Five folate-sensitive fragile sites have been characterized at the molecular level (FRAXA, FRAXE, FRAXF, FRA16A and FRA11B). Three of them (FRAXA, FRAXE and FRA11B) are associated with clinical problems, and two of the genes (FMR1 in FRAXA and CBL2 in FRA11B) have been identified. All of these fragile sites are associated with (CCG)n/(CGG)n triplet expansions that are hypermethylated beyond a critical size. FRAXE is a rare folate sensitive fragile site only recently recognized. Its cytogenetic expression was found to involve the amplification of a (CCG)n repeat adjacent to a CpG island. Normal alleles vary from 6 to 25 copies. Expansions of greater than 200 copies were found in FRAXE expressing males and their FRAXE associated CpG island was fully methylated. An association of FRAXE expression with concurrent methylation of the CpG island and mild non-specific mental handicap in males has been reported by several groups. The cloning and characterization of a gene (FMR2) adjacent to FRAXE is reported. Elements of FMR2 were initially identified from sequences deleted from a developmentally delayed boy. Loss of FMR2 expression is correlated with (CCG)n expansion at FRAXE, demonstrating that this is a gene associated with the CpG island adjacent to FRAXE and contributes to FRAXE-associated mild mental retardation (Gecz, 1996).
Five folate-sensitive fragile sites have been identified at the molecular level to date. Each is characterized by an expanded and methylated trinucleotide repeat CGG (CCG). Of the three X chromosome sites, FRAXA, FRAXE and FRAXF, the former two are associated with mental retardation in their expanded forms. FRAXA expansion results in fragile X syndrome due to down regulation of expression of the FMR1 gene, which carries the hypermutable CGG repeat in the 5' untranslated portion of its first exon. Mild mental retardation without consistent physical findings has been found associated with expanded CCG repeats at FRAXE. A large gene (FMR2) has been identified, transcribed distally from the CpG island at FRAXE, and down-regulated by repeat expansion and methylation. The gene is novel, expressed in adult brain and placenta, and shows similarity with another human protein, MLLT2, expressed from a gene at chromosome 4q21 involved in translocations found in acute lymphoblastic leukaemia (ALL) cells. Identification of this gene will facilitate further studies to determine the role of its product in FRAXE associated mental deficiency (Gu, 1996).
A novel human gene, LAF-4, was isolated from a subtracted cDNA library that showed strong sequence similarity to AF-4, a gene that is translocated in t(4;11)(q21;q23) acute lymphoblastic leukemias (ALLs). In t(4;11) ALL, the AF-4 gene at 4q21 is translocated into the MLL locus at 11q23, resulting in the expression of an MLL/AF-4 fusion protein that is the presumptive oncoprotein. AF-4 and LAF-4 are homologous throughout their coding regions, yet neither protein is related to previously cloned genes. Human LAF-4 readily hybridizes with genes in mouse and chicken, thus showing that this gene family has been highly conserved during vertebrate evolution. In mouse tissues, LAF-4 mRNA was found to be present at highest levels in lymphoid tissues, present at lower levels in brain and lung, and absent from other tissues. In human and mouse lymphoid cell lines, LAF-4 expression is highest in pre-B cells, intermediate in mature B cells, and absent in plasma cells, thus pointing to a potential regulatory role for LAF-4 in lymphoid development. Antibodies to LAF-4 showed it to be a nuclear protein that shows an uneven, granular immunofluorescence pattern. In vitro-translated LAF-4 is able to bind strongly to double-stranded DNA cellulose. Furthermore, both LAF-4 and AF-4 have domains that activate transcription strongly when fused to the GAL4 DNA-binding domain. Interestingly, the AF-4 transactivation domain is retained in the MLL/AF-4 fusion protein; thus, it may contribute to the transforming potential of the oncoprotein. Therefore, the cloning of LAF-4 has defined a new family of potential regulatory proteins that may function in lymphoid development and oncogenesis (Ma, 1996).
FMR2 is the gene associated with FRAXE mental retardation. It is expressed as an 8.7-kb transcript in placenta and adult brain. A fetal-specific FMR2 transcript of approximately 12 kb was detected in fetal brain and at a lower level in fetal lung and kidney. FMR2 is a large gene composed of 22 exons spanning at least 500 kb on Xq28. Alternative splicing involving exons 2, 3, 5, 7, and 21 is not tissue specific as tested on mRNA from human fetal and infant brain. FMR2 is translated into a 1311-amino-acid nuclear protein with putative transcription transactivation potential. Subcellular localization studies with green fluorescent protein as a reporter show that both nuclear addresses found in the FMR2 sequence are functional and direct the FMR2 protein into the nucleus. FMR2 together with AF4 and LAF4 forms a new family of nuclear proteins with DNA-binding capacity and transcription transactivation potential. BLAST searches of the dbEST database have revealed the presence of at least two other groups of nonoverlapping ESTs showing high similarity to the FMR2-related family of proteins. One of them, represented by the EST W26686, maps to chromosome 5q31. Amino acid similarity among the proteins encoded by members of the gene family is high in the NH2 terminus, low in the middle, and high again in the COOH end. Available information from members of the family shows that genomic organization is conserved. This FMR2-related gene family encodes nuclear proteins with involvement in mental retardation (FMR2), cancer (AF4), and lymphocyte differentiation (LAF4) or with unknown function (Gecz, 1997).
Acute leukemia with t(4;11)(q21,q23) translocation results from the in-frame fusion of the MLL to the AF4/FEL gene. AF4 transcripts are present in a variety of hematopoietic and nonhematopoietic human cells. To further study the wild-type and leukemia fusion AF4, glutathione S-transferase (GST)-fusion proteins were used as immunogens to produce rabbit polyclonal antibodies that are specific for normal and chimeric AF4 proteins. Using Western blotting analysis, it was demonstrated that the AF4 gene encodes proteins with apparent molecular weight of 125 and 145 kD. A 45-kD protein coprecipitates with AF4 protein in immunoprecipitation. Also, the anticipated MLL-AF4-encoded 240-kD protein is detected in all cell lines with t(4;11) translocations; fusion proteins are present in lesser quantity than the wild-type AF4. The proteins recognized by the antibodies are of the predicted sizes of the AF4 and MLL-AF4-encoded proteins. The MLL-AF4 fusion protein has a similar subcellular distribution as AF4. Both t(4;11) and non-t(4;11) leukemic cells show a similar pattern of punctate nuclear staining in all cell lines tested. AF4 antibodies should be useful for further elucidation of the function of AF4 in normal cellular physiology, as well as the function of MLL-AF4 in leukemogenesis. The antibodies should also be helpful for the diagnosis of the MLL-AF4 fusion proteins in t(4;11) leukemias (Li, 1998).
The expression of the FRAXE fragile site on the human X chromosome is associated with the expansion of a CCG repeat at the 5' end of the FMR2 gene. The repeat expansion results in transcriptional silencing of the gene and this event has been found to be associated with mild mental handicap in families. The gene is particularly abundantly expressed in the hippocampus and amygdala. The expression pattern of the homologous gene is demonstrated in adult mouse brain and early mouse embryos. High levels of fmr2 mRNA have been noted in the hippocampus, the piriform cortex, Purkinje cells and the cingulate gyrus. Expression of fmr2 occurs on, or before, day 7 in the embryo and reaches its highest levels at 10.5-11.5 days. A more detailed analysis shows that the fmr2 expression in the embryo at 11 days is more specific and evident in the roof of the hind brain and the lateral ventricle of the brain. The coding sequence of the mouse fmr2 gene shows very high conservation with 88% amino acid identity to the human FMR2 sequence (Chakrabarti, 1999).
The transcriptional silencing of the FMR2 gene has been implicated in FRAXE mental retardation. FRAXE individuals have been shown to exhibit learning deficits, including speech delay, reading and writing problems. FMR2 encodes a large protein of 1311 amino acids and is a member of a gene family encoding proline-serine-rich proteins that have properties of nuclear transcription factors. To characterize the expression of the fragile X mental retardation 2 (FMR2) protein, polyclonal antibodies were raised against two regions of the human FMR2 protein and used in immunofluorescence experiments on mouse brain cryosections. The FMR2 protein is localized in neurons of the neocortex, Purkinje cells of the cerebellum and the granule cell layer of the hippocampus. FMR2 staining is shown to colocalize with the nuclear stain DAPI confirming that FMR2 is a nuclear protein. The localization of FMR2 protein to the mammalian hippocampus and other brain structures involved with cognitive function is consistent with the learning deficits seen in FRAXE individuals (Miller, 2000).
Some chromosomal translocations in acute leukemias involve the fusion of the trithorax-related protein Mll (also called HRX, All1 or Htrx) with a variety of heterologous proteins. In acute lymphoblastic leukemia associated with the t(4;11)(q21;q23) translocation, the 4q21 gene that fuses with Mll is AF4. To gain insight into the potential role of AF4 in leukemogenesis and development, this gene was inactivated by homologous recombination in mice. As expected from the tissue distribution of the AF4 transcript, development of both B and T cells is affected in AF4 mutant mice. A severe reduction of the thymic double positive CD4/CD8 [CD4(+)/CD8(+)] population was observed; in addition most double- and single-positive cells express lower levels of CD4 and CD8 coreceptors. Most importantly, the reconstitution of the double-positive compartment by expansion of the double-negative cell compartment is severely impaired in these mutant mice. In the bone marrow pre-B and mature B-cell numbers are reduced. These results demonstrate that the function of the mAF4 gene is critical for normal lymphocyte development. This raises the possibility that the disruption of the normal AF4 gene or its association with Mll function by translocation may orient the oncogenic process toward the lymphoid lineage. This represents the first functional study using a knock-out strategy on one of the Mll partner genes in translocation-associated leukemias (Isnard, 2000).
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