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:

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 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  Due to the nature of FMRP, such a fractionation strategy may 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 ONM and RER contamination in isolated chromatin fractions is a 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).
The hypothesis that FMRP may bind histones dates 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:
Recently, Alpatov et al. and Zhang et al. reported the nuclear function of FMRP in replication stress-induced DNA damage response. As a well-known cytoplasmic protein functioning in pathogenesis of fragile X syndrome, FMRP's existence and function in nucleus should be cautiously considered. Here, the authors discuss the works in an alternative perspective.  Figure. 1. Models of FMRP nuclear functioning. (A) FMRP binds methylated histone lysine residues and functions in replication stress response. (B) Alternatively FMRP may cooperate with other partners via its Agenet domains to execute the same task.

VANTAGE POINTS
Qingzhong He, Wei Ge phenotype is reported to be caused by aberrantly proliferated Sertoli cells (Slegtenhorst-Eegdeman et al., 1998). Alpatov et al. report that FMRP mutants defective in the RS response do not influence the internalization of alphaamino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR), indicating that FMRP chromatin function is independent of its canonical mGluR function. However, as reported by Reeve et al. (Reeve et al., 2008), dFmr1 E68K mutation (corresponding to FMRP E66K) showed a similar dorsal axonal elaborations of ventral lateral neurons to dFmr1 homozygous null mutants (Reeve et al., 2008), while by homology, FMRP E-66 is an important residue that is involved in forming salt bridge that serve to stabilize Agenet domains. This phenomenon indicates that the Agenet domains may also contribute to the FMRP neuronal function.
Using in vitro pull-down assays, Alpatov et al. also demonstrated that N-terminus of FMRP binds histone H3 and is dependent on lysine methylation. However, FMRP did not display a strong preference for any of the individual methylation sites. We cannot rule out the possibility that FMRP requires other intracellular protein partners, such as nucleolin, to carry out its function.
Nucleolin is a well-characterized FMRP-interacting protein. The N-terminus of FMRP interacts with recombinant nucleolin via its methylated arginine-rich region (Taha et al., 2014). Knockdown of nucleolin suppresses the elevation of γH2AX in U2OS cells upon irradiation-induced DSB damage (Kobayashi et al., 2012).
In response to RS, γH2AX formation is mediated by ATR kinase. When γH2AX formation is suppressed by FMRP down-regulation, the potential impairment of ATR kinase recruitment or activation is worth considering. The authors showed that in FMR1 KO spermatocytes, ATR is abnormally loaded, which may partially explain the question, however further investigation is still needed.
As FMRP has been identified as chromatin-associated, chromatin immunoprecipitation followed by sequencing (ChIP-seq) is necessary to clarify its locus on chromatin (if ChIP-grade anti-FMRP is available). The data will provide deeper insight into the chromatin function of FMRP and also provide supporting information about whether or what histone modification recruits FMRP in vivo.
This year marks the 21 st year since the property of FMRP protein was initially characterized. As a cytoplasmic protein also functioning in chromatin, FMRP opens a new chapter of its story (Fig. 1).

FOOTNOTES
We are supported by National Natural Science Foundation of China (Grant No. 81373150). Qingzhong He and Wei Ge declare that there is no conflict of interest.

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