The Temporal Change Across the Class II/III Boundary 1 Is there a sharp switch in replication timing?

We detected a change in replication time across the GC/AT transition region separating the class II's L2 isochore from the H3 isochore of the class III region. This change, seen in both B-cells and fibroblasts, is consistent with that reported by Tenzen et al

(1997) in the same part of the MHC but identified using Southern analysis of nascent DNA from synchronized cells. Whilst both methods identify a temporal change, we would disagree with Tenzen's interpretation of the change and its significance.

Tenzen et a l (1997) used two rounds of synchronization with aphidicolin prior to releasing human myeloid leukaemia cells (HL60) into S-phase and labelling them with BrdU at Ih intervals for 6h. They isolated and quantitated the nascent DNA in each sample using competitive PCR of markers within the class H/m region's boundary. The PCR products were stained with a fluorescent dye and the relative intensities of the signals in each sample were presented graphically. However, even with the data presented in this way, some loci could easily be assigned the same replication time. One has the impression that the synchronization was not a tight, narrow peak passing quickly into and out of each fraction, but was broader and included contaminating cells from other stages of S-phase. This effect may be due to a proportion of the HL60 cells taking longer to enter S-phase after two rounds of aphidicolin.

It is important to note that the Ih interval separating the samples was, by definition, the limit of Tenzen et a l's temporal resolution. The assay could not, therefore, be expected to distinguish any difference between sequences of less than one hour. FISH does not have such a restriction because in the fixed samples it uses, time is essentially frozen and the entire S-phase is deliberately used to estimate the mean replication time of each locus. The only potential limitation with FISH is the time it takes for a replicated locus to resolve into a pair of fluorescent spots on the sister chromatids.

How long after replication it takes to detect doublets, and whether or not this time is constant for different sequences, has not been formally addressed by any investigators.

Tenzen et a l (1997) reported a difference of one hour between two primer sets just 16Kb apart in the GC/AT transition area separating the class II and HI regions (co­ ordinates -200-230 Kb, Figure 4.7). 3 O e O) c 2- 3 c O cd 4 g a . <P c . INT3A

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Figure 4 .7 . Adapted from Fig 4 in Tenzen et a l (1997). The y axis represents the replication timing; the time when S-phase was started by removing the aphidicohn from the medium (time 0). The interval corresponding to the highest peak of each graph is plotted as the replication time of the respective locus. Primer sets PCD and INT3A separated by -16Kb, are indicated. Markers used on our replication profiles are shown below the x axis (refer to Fig 4.6)

Although the primer sets PCD and INT3A appear clearly separated on Figure 4.7, this plot is of the single Ih interval with the highest intensity PCR product, and does not reflect the overlap between adjacent intervals so apparent in the raw data. When one looks at the distribution of the signal across all six Ih intervals and compares the distributions for PCD and INT3A, it is hard to believe that their replication times are really that different. In addition, the difference measured between PCD and INT3A is at the limit of the assay's temporal resolution. Therefore, Tenzen et a l's results do not

exclude the possibility that PCD and INT3A actually replicate within an hour of each other. Tenzen et al's raw data are more consistent with the view that PCD and INT3A have similar replication times because they are only 16Kb apart.

The apparent one hour's difference across 16Kb, was termed by Tenzen et a l a "precise switch" in replication timing. The word "switch" refers to the difference of an hour and "precise" to the suggestion that it occurs across just 16Kb in the region of GC/AT transition separating the isochores of the GC-poor class II and GC-rich class III regions. They suggested that the "precise switch" was evidence that the boundary between the class II and III regions is not merely between different isochores but different cytogenetic bands. Based on the previous observation of a small G-subband within 6p21.31 (Yunis, 1981), it was suggested that a chromosome band boundary existed between the class II and class III regions and - since R-bands replicate early and G-bands late - that this boundary would be "precisely assignable at the nucleotide level by identifying the early-to-late switch point for replication timing" (Fukagawa et a l, 1995). Tenzen et a l appear to have interpreted their replication timing data in order to support this hypothesis. Chapter 1 details why the structure, function and organization of chromosomes is more complicated than the popular cliché "GC-rich R- bands and GC-poor G-bands" suggests. We must, therefore, avoid interpreting replication timing data using this and other similarly out-moded ideas.

Aside from the difficulties with the hmited temporal resolution, it is difficult to subscribe to the idea - based on such experiments - that something as relatively massive as a chromosome band boundary can be described to within a few specific nucleotides. There seems httle sense in higher eukaryotes being so strict about where replication stops when they seem relatively relaxed about where it starts (Vaughan et a l, 1990; DePamphilis, 1999). Nor does it makes sense for the replication machinery to replicate the 1Mb class III region and then stop short of the class II region simply because it has a slightly lower GC content. Considering the wide range of functions

and properties attributable to isochores and chromosome bands, their physical boundaries are most likely composed of a number of elements including sequence motifs and GC changes, but also perhaps particular chromatin structures.

In document Replication Timing Analysis of the Major Histocompatibility Complex on Human Chromosome 6 (Page 91-94)