PubMed Health⌕ Search

Biomedical subjects

W H Strätling

Publications and source records attributed to W H Strätling.

At least 19 recordsLinked to original sources
PubMed2001

Methyl-CpG-binding protein 2 represses LINE-1 expression and retrotransposition but not Alu transcription.

In order to explore the defense mechanism by which retrotransposons are repressed, we assessed the ability of methyl-CpG-binding protein 2, MeCP2, to influence LINE-1 (L1) and Alu transcription and, furthermore, L1 retrotransposition. In transient transfection assays, targeting of the transcriptional-repression domain (TRD) of MeCP2 (via a linked Gal4 DNA-binding domain) to the transcriptional start site of L1 promoter-driven reporter constructs efficiently repressed transcription. The Gal4-linked TRD of the related methyl-CpG-binding protein MBD1 also repressed transcription but not that of MBD2. Furthermore, full-length MeCP2 effectively repressed transcription of a HpaII-methylated L1 reporter. Secondly, we used a genetic assay employing a full-length neo-marked L1 reporter construct to study L1 retrotransposition. We found the Gal4-linked TRD of MeCP2 to repress effectively L1 retrotransposition when targeted to the retrotransposition reporter. Retrotransposition was also reduced in response to in vitro HpaII methylation of the reporter and was further decreased by co-expressed full-length MeCP2. In striking contrast expression of the Gal4-linked TRD of MeCP2 had no inhibiting effect on transcription of an AluSx reporter tagged with a 7S-upstream sequence. Furthermore, full-length MeCP2 abrogated the methylation-induced repression of this reporter. Our results indicate that MeCP2 serves a role in repression of L1 expression and retrotransposition but has no inhibiting effect on Alu transcription.

Alu Elements↗
PubMed2000

Histone deacetylase-independent transcriptional repression by methyl-CpG-binding protein 2.

Methyl-CpG-binding protein 2 (MeCP2) contains a transcriptional repression domain (TRD), which can act by recruitment of a large transcriptional co-repressor complex containing histone deacetylases HDAC1 and 2. We demonstrate here that transient transcription from the SV40 enhancer/promoter or the SV40 promoter is strongly repressed in a histone deacetylase-independent manner, since repression is not alleviated by Trichostatin A (TSA). In a mutational analysis, repression depends on a conserved 30 residue sequence containing two clusters of basic amino acids. Mutation of the first of these clusters inhibits in vitro interaction between TRD and mSin3A. Furthermore, a subdomain of the TRD containing the conserved 30-residue sequence and 16 flanking amino acids was sufficient to compromise VP16-activated transcription. In summary, our results indicate an alternative, histone deacetylase-independent pathway of transcriptional repression by MeCP2.

3T3 Cells↗
PubMed2000

Comparative sequence analysis of the MECP2-locus in human and mouse reveals new transcribed regions.

Comparative sequence analysis facilitates the identification of evolutionarily conserved regions, that is, gene-regulatory elements, which can not be detected by analyzing one species only. Sequencing of a 152-kb region on human Chromosome (Chr) Xq28 and of the synthenic 123 kb on mouse Chr XC identified the MECP2/Mecp2 locus, which is flanked by the gene coding for Interleukin-1 receptor associated kinase (IRAK/Il1rak) and the red opsin gene (RCP/Rsvp). By comparative sequence analysis, we identified a previously unknown, non-coding 5' exon embedded in a CpG island associated with MECP2/Mecp2. Thus, the MECP2/Mecp2 gene is comprised of four exons instead of three. Furthermore, sequence comparison 3' to the previously reported polyadenylation signal revealed a highly conserved region of 8.5 kb terminating in an alternative polyadenylation signal. Northern blot analysis verified the existence of two main transcripts of 1.9 kb and approximately 10 kb, respectively. Both transcripts exhibit tissue-specific expression patterns and have almost identical short half-lifes. The approximately 10-kb transcript corresponds to a giant 3' UTR contained in the fourth exon of MECP2. The long 3' UTR and the newly identified first intron of MECP2/Mecp2 are highly conserved in human and mouse. Furthermore, the human MECP2 locus is heterogeneous with respect to its DNA composition. We postulate that it represents a boundary between two H3 isochores that has not been observed previously.

3' Untranslated Regions↗
PubMed1999

An origin of bidirectional DNA replication is located within a CpG island at the 3" end of the chicken lysozyme gene.

We previously identified a broad initiation zone of DNA replication at the chicken lysozyme gene locus. However, the existence of a highly preferred origin of bidirectional replication (OBR), often found in initiation zones, remained elusive. In order to re-examine this issue we used a competitive PCR assay to determine the abundance of closely spaced genomic segments in a 1 kb size fraction of nascent DNA. A sharp peak of nascent strand abundance occurred at the 3" end of the gene, where initiation events were 17 times more frequent than upstream of the gene. This primary initiation site, active in lysozyme expressing myelomonocytic HD11 cells and non-expressing hepatic DU249 cells, was found to reside within an unusually located CpG island. While most CpG islands are found at the 5" end of genes, the lysozyme gene island extends from the 3" end of the second intron and includes approximately 1.2 kb of 3" flanking DNA. As diagnosed by methylation-sensitive restriction enzymes, the island is largely non-methylated in HD11 cells, DU249 cells and inactive chicken erythrocytes. Furthermore, a DNase I hypersensitive site (HS) that is composed of two subsites separated by approximately 100 bp, was localised very close to the segment with the highest initiation activity. Our results suggest that the non-methylated CpG island and the HS provide an accessible chromatin structure for the lysozyme gene origin of replication.

Animals↗
PubMed1999

Origin and roles of nuclear matrix proteins. Specific functions of the MAR-binding protein MeCP2/ARBP.

HnRNP proteins are the major protein components of the nuclear matrix, and sites of nascent transcripts and RNA maturation are its main sources. The evidence for and the roles of functional and structural loops in interphase chromatin and metaphase chromosomes is discussed. Recent data suggest a specific role for the matrix attachment region (MAR)- and methyl-CpG-binding protein MeCP2/ARBP. This repressor protein binds to MARs and, through interaction with mSin3A, recruits a corepressor complex containing histone deacetylases. This in turn is thought to generate a localized silenced chromatin structure. Transfection experiments are presented in support of this model.

Animals↗
PubMed1998

Specific binding of Drosophila nuclear protein PEP (protein on ecdysone puffs) to hsp70 DNA and RNA.

The Drosophila protein PEP (protein on ecdysone puffs), a component hnRNP complexes, was previously immunocytologically localized on Drosophila giant chromosomes to puffs induced by ecdysone and to some heat shock-induced puffs (e.g. at the hsp70 locus at 87A7). Here, PEP was purified to homogeneity and characterized in its DNA and RNA binding features with specific reference to the hsp70 locus. In southwestern blotting assays, PEP was found to bind with high affinity to the hsp70 coding region, but not to a flanking region nor to the boundary elements scs and scs', and non-specifically to the intergenic hsp70 SAR. In UV cross-linking assays, PEP binds with even higher affinity to hsp70 transcripts, but not to transcripts of a flanking region or of a nearby gene, aurora . Finally, competition experiments indicate that PEP recognizes specific sequences within hsp70 mRNA; in these sequences two distinct motifs were found to be enriched. In summary, our results suggest the recognition of specific transcripts as a molecular basis for the association of the protein with specific hnRNP complexes.

Animals↗
PubMed1998

An initiation zone of chromosomal DNA replication at the chicken lysozyme gene locus.

The chicken lysozyme gene domain is distinguished by a broad knowledge of how its expression is regulated. Here, we examined the in vivo replication of the lysozyme gene locus using polymerase chain reaction amplification and competitive polymerase chain reaction of size-fractionated, nascent DNA strands. We found that DNA replication initiates at multiple sites within a broad initiation zone spanning at least 20 kilobases, which includes most of the lysozyme gene domain. The 5' border of this zone is probably located downstream of the lysozyme 5' nuclear matrix attachment region. Preferred initiation occurs in a 3'-located subzone. The initiation zone at the lysozyme gene locus is also active in nonexpressing liver DU249 cells. Furthermore, examining the timing of DNA replication at the lysozyme gene locus revealed that the gene locus replicates early during S phase in both HD11 and DU249 cells, irrespective of its transcriptional activity.

Animals↗
PubMed1997

Chicken MAR-binding protein ARBP is homologous to rat methyl-CpG-binding protein MeCP2.

Here, we describe the cloning and further characterization of chicken ARBP, an abundant nuclear protein with a high affinity for MAR/SARs. Surprisingly, ARBP was found to be homologous to the rat protein MeCP2, previously identified as a methyl-CpG-binding protein. A region spanning 125 amino acids in the N-terminal halves is 96.8% identical between chicken ARBP and rat MeCP2. A deletion mutation analysis using Southwestern and band shift assays identified this highly conserved region as the MAR DNA binding domain. Alignment of chicken ARBP with rat and human MeCP2 proteins revealed six trinucleotide amplifications generating up to 34-fold repetitions of a single amino acid. Because MeCP2 was previously localized to pericentromeric heterochromatin in mouse chromosomes, we analyzed the in vitro binding of ARBP to various repetitive sequences. In band shift experiments, ARBP binds to two chicken repetitive sequences as well as to mouse satellite DNA with high affinity similar to that of its binding to chicken lysozyme MAR fragments. In mouse satellite DNA, use of several footprinting techniques characterized two high-affinity binding sites, whose sequences are related to the ARBP binding site consensus in the chicken lysozyme MAR (5'-GGTGT-3'). Band shift experiments indicated that methylation increased in vitro binding of ARBP to mouse satellite DNA two- to fivefold. Our results suggest that ARBP/MeCP2 is a multifunctional protein with roles in loop domain organization of chromatin, the structure of pericentromeric heterochromatin, and DNA methylation.

Amino Acid Sequence↗
PubMed1996

Dissection of the ability of the chicken lysozyme gene 5' matrix attachment region to stimulate transgene expression and to dampen position effects.

The chicken lysozyme gene domain is flanked by nuclear matrix attachment regions (MARs) on each side. We have previously shown that bilaterally flanking 5'MARs in stably transfected artificial genetic units enhance expression of a reporter transgene and dampen position effects of the chromatin structure at the site of integration. The 5' MAR was now dissected into smaller fragments that were monitored for effects on transgene expression in mouse 3T3 cells by a similar assay. Fragments, which contain 1.32 and 1.45 kb and represent the upstream and the downstream half, respectively, of the 5' MAR, retained the ability to stimulate transgene expression as well as the ability to reduce the variation in the level of expression. However, a 452 bp subfragment (H1-HaeII), which still exhibits specific binding to nuclear matrices and contains two high-affinity binding sites for the abundant nuclear matrix protein ARBP, lost both of those abilities. A dimerized 177 bp sequence from fragment H1-HaeII, which also binds selectively to nuclear matrices and includes a duplicated ARBP binding site, was also unable to stimulate reporter gene expression. Furthermore, a 0.65 kb subfragment containing an intrinsically bent sequence did not affect an elevated reporter gene expression and its dampening. Our results show that the ability of MAR fragments to bind to nuclear matrices is not sufficient to enhance and insulate transgene expression in stably transfected cells.

3T3 Cells↗
PubMed1996

The chicken lysozyme gene 5' MAR and the Drosophila histone SAR are electroelutable from encapsulated and digested nuclei.

Cultured chicken cells were encapsulated in agarose microbeads, lysed in a near-physiological buffer and resulting encapsulated nuclei were digested with a restriction enzyme and electroeluted. After removal of approximately 97% of the chromatin, the nuclear lamina, residual nucleoli and an internal nuclear network remained. The majority of nascent RNA was also recovered in digested and electroeluted nuclei. Surprisingly, however, the chicken lysozyme gene 5' MAR was quantitatively electroeluted from digested nuclei of expressing and non-expressing cells, as well as the promoter region and the coding sequence. When encapsulated nuclei were digested partially, the proportion of elutable 5' MAR chromatin was comparable to that of elutable bulk chromatin. Furthermore, after digestion of encapsulated nuclei from Drosophila Kc cells, the histone SAR was electroeluted to the same extent as bulk chromatin. We conclude that the lysozyme gene 5' MAR and the histone SAR are not permanently attached to a nuclear matrix or scaffold.

Animals↗
PubMed1995

Nuclear matrix protein ARBP recognizes a novel DNA sequence motif with high affinity.

ARBP is a nuclear protein that specifically binds to matrix/scaffold attachment regions (MARs/SARs). Here we characterize by DNase I footprinting, dimethyl sulfate protection, and mobility shift assays two binding sites for ARBP within a chicken lysozyme MAR fragment. Our results indicate that ARBP recognizes a novel DNA sequence motif containing the central sequence 5'-GGTGT-3' and flanking AT-rich sequences. Binding occurs through major groove contacts to two guanines of the central sequence. Collective and single-base substitutions in the 5'-GGTGT-3' core motif result in loss or significant reductions of ARBP binding, underscoring the importance of the GC-rich core sequence. Structural elements of the sequence motif are probably also recognized. The affinity of ARBP to both binding sites is surprisingly high [KD = (2-6) x 10(-10) M]. High-affinity recognition of the identified DNA motif in MARs/SARs by ARBP is likely an important feature in the domain organization of chromatin.

Animals↗
PubMed1994

Characterization of a myeloid-specific enhancer of the chicken lysozyme gene. Major role for an Ets transcription factor-binding site.

Expression of the lysozyme gene in chicken macrophages is regulated by an enhancer located 2.7 kilobases upstream of the transcription start site. The organization of this enhancer was analyzed by in vitro assays (DNase I footprinting, dimethyl sulfate methylation protection, and band shift assays) and in vivo footprinting experiments. The results show that the enhancer contains four regions (I-IV) of protein-DNA interactions. First, transient gene transfer experiments demonstrate that region II contributes most to the enhancer function. This region contains a recognition site for the macrophage- and B cell-specific transcription factor PU.1, a member of the Ets transcription factor family. This site plays a major role in the enhancer since a mutation of this site abolishes enhancer activity. Second, in competition band shift experiments, we show that the only myeloid-specific complexes in regions I and II with nuclear factors are formed on the PU.1 recognition site by a member of the Ets transcription factor family. Third in in vivo footprinting experiments, the only sign of a protein-DNA interaction is a dimethyl sulfate-hyperreactive guanine 3'-adjacent to the PU.1 recognition site.

Animals↗
PubMed1994

Chicken MAR binding protein p120 is identical to human heterogeneous nuclear ribonucleoprotein (hnRNP) U.

We have previously identified two proteins from chicken oviduct nuclei that specifically bind to matrix/scaffold attachment regions (MARs/SARs). Here one of these proteins, named p120 due to its apparent molecular weight, is purified to near homogeneity and shown to be identical to a previously described component of heterogeneous nuclear ribonucleoprotein particles, hnRNP U, on the basis of amino acid sequence analysis of tryptic peptides. p120 binds to multiple MAR fragments provided they have a minimal length of approximately 700 bp. Binding of MAR fragments is specifically competed by homoribopolymers poly(G) and poly(I), which form four-stranded structures. Our results suggest that p120/hnRNP U may serve a dual function, first as a component of hnRNP particles, and second as an element in the higher-order organization of chromatin.

Amino Acid Sequence↗
PubMed1994

Biochemical properties of attachment region binding protein ARBP.

ARBP (attachment region binding protein) is an abundant nuclear protein that specifically binds to matrix/scaffold attachment regions (MARs/SARs). Here we show by gel filtration and gradient sedimentation that ARBP has an elongated shape. The sedimentation coefficient was determined as only 2.1 S. Furthermore, limited proteolysis of ARBP in situ (in isolated nuclei) with several proteases generated limiting resistant peptides from 14.5 to 18 kDa, that retained the ability to bind MARs specifically. This indicates that these peptides encompass the DNA binding domain of ARBP.

Animals↗
PubMed1992

Structure analysis of purified histone H5 and of H5 in nuclei by limited proteolysis.

The structure of purified histone H5 in 1 M-NaClO4 and of H5 in nuclei was analysed by digestion with either one of three endoproteinases, papain, subtilisin or elastase, which preferentially cleave unstructured protein regions (and additionally with trypsin). Digestion with papain and subtilisin produced 'limiting' resistant peptides (p1 and s1) that contain the central region between residues 18-20 and residue 114. Digestion of purified H5 with elastase generated resistant peptides e1 and e2, and that of H5 in nuclei, peptide e2. Peptides e1 and e2 contain the region from residues 22 to 114 and 109 respectively. These results show that a central region of H5 encompassing the sequence between residues 18-22 and residue 114 is folded into a compact structure. A central structured 'core' domain ranging from residues 22 to 100 is defined by the limit trypsin peptide t3, which is identical to the previously described fragment GH5 [Aviles et al. (1978) Eur. J. Biochem. 88, 363-371]. Generation of peptides e2 and t3, as well as of resistant peptides of lower abundance, shows that the sites near Lys-100 and Lys-109 exhibit some proteolytic sensitivity, which may result either from an exposed location or from a locally less compact conformation. Significantly, all these structural features of H5 are manifested in the purified form as well as in nuclei. A role of the structured region from residues 101 to 114 for the interaction with linker DNA and the determination of its path is discussed.

Amino Acid Sequence↗