Cytogcnet. Cell Genet. 22: 527-530 (1978)
Partial reactivation of a human inactive X chromosome in human-mouse somatic cell hybrids B. H ellkuhl and K.-H. G rzfschik Institut für Humangenetik der Universität Münster
Supported by the Deutsche Forschungsgemeinschaft (GR 373/8).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/19/2018 3:35:05 PM
Several authors have put forward theories explaining the mechanism of inactivation of one X chromosome in mammals that underlies the Lyon hypothesis.1 In these theories, more attention is given to the question of which of the two X chromosomes is chosen for inactivation than to the problem of how, once the decision has been made, inactivation of the same chromosome is maintained in somatic cells. In order to investigate factors which could influence the maintenance of inactivation, we tried to reactivate inactive human X chromosomes present in human-mouse somatic cell hybrids in an approach slightly different from the one followed by K ahan and D f.M ars .2 In human-mouse cell hybrids involving human female cells, the human inactive X chromosome is frequently lost early in hybrid develop ment, and there is no possibility of selecting for this “silent” chromo some. Examining our collection of hybrids between mouse cells and human female fibroblasts for the retention of inactive X chromosomes, we found two hybrids (194RAG5 and 194RAG6) that were both fusion products of mouse HPRT- RAG cells and human fibroblasts from an (X;3) translocation carrier (GM 194; Human Genetic Mutant Cell Repository, Camden, N.J.). In some of the parental cells the structurally normal X, and in the others the (X;3) translocation chromosome, was late-replicating. The two hybrid populations mentioned above represent both cases. We received a third hybrid with an inactive human X chro mosome (89A99c) from Dr. M. Siniscaixo, New York, N.Y.
H ellkuiii ., G rzesciiik
Regional mapping of X chromosome
This hybrid line was a fusion product of mouse HPRT~, APRT A9 cells and human fibroblasts from an X/19 translocation carrier (GM 89). In the GM 89 parental cells, the normal X was late-replicating. In all three hybrid lines the frequency of metaphases in which the inactive X chromosome (Xi) was the only X present was too low to isolate clones of this cell type by cloning the mixed population. Therefore, mass cultures of all three hybrid lines were treated with 8-azaguanine (8-AG) for 4 days. They were then cloned, and the clones were analyzed by Q-banding for their chromosome content and by Cellogel electro phoresis for the expression of human X-chromosomal enzyme markers (G6PD, PGK, and HPRT). From each hybrid line, two clones (Xi * clones) were isolated with an inactive X chromosome and without the active X or the active fragments of the X and, as controls, one parallel clone without an inactive X and without the translocated part known to carry the X-chromosomal gene HPRT (Xi- clone). The Xi* and Xi* clones from all three hybrid lines were subjected to selection in HAT medium (10-4 m hypoxanthine, 4X 10~5 m azaserine, and 1.6 X 10-5 M thymidine). In the hybrid line 89A99c, HAT-resistant subclones appeared from both Xi" clones with a frequency of 10 °, and no HAT-resistant subclones could be detected in 6 X l()l! cells of the parallel Xi - clone. In a different set of experiments, one HATresistant subclone was found in the Xi clone of hybrid line 89A99c after treatment of 1.2 X 10fi cells with a mutagen (ethyl methanesulphonate). In the two hybrid lines 194RAG5 and 194RAG6, containing, respec tively, a structurally normal inactive X chromosome and an inactive (X;3) translocation chromosome, no HAT-resistant subclones were found in 8.5 X 10° cells each. The HAT-resistant subclones of hybrid line 89A99c were analyzed for X-chromosomal enzyme markers and human chromosomes. Twentythree HAT-resistant subclones of both Xi* clones and the only HATresistant subclone of the Xi clone were tested for human HPRT, G6PD, and PGK. In electrophoresis for HPRT,1 all 23 subclones of both Xi* clones showed mobility of the human HPRT protein, and the one subclone of the Xi- clone showed mobility of the mouse protein. No human G6PD and PGK activities could be detected in the subclones tested. In a backselection system, 8-AG-resistant subclones of HAT-resistant (HPRT*) X i+ clones and the HPRT* Xi- clone grew with a fre
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/19/2018 3:35:05 PM
528
Regional mapping of X chromosome
529
quency of 5 X 10-3 for the X i+ clones and 10-5 for the Xi- clone. The frequency of 8-AG-resistant subclones of the HPRT+ Xi- clone can be explained by mutational events. The 8-AG-resistance in the HPRT+ Xi + clones is more frequent than would be expected if it were caused by mutational events. Instead, it might be consistent with the well-known segregation of a human chromosome carrying the HPRT gene from the human-mouse cell hybrids. The electrophoretic mobility of the HPRT enzyme in the X if and Xi- clones and the behavior of the HPRT- clones in 8-AG-selection support the assumption that the HPRT enzyme in the X i+ clones is of human origin. This explanation is further supported by chromosome segregation analysis: Nine HPRT+ subclones of both X i+ clones were analyzed before and after backselection in 8-AG medium for the presence of human chromo somes by Q-banding. In HAT-medium they all retained an obviously unmodified human X chromosome, but they all lost this human X chro mosome after the 8-AG treatment. The autosome pattern in the 8-AGtreated cell lines was also altered, but all autosomes present in any of the HPRT- clones were found in at least one of the 8-AG-treated cell lines. These results provide evidence of a strong cosegregation of the HPRT enzyme activity only with the human "inactive” X chromosome in the X i+ clone derivatives. The data derived from the experiments with the 89A99c hybrid line might be explained by a mutation of the HPRT gene of the active X chromosome to an enzymatically inactive form and by translocation of the little piece of this chromosome carrying the mutated HPRT gene but not the G6PD gene onto just the inactive X chromosome. This piece presumably would be too tiny to be detected in the chromosomal analysis. The more likely explanation, however, is a partial reactivation of the inactive human X chromosome. The failure to find this phenomenon in the human-mouse RAG hybrids can be explained in several ways. The human 89A99c and the two 194RAG hybrids are different in both fusion partners, and they are different in their human chromosomal content. Studies to detect the crucial difference are in progress. Our results support the previous report of K ahan and D e M ars- on the possible reactivation of part of an inactive X chromosome in humanmouse somatic cell hybrids. This possibility should be taken into con sideration when X chromosome mapping is performed using somatic cell hybrids.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/19/2018 3:35:05 PM
H ellkuiil , G rzeschik
530
H ellkuiil , G rzesciiik
Regional mapping of X chromosome
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/19/2018 3:35:05 PM
1 L yon, M .F.: Gene action in the X-chromosome of the mouse. Nature, Lond. 190: 372-373 (1961). 2 K ahan, B. and D eM ars, R.: Localized derepression on the human inactive X chromosome in mouse-human cell hybrids. Proc. natn. Acad. Sci. USA 72: 1510-1514 (1975). 3 van D iggelen , O.P. and Shin , S.: A rapid fluorescence technique for electro phoretic identification of hypoxanthine phosphoribosyltransferase allozymes. Biochem. Genet. 12: 375-384 (1974).
Partial reactivation of a human inactive X chromosome in human-mouse somatic cell hybrids B. H ellkuhl and K.-H. G rzfschik Institut für Humangenetik der Universität Münster
Supported by the Deutsche Forschungsgemeinschaft (GR 373/8).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/19/2018 3:35:05 PM
Several authors have put forward theories explaining the mechanism of inactivation of one X chromosome in mammals that underlies the Lyon hypothesis.1 In these theories, more attention is given to the question of which of the two X chromosomes is chosen for inactivation than to the problem of how, once the decision has been made, inactivation of the same chromosome is maintained in somatic cells. In order to investigate factors which could influence the maintenance of inactivation, we tried to reactivate inactive human X chromosomes present in human-mouse somatic cell hybrids in an approach slightly different from the one followed by K ahan and D f.M ars .2 In human-mouse cell hybrids involving human female cells, the human inactive X chromosome is frequently lost early in hybrid develop ment, and there is no possibility of selecting for this “silent” chromo some. Examining our collection of hybrids between mouse cells and human female fibroblasts for the retention of inactive X chromosomes, we found two hybrids (194RAG5 and 194RAG6) that were both fusion products of mouse HPRT- RAG cells and human fibroblasts from an (X;3) translocation carrier (GM 194; Human Genetic Mutant Cell Repository, Camden, N.J.). In some of the parental cells the structurally normal X, and in the others the (X;3) translocation chromosome, was late-replicating. The two hybrid populations mentioned above represent both cases. We received a third hybrid with an inactive human X chro mosome (89A99c) from Dr. M. Siniscaixo, New York, N.Y.
H ellkuiii ., G rzesciiik
Regional mapping of X chromosome
This hybrid line was a fusion product of mouse HPRT~, APRT A9 cells and human fibroblasts from an X/19 translocation carrier (GM 89). In the GM 89 parental cells, the normal X was late-replicating. In all three hybrid lines the frequency of metaphases in which the inactive X chromosome (Xi) was the only X present was too low to isolate clones of this cell type by cloning the mixed population. Therefore, mass cultures of all three hybrid lines were treated with 8-azaguanine (8-AG) for 4 days. They were then cloned, and the clones were analyzed by Q-banding for their chromosome content and by Cellogel electro phoresis for the expression of human X-chromosomal enzyme markers (G6PD, PGK, and HPRT). From each hybrid line, two clones (Xi * clones) were isolated with an inactive X chromosome and without the active X or the active fragments of the X and, as controls, one parallel clone without an inactive X and without the translocated part known to carry the X-chromosomal gene HPRT (Xi- clone). The Xi* and Xi* clones from all three hybrid lines were subjected to selection in HAT medium (10-4 m hypoxanthine, 4X 10~5 m azaserine, and 1.6 X 10-5 M thymidine). In the hybrid line 89A99c, HAT-resistant subclones appeared from both Xi" clones with a frequency of 10 °, and no HAT-resistant subclones could be detected in 6 X l()l! cells of the parallel Xi - clone. In a different set of experiments, one HATresistant subclone was found in the Xi clone of hybrid line 89A99c after treatment of 1.2 X 10fi cells with a mutagen (ethyl methanesulphonate). In the two hybrid lines 194RAG5 and 194RAG6, containing, respec tively, a structurally normal inactive X chromosome and an inactive (X;3) translocation chromosome, no HAT-resistant subclones were found in 8.5 X 10° cells each. The HAT-resistant subclones of hybrid line 89A99c were analyzed for X-chromosomal enzyme markers and human chromosomes. Twentythree HAT-resistant subclones of both Xi* clones and the only HATresistant subclone of the Xi clone were tested for human HPRT, G6PD, and PGK. In electrophoresis for HPRT,1 all 23 subclones of both Xi* clones showed mobility of the human HPRT protein, and the one subclone of the Xi- clone showed mobility of the mouse protein. No human G6PD and PGK activities could be detected in the subclones tested. In a backselection system, 8-AG-resistant subclones of HAT-resistant (HPRT*) X i+ clones and the HPRT* Xi- clone grew with a fre
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/19/2018 3:35:05 PM
528
Regional mapping of X chromosome
529
quency of 5 X 10-3 for the X i+ clones and 10-5 for the Xi- clone. The frequency of 8-AG-resistant subclones of the HPRT+ Xi- clone can be explained by mutational events. The 8-AG-resistance in the HPRT+ Xi + clones is more frequent than would be expected if it were caused by mutational events. Instead, it might be consistent with the well-known segregation of a human chromosome carrying the HPRT gene from the human-mouse cell hybrids. The electrophoretic mobility of the HPRT enzyme in the X if and Xi- clones and the behavior of the HPRT- clones in 8-AG-selection support the assumption that the HPRT enzyme in the X i+ clones is of human origin. This explanation is further supported by chromosome segregation analysis: Nine HPRT+ subclones of both X i+ clones were analyzed before and after backselection in 8-AG medium for the presence of human chromo somes by Q-banding. In HAT-medium they all retained an obviously unmodified human X chromosome, but they all lost this human X chro mosome after the 8-AG treatment. The autosome pattern in the 8-AGtreated cell lines was also altered, but all autosomes present in any of the HPRT- clones were found in at least one of the 8-AG-treated cell lines. These results provide evidence of a strong cosegregation of the HPRT enzyme activity only with the human "inactive” X chromosome in the X i+ clone derivatives. The data derived from the experiments with the 89A99c hybrid line might be explained by a mutation of the HPRT gene of the active X chromosome to an enzymatically inactive form and by translocation of the little piece of this chromosome carrying the mutated HPRT gene but not the G6PD gene onto just the inactive X chromosome. This piece presumably would be too tiny to be detected in the chromosomal analysis. The more likely explanation, however, is a partial reactivation of the inactive human X chromosome. The failure to find this phenomenon in the human-mouse RAG hybrids can be explained in several ways. The human 89A99c and the two 194RAG hybrids are different in both fusion partners, and they are different in their human chromosomal content. Studies to detect the crucial difference are in progress. Our results support the previous report of K ahan and D e M ars- on the possible reactivation of part of an inactive X chromosome in humanmouse somatic cell hybrids. This possibility should be taken into con sideration when X chromosome mapping is performed using somatic cell hybrids.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/19/2018 3:35:05 PM
H ellkuiil , G rzeschik
530
H ellkuiil , G rzesciiik
Regional mapping of X chromosome
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/19/2018 3:35:05 PM
1 L yon, M .F.: Gene action in the X-chromosome of the mouse. Nature, Lond. 190: 372-373 (1961). 2 K ahan, B. and D eM ars, R.: Localized derepression on the human inactive X chromosome in mouse-human cell hybrids. Proc. natn. Acad. Sci. USA 72: 1510-1514 (1975). 3 van D iggelen , O.P. and Shin , S.: A rapid fluorescence technique for electro phoretic identification of hypoxanthine phosphoribosyltransferase allozymes. Biochem. Genet. 12: 375-384 (1974).
