Yoshito Matsui

An ERG (ets-related gene)-associated histone methyltransferase interacts with histone deacetylases 1/2 and transcription co-repressors mSin3A/B.

Covalent modifications of histone tails play important roles in gene transcription and silencing. We recently identified an ERG ( ets -related gene)-associated protein with a SET (suppressor of variegation, enhancer of zest and trithorax) domain (ESET) that was found to have the activity of a histone H3-specific methyltransferase. In the present study, we investigated the interaction of ESET with other chromatin remodelling factors. We show that ESET histone methyltransferase associates with histone deacetylase 1 (HDAC1) and HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A and mSin3B. Deletion analysis of ESET reveals that an N-terminal region containing a tudor domain is responsible for interaction with mSin3A/B and association with HDAC1/2, and that truncation of ESET enhances its binding to mSin3. When bound to a promoter, ESET represses the transcription of a downstream luciferase reporter gene. This repression by ESET is independent of its histone methyltransferase activity, but correlates with its binding to the mSin3 co-repressors. In addition, the repression can be partially reversed by treatment with the HDAC inhibitor trichostatin A. Taken together, these data suggest that ESET histone methyltransferase can form a large, multi-protein complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in multiple pathways of transcriptional repression.

Progressive degeneration of articular cartilage and intervertebral discs : an experimental study in transgenic mice bearing a type IX collagen mutation

Summary. Transgenic mice expressing mutant α1(IX) collagen were produced and found to develop progressive joint degeneration with age, as well as accelerated intervertebral disc degeneration. Radiological and histological studies showed that cervical and lumbar disc degeneration was more advanced in the transgenic mice than in control litter-mates. The changes included shrinkage or disappearance of the nucleus pulposus, and fissures in the annulus fibrosus which sometimes lead to herniation of disc material and slight osteophyte formation. These findings suggest that mutations of the type IX collagen may cause certain forms of degenerative disease in the spine as well as in joints.Résumé. Nous avons produit des souris transgéniques experimant un collagène mutant α1(IX) et constaté que ces sujets développaient avec l’âge une dégénérescence progressive des articulations alliée à une dégénérescence accélérée des disques intervertébraux. Les examens radiologiques et histologiques ont montré que ce processus e’tait plus avancé sur les souris transgéniques que sur les sujets de référence. La dégénérescence prend la forme du rétrécissement jusqu’à disparition du tissu pulpeux du noyau cellulaire et d’une fissuration du tissue fibreux annulaire avec, dans certains cas, herniation de la matière discale et légère formation d’ostéophytes. Ces constatations nous suggèrent que les mutations de type collagène IX pourraient entraîner certaines affections dégénératives du rachis et des articulations.

Collagen polymorphisms of the intervertebral disc

The mechanical function and the collagen phenotype of the disc are complex, each a hybrid of elements of ligament and cartilage. In detail, the collagen properties are unique. Collagens I and II provide the bulk of the tissue fabric interwoven in opposing radial concentration gradients. From analysis of isolated cross-linked peptides, some degree of commingling of these major fibrillar collagens occurs down to the molecular level. Collagens V, VI, IX, XI, XII and XIV all contribute to the matrix. Collagen IX is the short molecular form that lacks a non-collagenous (NC)4 domain, not the long form found in most hyaline cartilages. Protein sequence and reverse transcriptase-PCR analysis confirmed this was the result of expression from the alternative transcription start site, not proteolysis of the long form. In view of recent reports that common single nucleotide polymorphisms in COL9A2 and COL9A3 are linked to chronic sciatica associated with disc pathology, the specific interactions and role of collagen IX in disc tissue are important to define.

Genotype phenotype correlation in achondroplasia and hypochondroplasia

Recent studies of the fibroblast growth factor receptor 3 (FGFR3) gene have established that achondroplasia and hypochondroplasia are allelic disorders of different mutations. To determine whether the genotype could be distinguished on the basis of the phenotype, we analysed height, arm span, and skeletal radiographs from 23 patients with achondroplasia and the G380R mutation of FGFR3 and eight with hypochondroplasia and the N540K mutation. Both conditions share the classical pathological features of micromelic short stature, reduced or unchanged interpedicular distances in the lumbar spine, disproportionately long fibulae, and squared and shortened pelvic ilia. These were significantly more severe in the G380R patients than in the N540K patients. Our findings have shown a firm statistical correlation between the genotype and the phenotype, although there were a few exceptional cases in which there was phenotypic overlap between the two conditions.

Sp1 Family of Transcription Factors Regulates the Human α2 (XI) Collagen Gene (COL11A2) in Saos-2 Osteoblastic Cells†

Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos‐2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast‐specific mechanisms of collagen gene regulation.

Differential in situ expression of α2(XI) collagen mRNA isoforms in the developing mouse

Abstract Type XI collagen is an essential structural component of the extracellular matrix of cartilage and plays a role in collagen fibril formation and skeletal morphogenesis. The expression of all three type XI collagen genes is not restricted to cartilage. In addition, alternative exon usage seems to increase the structural diversity and functional potential of type XI collagen during development. In order to investigate type XI collagen expression during development, we have examined α2(XI) and α1(XI) collagen genes by in situ hybridization in mice. Transcripts of the α2(XI) collagen gene were first detected in the notochord of mouse embryos after 11.5 days of gestation. Subsequently, α2(XI) mRNA was mainly found in the cartilaginous tissues of the developing limbs and axial skeleton together with transcripts of the α1(XI) gene. The α2(XI) transcripts seemed to be alternatively spliced isoforms lacking exons 6–8, which code for an acidic domain. Expression of α2(XI) outside the cartilage was relatively restricted, whereas expression of the α1(XI) gene was widespread. However, expression of α2(XI) transcripts containing exons 6–8 was found in non-chondrogenic tissues, including the calvarium and periosteum where intramembranous ossification occurs. These results indicate that α2(XI) mRNA isoforms are differentially expressed in various tissues during development. In addition, α2(XI) mRNA isoforms containing alternative exons are present in osteogenic cells, and their expression may be closely related to the formation of bone or cartilage.

COL11A2 Collagen Gene Transcription Is Differentially Regulated by EWS/ERG Sarcoma Fusion Protein and Wild-type ERG

A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewings sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG andCOL11A2 genes are expressed in the Ewings sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG inCOL11A2 gene expression, we characterized theCOL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction betweenCOL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.

Matrix deposition of tryptophan-containing allelic variants of type IX collagen in developing human cartilage

Genetic polymorphisms that encode a tryptophan (Trp) residue in the triple-helical domain of the alpha2 (Trp2) or alpha3 chain (Trp3) of human type IX collagen have been linked to risk of degenerative intervertebral disc disease. To determine whether these two allelic variants express protein that may affect the extracellular matrix of cartilage in vivo, we examined the properties of resident type IX collagen in an anonymous collection of embryonic and fetal human cartilage samples screened for Trp genotypes. No difference was found in the yield and electrophoretic properties of pepsin-solubilized type IX collagen between Trp2, Trp3 and non-Trp cartilage samples. On Western blot analysis, a polyclonal antiserum raised against a synthetic peptide matching the immediate Trp-containing sequence of the Trp3 allele reacted specifically with the alpha3(IX) chain prepared from Trp3 cartilage samples. Two-dimensional peptide mapping of type IX collagen in CNBr-digests of whole tissue gave indistinguishable fingerprints for Trp2, Trp3 and control tissues, including the yield of cross-linked peptides. Analysis of one cartilage sample that was homozygous for the Trp2 allele also gave a normal yield of collagen IX, including its alpha2 chain and a normal profile of cross-linked peptides. Together, the findings indicate that both Trp2 and Trp3 allelic products are incorporated into the cross-linked fibrillar network of developing human cartilage apparently normally. Any pathological consequences are likely, therefore, to be long-term and indirect rather than from overt misassembly of matrix.

Intrapatellar tendon lipoma with chondro-osseous differentiation: detection of HMGA2-LPP fusion gene transcript

A 54 year old man developed an unusual lipoma in the patellar tendon, consisting of a fibro-adipose component and a chondro-osseous component. The fibro-adipose component contained mature adipocytes, lipoblasts, and fibroblasts; the chondro-osseous component showed typical endochondral bone formation. Molecular analysis showed that the identical HMGA2-LPP fusion transcript—characteristic of lipoma, parosteal lipoma, and pulmonary chondroid hamartoma—was detectable in the both components.

Splicing patterns of type XI collagen transcripts act as molecular markers for osteochondrogenic tumors

Primary transcripts for three distinct alpha chains of the type XI collagen molecule (alpha1(XI), alpha2(XI) and alpha3[XI]) undergo tissue-specific alternative splicing during the process of osteochondrogenesis. In the present study, we analyzed the splicing patterns of type XI collagen genes in osteochondrogenic tumors as well as in various normal tissues using the reverse transcription-polymerase chain reaction method. Analysis of normal subjects revealed the coordinated expression of short alpha1(XI), alpha2(XI) and alpha3(XI) transcripts in the normal differentiated cartilage. Osteochondroma followed this pattern, reflecting the highly chondrogenic phenotype of this benign tumor. Another benign tumor, chondroblastoma, exclusively expressed the long alpha1(XI) transcript, probably reflecting the lack of a chondrogenic nature. Among malignant chondrogenic tumors, the splicing patterns of type XI collagen transcripts were more complex, showing dissociated expression of long alpha1(XI) and short alpha2(XI) mRNAs. This expression pattern may reflect heterogeneous cell populations and may also reflect various levels of cell differentiation in malignant tumors. In addition, short alpha3(XI) expression switched to the long transcript as chondrosarcomas became more aggressive. Thus, the alternative splicing of type XI collagen genes seems to be oncodevelopmentally regulated and splicing analysis may therefore be a useful marker for chondrogenic tumors.