Since the 1960s, when inter-specific somatic cell hybrids were first described [21
], the progressive loss of human chromosomes in murine cells has represented a puzzling problem. In some of the hybrids, most chromosomes were lost and only a few displayed a higher stability without acquiring murine DNA [22
]. More recently, studies on artificially size reduced minichromosomes and HAC in rodent cells, have shown large differences in stability between clonal lines, even when they are derived from the same parent [6
]. In some cases [7
], the minichromosomes lacked essential centromere and kinetochore components, thus explaining the high loss rate observed in murine cells in absence of selection. In most other cases, however, the essential proteins CENP C and CENP A were present at apparently normal levels, but the HAC or minichromosome still displayed high levels of instability [6
In this study, we analyzed the chromatin organization and segregation behavior of two HAC in human and murine cells, with the aim of identifying factors important for correct chromosome function. The human HAC AG6-1, containing 17α DNA and the HPRT
genomic locus [16
], and LJ2-1, containing 17α DNA only [17
] were transferred to murine cells by MMCT. HAC Sag 1.1, Sag 1.2, Sag 2.2, and Sag 2.3 (derived from HAC AG6-1) and SM1-1 (C6) (derived from HAC LJ2-1) respectively were generated. To rule out that differences between HAC in murine cells could be the effect of biochemical variations between cell lines, STO cells were used for both experiments. They are immortalized embryonal fibroblast cells derived from the SIM mouse, and are often used as feeder cells for murine and human ES cell culture [23
To determine the possible causes for the differential stability displayed by the HAC in the murine cells, we first investigated if the HAC bound CENP A, a protein that plays a key role in centromere formation and chromosome segregation. All the HAC bound CENP A in an amount similar to each other and to the human or murine endogenous chromosomes. We then characterized the histone modification associated with either euchromatin (H3diK4) or heterochromatin (H3triK9). All HAC, in both human and murine cells, were euchromatic in composition, but also contained a heterochromatic domain. Similarly, the histone H3 modifications associated with chromosome condensation prior to mitosis (H3phosphoSer10/Ser28) were also present on all HAC.
We investigated the nuclear positioning of the HAC in murine cells as it is important for key processes such as DNA replication, and chromosome segregation. A statistical analysis of the HAC position in the nucleus of STO cells was undertaken on SM1-1 (C6), Sag1.1, Sag1.2, Sag 2.2, and Sag 2.3. In all the murine clones analyzed, the HAC displayed a differential level of stability, even when deriving from the same parental HAC (Sag clones). The HAC Sag 1.1 was the most stable (loss rate 0.92%) and SM1-1 (C6) was the least stable (loss rate 5.2%). The position of the HAC in the nucleus, and more specifically the association with the chromocenters correlated with HAC stability. The HAC that associated more frequently with the chromocenters i.e. Sag 1.1, Sag 2.2 and Sag 2.3, have a lower loss rate compared to SM1-1 (C6), which was not localized with the chromocenter. The data show that the positioning of HAC in the nucleus of murine cells was important for correct chromosome segregation. Also, the HAC that colocalize less frequently with chromocenters displayed intraclonal variable levels of H3triK9 in different cells. The H3triK9 variability in turn, correlated significantly with the loss rate. The H3trik9 modification is fundamental for maintenance of chromatid pairing at the centromere [7
], and it is linked to determination of the replication timing. The frequency of segregation errors (non-disjunction events) in the murine HAC clones correlated significantly with the H3triK9 variability. The HAC in murine cells tended to replicate late in the S phase, which correlated with increased HAC loss rate. It is possible that this behavior is linked to the incorrect epigenetic marking of the HAC, however there is no direct evidence to support this. It may be that the human HAC replication origins are not fully functional in murine cells, or are recognized less efficiently. The murine origins of replication would compete for the replication factors machinery, that hence would become accessible to the HAC sequences only when most of the host DNA has been replicated.
Although a significant statistical correlation does not indicate that there is a causality link between two variables, based on the data we obtained, it seems likely that when a high percentage of HAC do not associate with the chromocenter, they are removed from the nuclear environment that allow the deposition of the correct heterochromatic marker. The resulting variability in the H3triK9 deposition is responsible for the high loss rate displayed by HAC in murine cells, due to segregation errors and/or incorrect replication timing. On the other hand, it is possible that the more stable HAC are the ones that in the initial replications, after the MMCT, assembled the correct levels of heterochromatin, and because of that they then localized in the heterochromatin rich chromocenter. However, recent publications, that show the existence of a pericentric heterochromatin duplication body [24
], lend support to the theory that it is necessary for the HAC to be localized within the chromocenter to guarantee that they are replicated at the correct time, and are modified with the appropriate heterochromatin markers.
But what is the mechanism that leads some of the HAC to localize within a chromocenter while others do not? The chromocenter structure is re-formed, after each cell division, in the early G1 phase [24
]. It is possible then that the different murine HAC clones were generated by MMCT in cells in different phases of the cell cycle, and synchronization of the receiving murine cells may be important in determining if this is the case. Alternatively, other unknown factors may be responsible for the difference in nuclear positioning of the HAC. The HAC SM1-1 (C6) characterized by the highest loss rate, is composed mostly of 17α DNA, while the other murine HAC, derived from AG6-1 also contains large non-satellite sequences corresponding to the human HPRT genomic locus. The different composition may play a role in determining the localization of the HAC in the nucleus. For larger human chromosomes, there seems to be a preferential positioning in the murine nucleus related to gene richness [26
], or to mimic the position of the mouse syntenic chromosomes [27
]. It is possible that the HAC analyzed in this study are too small, or have no syntenic correspondent in the mouse genome and so are randomly allocated in the nuclear structure. Also, we cannot rule out that rearrangements in the HAC chromosome structure during the MMCT procedure (such as in HAC SM1-1 (C6) following transfer from LJ2-1) may interfere with epigenetic markings conferring its heterochromatic characteristics to the HAC pericentromeric region, and as a result the HAC are not correctly positioned in the mouse nucleus. All of the HAC characterized in this study are most likely circular [16
]. Thus, topological constraints may have an effect on their stability.
Finally, it is possible that protein components of the murine kinetochore are not fully capable of recognizing the human satellite sequences, and thus formed a partially functional centromere which was responsible for the HAC loss. However, the data obtained by the direct generation of HAC in murine cells [2
], suggested that, at least in this case, the HAC were able to form a centromere/kinetochore structure and segregate correctly. On the other hand, the stability of these HAC constructs could be due to unknown, specific characteristic of the murine receiving parental. In this light, other reports [9
] that show differential stability of HAC transferred to murine ES cells could be explained by genetic differences in the mouse strain from which the ES derive.