Telomeric Repeat-Binding Factor Homologs in Entamoeba histolytica: New Clues for Telomeric Research.

Francisco Javier Rendón-Gandarilla,Esther Orozco,Esther Orozco,Helios Cárdenas-Hernández,Lilia López-Canovas,Elisa Azuara-Liceaga,Anel Lagunes-Guillen,Rosa Elena Cárdenas-Guerra,Víctor Álvarez-Hernández,Bibiana Chávez-Munguía,Bibiana Chávez-Munguía,Abigail Betanzos,Abigail Betanzos,Abigail Betanzos,Jesús Valdés,Jesús Valdés,Elizabeth J Castañeda-Ortiz

doi:10.3389/fcimb.2018.00341

Abstract

Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.

Highlights

Telomeres are specialized protein-DNA complexes localized at the end of eukaryotic chromosomes (Blackburn and Gall, 1978; Meyne et al, 1989; Giraud-Panis et al, 2013)
Anti-Actin antibody was used as loading control. (D) Transfected trophozoites were processed for immunofluorescence and incubated with pre-immune serum (PS), α-EhTRF-like I or α-Myc antibodies, followed by the α-rabbit FITC-coupled secondary antibody
In silico analysis showed that Entamoeba histolytica has three genes coding for Telomeric Repeat Binding Factors (TRFs)-like proteins

Summary

Introduction

Telomeres are specialized protein-DNA complexes localized at the end of eukaryotic chromosomes (Blackburn and Gall, 1978; Meyne et al, 1989; Giraud-Panis et al, 2013). Proteins that bind to telomeric DNA play critical roles in telomere length regulation and chromosomal end protection in eukaryotic organisms They conform a machinery known as Telosome or Shelterin complex (Palm and de Lange, 2008). TRF proteins are architectural nuclear proteins involved in diverse roles, such as telomere length regulation, chromosome end protection, prevention of chromosomes fusion, sense of DNA damage, and regulation of senescence (de Lange, 2005; Palm and de Lange, 2008). These proteins are conserved from lower eukaryotes, to plants and mammals (Horvath, 2000–2013). In Trypanosomes besides the TRF2, a homolog of Rpa-1 has been identified, suggesting that the telomeric function is conserved and that the telomeric machinery evolved early in eukaryotes (Lira et al, 2007)

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