Replication of the MHC in B-cells

4.3.1.1. The MHC is not entirely early replicating in B-cells

Figure 4.1 shows the replication profile for the entire MHC in lymphoblastoid B-cells. We can see that our original hypothesis - that the MHC will be entirely early

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Figure 4.1. The replication profile of the entire MHC in B-cells. The data points are the mean relative replication time (expressed as % singlets) with the bars representing +/- 2 standard errors. The internal controls PYGM (early) and A9.5 (late) are indicated by the dotted lines. The approximate positions of a number of landmark MHC loci are indicated below the graph.

replicating in B-cells - is not supported by these data. The extended class II, the class III and the class I regions all replicated relatively early as predicted. Looked at as distinct classes, the mean replication times across their lengths were 0.36 +/- 0.01, 0.34 +!- 0.05 and 0.40 +!- 0.04, respectively. However, the classical class II region confounded our prediction. The first -200Kb of the classical class II region replicated early (300-500Kb on Fig 4.1); it then underwent a transition to later replication (500- 700Kb on Fig 4.1) which was maintained through its remaining 200Kb. As a distinct class, its overall replication time was later than the adjacent classes at 0.51 +/- 0.11. And unlike its neighbours, whose replication times remained relatively constant, the mean replication time of the classical class II region ranged from 0.32 to 0.66, which is reflected in its greater standard deviation.

4.3.1.2. B-cells have a temporal boundary in their MHC

The late replication of the HLA-DQ/DR locus contrasts the early replication seen at the proximal end of the class III region (~ 1200Kb on Fig 4.1 (though it may be seen in greater detail in Fig 4.6). Finding a temporal boundary between the classical class II and class III regions in B-cells was unexpected, since we had expected the entire classical class II region to be early replicating. The transition from early to late replication timing was coincident with the region of GC/AT transition that separates the isochores making up the two classes (Fig 1.2). One mechanism to account for this progressive change in replication time would be the sequential activation of replicons within the transition region. Another would involve a single replication fork progressing from the last replicon in the proximal class III region into the distal end of the classical class II region.

The duration of S-phase could not be determined in the B-cells because they were refractory to ceU synchronization. However, if we accept that mammalian S-phase is between 6-8 hours, we can assign replication times in terms of hours and minutes and

estimate the approximate rate of replication. The last probe of the classical class II (LH l, see Fig 4.1 or detailed in Fig 4.6) and the first in the class III (C47, see Fig 4.6) are separated by ~ 125Kb and 18% singlets - or 18% of S-phase. This equates to between 1 and 1.5 hours. This suggests that replication has occurred from C47 to LHl at a rate of about 125 kb/h or between 1.5-2 Kb/min, well within the accepted range of 0.3-6 Kb/min for mammahan replication fork movement (Edenberg & Huberman, 1975). Alternatively, this ~ 125Kb region could be rephcated by numerous minute replicons, with the first initiating around C47 and the rest firing sequentially in the direction of LH l.

Within the classical class II, the difference between its early portion (average of probes Mann3.6 to HA14, 0.38 +/- 0.05) and its late portion (FI 121 to L H l, 0.62 +!- 0.03) is approximately 1.5 to 2h (see Fig 4.1). Therefore, a single fork may have taken 1.5 to 2h to replicate -250Kb. If so, -250Kb in 90-120 minutes (i.e. 2-2.7 Kb/min) is again consistent with the estimate of Edenburg & Huberman (1975). It is important to note that this transition in rephcation time in the proximal half of the classical class II region is not coincident with the isochore boundary separating the classical class II and extended class II regions. This transition in GC content is approximately 400Kb more centromeric (see Fig 1.2).

To summarize, the class II region's LI isochore had both early and late rephcating portions in B-cells. There was a change from early to late replication from the class III region into the distal end of the classical class II region. A similar change in replication time from early to late was seen across the middle of the classical class II region itself; however, this did not coincide with the isochore boundary known to exist between the classical and extended class II regions. In both instances the observed rate of change in replication time linking the early and late replicating portions was consistent with the previously reported rate for eukaryotic replication forks (Edenberg & Huberman,

4.3.1.3. B-cells express class II molecules

S ince tissu e -sp e c ific gen es are th o u g h t to re p lic a te e a rly w h en they are e x p re sse d , th ere

w ere tw o p o ssib le e x p la n a tio n s fo r the re la tiv e ly late re p lic a tio n o f th e H L A -D Q /D R

locus. E ith e r the B -cells w e re not ex p re ssin g th e H L A -D Q / D R m o le c u le s, o r w e h ad

an ex c e p tio n to the ru le that tissu e -sp e c ific g en e c lu ste rs re p lic a te ea rly w h e n

tran sc rib ed . N o rth e rn a n a ly sis and im m u n o flu o re sc e n c e d e te c tio n w e re u sed to te st the

B -cells fo r ex p ressio n o f th e D R A gen e (e n c o d in g the a su b -u n it o f th e H L A -D R h e te ro d im e r)a s d e sc rib e d in C h a p te r 2. AHB MRC5 n a 24 24 - 48 48 IFN-g (h) - 48 - - 48 C h a s e na «

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Figure 4.2 . N o rth e rn a n a ly sis o f D R A m R N A in B -ce lls and fib ro b la sts. L a n e 1 d e m o n stra te s the c o n stitu tiv e ex p ressio n o f the H L A -D R A g en e in B -cells. L a n e 4 show s th at H L A -D R A is n o t tra n sc rib e d in th ese fib ro b la sts (see 4 .3 .2 .2 ). T h e u b iq u ito u sly ex p ressed p -a c tin gen e w as used as a co n tro l. T h e sig n a ls in the re m a in in g la n e s w ill be d isc u sse d later.

T h e p o sitiv e N o rth e rn resu lt in lane 1 (F ig 4 .2 ) sh o w s th at the B -ce lls w ere ac tiv e ly

ex p ressin g the D R A gene. F ig u re 4.3 (b e lo w ) illu stra te s th at the resulting H L A -D R m o le c u le w a s ex p re sse d on th e su rface o f th e B -cell. H ere B -ce lls w ere in cu b ate d w ith

a F IT C -la b e lle d an tib o d y d ire c te d ag a in st the a - s u b u n it o f the D R m o le c u le (see

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Figure 4.3 . Im m u n o flu o re sc e n c e an a ly sis o f D R A ex p ressio n on B-cells. Red: n e g a tiv e c o n tro l, B -ce lls w ith o u t a n ti-D R A a n tib o d y . B lue: B -cells in cu b ate d w ith anti- D R A -F IT C are sh ifted alo n g the lo g a rith m ic flu o re sc e n c e scale d ue to sp ec ific bin d in g o f la b e lle d a n tib o d y .

It is c le a r fro m th e ab o v e resu lts th at the B -c e lls used in th ese e x p e rim e n ts not only

tra n sc rib e d th e //L A -D /? A gene but, as p ro fessio n al a n tig e n -p re se n tin g c e lls, tran slate d

it into an e x p re sse d D R m o lecu le. S uch p o sitiv e re su lts c o n tra st w ith the re p lic atio n

tim e p ro file fo r th e H L A -D Q /D R lo c u s, w h ich sh o w s it to be re la tiv e ly late re p lic atin g

and, th e re fo re , an ex c ep tio n to the rule th at tissu e -sp e c ific gen e c lu ste rs re p lic a te e a rly

w hen e x p re sse d (H a tto n e t a i, 1988).

In document Replication Timing Analysis of the Major Histocompatibility Complex on Human Chromosome 6 (Page 76-81)