VANTAGE POINTS FMRP: a new chapter with chromatin

Deficient expression of fragile X mental retardation protein (FMRP) underlies the molecular mechanism of fragile X syndrome (FXS). Traditionally, FMRP is classified as a cytoplasmic RNA-binding protein and functions as a translational repressor in the metabotropic glutamate receptor (mGluR) pathway in FXS pathogenesis. In certain contexts (Blonden et al., 2005; Feng et al., 1997; Kim et al., 2009; Van ‘t Padje et al., 2005), nuclear FMRP is also detected, yet its nuclear role remained elusive. Recently, two groups have reported that FMRP is important in the replication stress (RS) response and may play a role in meiosis (Alpatov et al., 2014; Zhang et al., 2014). Alpatov et al. demonstrated that in mammalian cells, H2A. X S-139 phosphorylation (γH2A.X) in response to RS, rather than double strand break (DSB), is suppressed when endogenous FMRP is down-regulated. This suppression can be rescued by exogenous expression of wild-type FMRP but not by its nucleosome-binding-deficient mutant T102A or Y103L. Similar phenomena were also observed by Zhang et al. in a Drosophila model, where the addition of KH domains of dFmr1 is also required for the function. Zhang et al. found that dFmr1 increases at both the mRNA and protein levels in replication-stressed Drosophila S2 cells, while Alpatov et al. demonstrated that Fmrp levels in total lysate of mouse embryonic fibroblasts (MEFs) reduces slightly upon aphidicolin (APH) treatment. Using fractionation and immunofluorescence (IF) data, they both conclude that FMRP is recruited to chromatin upon RS. Due to the nature of FMRP, such a fractionation strategymay not be suitable for researching the intracellular localization of the protein. It has been well-established that FMRP is tightly associated with the ribosome and the rough endoplasmic reticulum (RER) (Corbin et al., 1997; Feng et al., 1997; Khandjian et al., 1996), and the outer nuclear membrane (ONM) is rich in ribosome and continuous with the RER. Therefore, eliminating ONMandRERcontamination in isolatedchromatin fractions isa prerequisite for investigating chromatin association of FMRP; otherwise, FMRP readily appears in the nuclear fraction. In consideration of these issues, either the use of micrococcal nuclease for chromatin digestion in order to observe co-release of FMRP and nucleosomes, or immunoelectron microscopy (Feng et al., 1997) may provide a more rigorous analysis. Both Alpatov et al. and Zhang et al. have used Leptomycin B (LPB) to facilitate IF detection of nuclear FMRP. Zhang et al. demonstrated that dFmr1 accumulate in an S2 nucleus treated with combination of hydroxyurea (HU) and LPB, but not with HU or LPB alone, and that the dFmr1 signal concentrates in the Hoechst dull staining area. In MEFs, Fmrp staining is proximal to DAPI-condensed chromocenters, reminiscent of the centromere localization of PARP-1, which has been reported to interact with FMRP (Isabelle et al., 2010). Thehypothesis that FMRPmaybind histonesdates back to bioinformatic analyses by Maurer-Stroh et al. (Maurer-Stroh et al., 2003). They identified that the N-terminus of FMRP contains two tandem Agenet domains of the Tudor superfamily (Maurer-Stroh et al., 2003). Subsequently, Ramos et al. prove that the Agenet domains bind methylated lysine but not arginine (Ramos et al., 2006). Destabilizing the Agenet domains does not influence the subcellular localization of FMRP cytoplasmic isoform 7, but causes its nuclear isoform 12 to lose perinucleolar localization (Ramos et al., 2006). EDITOR’S NOTE: