Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes

Abstract

Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging.

FIG. 1

Analyses of MORF 4-transfected cells. Senescence-associated β-galactosidase staining of a stably transfected clone of HeLa+MORF 4 cells at 20 PD (a), 30 PD (b), and 35 PD (c), when the cells had ceased proliferation. (d) Four weeks after the cells had stopped dividing. (e to g) Mortalin staining of HeLa cells (e), normal human fibroblasts (f), and a stably transfected HeLa+MORF 4 cell that had ceased division (g). (h) Fluorescence 8 h after transfection of EJ cells transiently transfected with the MORF 4 green fluorescent protein-tagged construct. (i) Fluorescence 12 h after transfection.

FIG. 2

Comparison of the protein sequences and predicted motifs in the expressed MRG and MORF genes. The various predicted motifs are indicated. +, potential cyclic AMP (cAMP) phosphorylation site; ‡, protein kinase C phosphorylation site; ¥, a tyrosine phosphorylation site. The three regions of homology to the E. crassus telomere binding subunit p51 within the HLH domain are underlined.

FIG. 3

Analysis of RNA levels of MRG 15 and MRG X by using probes specific for each gene. (A) Dot blot analysis of BAC and cDNA encoding MRG 15 or MRG X probed with 132 bp from the 5′ end of each gene demonstrates the specificity of the probes. (B) RNA levels of MRG 15 and MRG X with increasing PD. (C) RNA levels of MRG 15 and MRG X in normal human fibroblast cells (PD 23) made quiescent by removal of serum growth factors for 1 week and then stimulated with 10% serum. The RNA was harvested 0, 4, 8, 18, and 28 h after serum stimulation. (D) RNA levels of MRG 15 and MRG X in various tissues. Poly(A)⁺ RNA blots were obtained from Clontech. Lanes from left to right: heart (h), brain (br), placenta (pl), lung (lu), liver (li), skeletal muscle (sk), pancreas (pa), spleen (s), thymus (th), prostate (pr), testis (te), ovary (ov), small intestine (si), colon (co), and peripheral blood lymphocytes (pbl). In panels B and C, total RNA was used and 28S RNA was the loading control.