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Hypospermatogenesis in men: Investigation regarding HENMT1 expression and pathway function
thesisposted on 06.02.2017, 01:48 by Anna-Lena Hempfling
HENMT1 is a testis-specific methyltransferase which catalyses the addition of a 2-O-methyl group to the 3' end of piRNAs. This methylation protects the piRNAs from premature degradation. Piwi proteins, such as PIWIL1, bind piRNAs as sequence-specific guides to recognize their target mRNA sequences, which are subsequently cleaved and thus destroyed. This mechanism is of great importance in the control of retrotransposon sequences, which are mobile DNA sequences, able to move around the genome via a copy and paste mechanism that includes an mRNA intermediate. This can have a destabilising effect on the genome and can lead to infertility if it occurs in germ cells. In addition, protein-coding sequences can also be affected by PIWI/piRNA-mediated cleavage.
While the function of HENMT1 has been investigated in many species from flies to mouse, and was found to be critical for male fertility and the control of retrotransposon expression and the spermiogenic program in the mouse, the role of HENMT1 in human fertility has not been investigated. This thesis aims at characterising the expression and function of HENMT1 in human testis, under the hypothesis that HENMT1 and the piRNA pathway play a role in human spermatogenic disorders and testis cancer by regulating transposon expression and/or the spermatogenic program. In addition, I have expanded the known knowledge of HENMT1 in mouse testis through an analysis of the ultrastructure of spermatogenesis in a Henmt1 mouse mutant and by integrating HENMT1 into a network of interacting partners.
Firstly, the localisation of HENMT1 and PIWIL1 was defined using a range of immunochemical methods and RT-PCR after microdissection of germ cell populations. HENMT1 was found in all phases of germ cell development and in Sertoli cells, while PIWIL1 was only produced from late pachytene to the elongating spermatid stage.
The function of HENMT1 in human testis was inferred through a RT-qPCR analysis of testis biopsies for expression of HENMT1, PIWIL1/2/4 and active L1 retrotransposon sequences. My data showed that L1s are overexpressed in a sub-set of infertile men, and that overexpression can occur concurrently with histologically normal spermatogenesis. However, L1 expression was significantly lower in patients expressing HENMT1 compared to patients where HENMT1 expression was not detected. A similar effect on transposon expression was found in the case of absent expression of PIWIL1, 2 or 4. Taken together, these data support a role for the piRNA pathway in retrotransposon control in human testis.
In line with the patient results, overexpression of HENMT1 in a TCam2 seminoma cell line led to a downregulation of retrotransposon expression. Interestingly, proliferation and invasion assays revealed cells overexpressing HENMT1 to be more proliferative and invasive compared to empty vector-transfected or wild type cells.
Interaction of PIWIL1 and HENMT1 in mouse testis was demonstrated using a proximity ligation assay and was localized to particulate structures in the cytoplasm of spermatocytes and spermatids. Previously unknown interacting partners of HENMT1 were discovered by mass spectrometry after HENMT1 co-immunoprecipitation, and confirmed in reciprocal pull-downs and a yeast two-hybrid screen. Novel binding partners included proteins involved in alternative splicing, histone modifications and the miRNA pathway, but also proteins regulating intracellular trafficking, energy homeostasis, and kinases that could potentially modify HENMT1. The majority of the interacting candidates have been reported as either tumour enhancers or tumour suppressors, and thus may contribute to the tumourigenic effect of HENMT1 overexpression in seminoma cells.
The work in this thesis has defined, for the first time, HENMT1 and PIWIL1 expression in human testis and demonstrated an influence of HENMT1 on transposon expression in human testis. In addition, HENMT1 overexpression influenced invasive and proliferative characteristics of seminoma cells. The current mammalian piRNA pathway model was refined by demonstrating interaction between HENMT1 and PIWIL1, which had been assumed but not proven. Finally, novel interacting partners of HENMT1 were identified, potentially expanding the known function of HENMT1 beyond the piRNA pathway.