The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery.

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.

DNA binding of methyl-CpG-binding protein MeCP2 in human MCF7 cells.

MeCP2 has been identified as a chromatin-associated protein that recognizes MAR elements as well as methyl-CpGs. To characterize target sequences of MeCP2 in human cells, we employed two complementary methods. First, by use of a preparative chromatin immunoprecipitation protocol, we created from MCF7 cells a library enriched with sequences bound to MeCP2. A total of 154 representative clones were sequenced and analyzed. A large fraction of clones was found to be associated with retrotransposons, mostly with Alu repeats. A subgroup of four clones is derived from putative MARs; one clone is associated with a CpG island, and four clones contain alphoid repeats. Classical satellite DNAs II and III are not represented among clones, although they are heavily methylated. Second, using indirect immunofluorescence microscopy, we show that MeCP2 staining of human metaphase chromosomes has a dotted to knobby appearance with a reduced level of staining of centromeric regions of some chromosomes. On the other hand, an anti-5-methylcytosine antibody preferentially stained the juxtacentromeric regions of chromosomes 1, 9, and 16, which habor highly methylated, classical satellite DNAs, and methylated alphoid sequences in centromeric regions of several other chromosomes with reduced intensity. In interphase MCF7 cells, the distribution of MeCP2 exhibits a granular appearance throughout the nucleus. This distribution does not parallel that of methylated cytosine and heterochromatin. The selective binding behavior of MeCP2 revealed by these results (preference for murine major satellite DNA, Alu sequences, MARs, and CpG islands) is explained by its ability to recognize the sequence information (guanine bases) adjacent to CpG (TpG) as demonstrated in previous footprinting experiments.

Cell type-specific expression of LINE-1 open reading frames 1 and 2 in fetal and adult human tissues.

The LINE-1 (L1) family of non-long terminal repeat retrotransposons is a major force shaping mammalian genomes, and its members can alter the genome in many ways. Mutational analyses have shown that coexpression of functional proteins encoded by the two L1-specific open reading frames, ORF1 and ORF2, is an essential prerequisite for the propagation of L1 elements in the genome. However, all efforts to identify ORF2-encoded proteins have failed so far. Here, applying a novel antibody we report the presence of proteins encoded by ORF2 in a subset of cellular components of human male gonads. Immunohistochemical analyses revealed coexpression of ORF1 and ORF2 in prespermatogonia of fetal testis, in germ cells of adult testis, and in distinct somatic cell types, such as Leydig, Sertoli, and vascular endothelial cells. Coexpression of both proteins in male germ cells is necessary for the observed genomic expansion of the number of L1 elements. Peptide mass fingerprinting analysis of a approximately 130-kDa polypeptide isolated from cultured human dermal microvascular endothelial cells led to the identification of ORF2-encoded peptides. An isolated approximately 45-kDa polypeptide was shown to derive from nonfunctional copies of ORF2 coding regions. The presence of both ORF1- and ORF2-encoded proteins in vascular endothelial cells and its apparent association with certain stages of differentiation and maturation of blood vessels may have functional relevance for vasculogenesis and/or angiogenesis.

A WW domain binding region in methyl-CpG-binding protein MeCP2: impact on Rett syndrome.

Rett syndrome is a dominant neurological disorder caused by loss-of-function mutations of methyl-CpG-binding protein 2 (MeCP2). MeCP2 is an abundant chromatin-associated protein that contains two well characterized domains. Through an N-terminal domain it recognizes methyl-CpGs and binds to nonmethylated DNA. A domain in the middle of the protein can act as a transcriptional repressor in transient transfection studies. The C-terminal region of the protein is equally essential for the function of MeCP2, as documented by recurrently found frameshift mutations. However, little is known about its functional role. Here we mapped a domain within MeCP2 capable of binding specifically to Group II WW domains of splicing factors formin-binding protein (FBP) 11 and HYPC. Binding was assessed by glutathione S-transferase pull-down assays and coimmunoprecipitation assays. The Group II WW domain binding region was localized from residue 325 to the C-terminus, with the interacting proline-rich sequence at its center. We then used comparison with genotype-phenotype studies in Rett syndrome patients to evaluate the relevance of Group II WW domain interactions of MeCP2 for pathogenesis. Truncation of the WW domain binding region by 48 C-terminal amino acids (to residue 438), causing Rett syndrome, resulted in reduced or loss of WW domain binding activity. Truncation to residue 400, representing a large group of frameshift mutations accounting for approx. 10% of Rett syndrome cases, abolished WW domain binding activity completely. On the other hand, two benign missense mutations did not affect binding. Furthermore, a short C-terminal truncation and an internal deletion, both causing mild to moderate mental retardation in males, were associated with weak or loss of WW domain binding activity.

Solution structure of the matrix attachment region-binding domain of chicken MeCP2.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional protein involved in chromatin organization and silencing of methylated DNA. MAR-BD, a 125-amino-acid residue domain of chicken MeCP2 (cMeCP2, originally named ARBP), is the minimal protein fragment required to recognize MAR elements and mouse satellite DNA. Here we report the solution structure of MAR-BD as determined by multidimensional heteronuclear NMR spectroscopy. The global fold of this domain is very similar to that of rat MeCP2 MBD and MBD1 MBD (the methyl-CpG-binding domains of rat MeCP2 and methyl-CpG-binding domain protein 1, respectively), exhibiting a three-stranded antiparallel beta-sheet and an alpha-helix alpha1. We show that the C-terminal portion of MAR-BD also contains an amphipathic helical coil, alpha2/alpha3. The hydrophilic residues of this coil form a surface opposite the DNA interface, available for interactions with other domains of MeCP2 or other proteins. Spectroscopic studies of the complex formed by MAR-BD and a 15-bp fragment of a high-affinity binding site from mouse satellite DNA indicates that the coil is also involved in protein.DNA interactions. These studies provide a basis for discussion of the consequences of six missense mutations within the helical coil found in Rett syndrome cases.

Targeted disruption of the GAS41 gene encoding a putative transcription factor indicates that GAS41 is essential for cell viability.

The glioma-amplified sequence (GAS) 41 protein has been proposed to be a transcription factor. To investigate its functional role in vivo, we attempted to knock out the GAS41 gene by targeted disruption in the chicken pre-lymphoid cell line DT40. Heterozygous GAS41+/- cell lines generated by the first round of homologous recombination express approximately half the normal level of GAS41 mRNA. However, a homozygous GAS41-/- cell line with both GAS41 alleles disrupted was not obtained following the second round of transfection, indicating that the GAS41 gene is essential for cell viability. Indeed, homozygous GAS41-/- cell lines with two disrupted GAS41 alleles can be generated following substitution of the endogenous gene by stable integration of GAS41 cDNA controlled by a tetracycline-regulated CMV promoter. Inactivation of this promoter by tetracycline withdrawal results in rapid depletion of GAS41, causing a significant decrease in RNA synthesis and subsequently cell death. Thus, our results indicate that GAS41 is required for RNA transcription.