fertility. Alternatively though, H2A.Lap1 may be critical but compensatory epigenetic mechanisms may come into play in its absence. To test this hypothesis, I examined the immunofluorescence distribution of several active promoter-associated histone post translational modifications (PTMs) in germ cells, as well as investigating another H2A.Lap1 like histone variant, H2A.L.2 (expressed at latter spermatogenesis stages compared to H2A.Lap1) in H2A.Lap1 -/Y mice and their wt siblings. The immunostaining analyses showed that, in comparison to the wt mice, H3K4me3 become more enriched in the euchromatin of round spermatids of KO mice, implying a possible involvement of this PTM in a compensation mechanism. In addition, and most interestingly, H2A.L.2 changed its timing of expression being expressed earlier in the round spermatid stage thus also possibly compensating for the loss of H2A.Lap1. Another striking observation in this thesis is that, in the absence of H2A.Lap1, the nuclei of round spermatids become significantly compacted consistent with its in vitro ability to decompact chromatin. Further, the nuclei of round spermatids in KO males showed RNA Polymerase II (RNA Pol II) no longer occupies microscopically distinct transcription hubs. Significantly, RNA-Seq data revealed an increase in intron retention in the absence of H2A.Lap1. Therefore, despite possible compensatory mechanisms, clear changes to the functional organisation of the nucleus and splicing are observed. During meiosis, sex chromosome becomes highly condensed and silenced to ensure that there is no recombination between the X and Y chromosomes. This inactivation continues post-meiotically, which is referred to post meiotic sex chromosome (PMSC). However, as early round spermatids (ERS) develop into late round spermatids (LRS), approximately 20 % of X-chromosome genes required for spermiogenesis become activated. In the absence of H2A.Lap1, the expression of several X-linked genes (as well as examined autosomal genes) is significantly reduced. Another interesting observation arising from the immunostaining of active and repressive marks are the dynamic epigenetic changes between ERS and LRS, which has not been observed before. The repressive mark H3K9me3 becomes more enriched in LRS on PMSC which coincides with a depletion in the active marks H4K8Ac and Kcr. At euchromatin, we also observed other active marks (H3K36me3 and H3K4me3) become depleted in LRS in wt mice. This reduction of active marks as round spermatids differentiate suggests that these cells are preparing for a global transcription shut down, which occurs in subsequent developmental stages. Finally, we examined a possibly phagocytic role for H2A.Lap1 in Sertoli cells. Microscopic analysis of seminiferous tubules of the testes showed that the KO mice have an increased number of clogged tubules indicating an inefficient clearance of residual bodies (RBs) by Sertoli cells, which could also explain why H2A.Lap1 KO mice are subfertile. Show more