RNAmetasome network for macromolecule biogenesis in human cells.

RNA plays a central role in macromolecule biogenesis for various pathways, such as gene expression, ribosome biogenesis, and chromatin remodeling. However, RNA must be converted from its nascent to functional forms for that role. Here, we describe a large RNA metabolic network (RNAmetasome network) for macromolecule biogenesis in human cells. In HEK293T, the network consists of proteins responsible for gene expression, splicing, ribosome biogenesis, chromatin remodeling, and cell cycle. Reciprocal immunoprecipitations show that MKI67, GNL2, MDN1, and ELMSAN1 are core proteins of the network, and knockdown of either MKI67 or GNL2 affects the state of the other protein, MDN1, and some other network members. Furthermore, GNL2 knockdown retards cell proliferation. Several proteins of the RNAmetasome network are diminished in Hela.cl1, and this diminishment is associated with low expression of MDN1 and elevated MKI67 degradation. These results together suggest that the RNAmetasome network is present in human cells and associated with proliferation, and that MKI67, GNL2, and MDN1 play an important role in organizing the RNAmetasome network.

Lysine-specific demethylase 2A enhances binding of various nuclear factors to CpG-rich genomic DNAs by action of its CXXC-PHD domain.

The lysine-specific demethylase 2A gene (KDM2A) is ubiquitously expressed and its transcripts consist of several alternatively spliced forms, including KDM2A and the shorter form N782 that lacks the 3' end encoding F-box and LRR. KDM2A binds to numerous CpG-rich genomic loci and regulates various cellular activities; however, the mechanism of the pleiotropic function is unknown. Here, we identify the mechanism of KDM2A played by its CXXC-PHD domain. KDM2A is necessary for a rapid proliferation of post-natal keratinocytes while its 3' end eclipses the stimulatory effect. EGFP-N782 binds to chromatin together with the XRCC5/6 complex, and the CXXC-PHD domain regulates the CpG-rich IGFBPL1 promoter. In vitro, CXXC-PHD enhances binding of nuclear extract ORC3 to the CpG-rich promoter, but not to the AT-rich DIP2B promoter to which ORC3 binds constitutively. Furthermore, CXXC-PHD recruits 94 nuclear factors involved in replication, ribosome synthesis, and mitosis, including POLR1A to the IGFBPL1 promoter. This recruitment is unprecedented; however, the result suggests that these nuclear factors bind to their cognate loci, as substantiated by the result that CXXC-PHD recruits POLR1A to the rDNA promoter. We propose that CXXC-PHD promotes permissiveness for nuclear factors to interact, but involvement of the XRCC5/6 complex in the recruitment is undetermined.

Lysine-specific demethylase 2A (KDM2A) normalizes human embryonic stem cell derived keratinocytes.

Studies on human lysine-specific demethylase 2A (KDM2A) by others have recently begun. To date, the demethylase activity has been known to reduce expression of genes and eventually inhibit proliferation of cells. However, while attempting to improve proliferation of hES-cell-derived Nod keratinocytes, which grow poorly and have a short life span, we found that high expression of the KDM2A gene improves the poor proliferation of the cells. Of the four isomer cDNAs that we prepared from alternatively spliced KDM2A transcripts, only one stimulates the proliferation. This (KDM2A-N782) encodes the 782AA protein containing the JmjC, CXXC, and Ring domains, but not the F-box and AMN1 domains, unlike KDM2A, which has been studied by other groups. Our results not only show that differently spliced transcripts from a gene result in totally opposite outcomes, but also present critical evidence of the complicated activities of KDM2A, which contains all of the five domains.

An immortalized drug-resistant cell line established from 12-13-day mouse embryos for the propagation of human embryonic stem cells.

Human embryonic stem (ES) cells are usually co-cultivated with supporting cells consisting of short-term cultures of fibroblasts (not an immortalized line) in a medium lacking serum. This method has promoted important progress in the field, but suffers from certain disadvantages. By serial cultivation for 27 consecutive transfers and about 63 cell generations, we have evolved an immortalized line from fibroblastic cells of 12-13-day mouse embryos. This line (MMM) supports the multiplication of H9 cells better than the 3T3 line. It supports the growth of H9 cells as well as do available short-term fibroblast cultures, but maintains more effectively the stem cell character of the H9 cells, judging by their better retention of Oct4. We have made MMM cells resistant to blasticidin and zeocin, the most efficient antibiotics for selection of stable transformants. In the presence of zeocin, the resistant MMM were able to support multiplication and selection of ES cells transfected with an exogenous gene encoding zeocin resistance.

Immortalized keratinocyte lines derived from human embryonic stem cells.

Cells of the human embryonic stem (hES) cell line H9, when cultured in the form of embryoid bodies, give rise to cells with markers of the keratinocyte of stratified squamous epithelia. Keratinocytes also form in nodules produced in scid mice by injected H9 cells; the hES-derived keratinocytes could be recovered in culture, where their colonies underwent a peculiar form of fragmentation. Whether formed from embryoid bodies or in nodules, hES-derived keratinocytes differed from postnatal keratinocytes in their much lower proliferative potential in culture; isolated single keratinocytes could not be expanded into mass cultures. Although their growth was not improved by transduction with the hTERT gene, these keratinocytes were immortalized by transduction with the E6E7 genes of HPV16. Clonally derived lines isolated from E6E7-transduced keratinocytes continued to express markers of the keratinocyte lineage, but the frequency with which they terminally differentiated was reduced compared with keratinocytes cultured from postnatal human epidermis. If other hES-derived somatic cell types also prove to be restricted in growth potential, not identical to the corresponding postnatal cell types, and to require immortalization for clonal isolation and expansion, these properties will have to be considered in planning their therapeutic use.

Marker succession during the development of keratinocytes from cultured human embryonic stem cells.

Human embryonic stem cells injected into scid mice produce nodules containing differentiated somatic tissues. From the trypsinized cells of such a nodule, we have recovered keratinocytes that can be grown in cell culture. The method of recovery is sensitive enough to detect small numbers of keratinocytes formed in the nodule, but for purposes of analysis, it is preferable to study the development of the entire keratinocyte lineage in culture. The principle of our analysis is the successive appearance of markers, including transcription factors with considerable specificity for the keratinocyte (p63 and basonuclin) and differentiation markers characteristic of its final state (keratin 14 and involucrin). We have determined the order of marker succession during the time- and migration-dependent development of keratinocytes from single embryoid bodies in cell culture. Of the markers we have examined, p63 was the earliest to appear in the keratinocyte lineage. The successive accumulation of later markers provides increasing certainty of emergence of the definitive keratinocyte.