Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...

M2F:Co Crrt6 Cdrtot ot H4G.n. fn.piDtion
we ntrodlced 7 d srinct nlcleo de subslturion mdar ons (mutanl Mc-7) i|to
Sne rl. These subsriruions are locared ar ksy cdlseMd .ucleotad6s between
.r .97 and .77, and 6nconpass a seies oI /n vpo G residus proleln/DNA
conracrs fial hav€ been derecr€d in jn6ct c€lls (Paullel a/., 1987). Mosr ol
rhese murations oc.ur at methylaiion inrgrlgrence contacls ior HiNF-M/|FF-2,
HiNF-P/H4.TF2 and/orHiNF-D (Figs.5 and 6).
Gel shitt compelltion assays show lhat these mdalons when
i.corporared n a sir€ lr origonlcigoride (Mc.7) abolish bindng ot H NF-M/rFF-
2 and HiNF P/Ha-TF2, as wellas sev€rely r€duce bindlng o, HjNF.D to Sle ll
(Frg. 25) Honce. wrren lhis ori9onucl6o da is cron€d inlo a chrmeric H4
promorer/cAT construct rhe coresponding murant reponer gene consrrlcl
(MC'7ICAT) repr€sen$ a rripl€ mula wirh respecl to rhe binding o' these
werL characte zed Sile 'l bind ng prol€ins. For di.6cl companson, we also
i.corporated a w ldtype Sr€ ll oligonucleotide OM-3; see Mar€ es and
Melhods) inlo lhe same h4 p.omote./CAT veclor. The wildlype TM.3 plobe
medrales he exp€cled bindng ol HiNF M,.P and -O in gelshitl assays in Mo
ditferenl .unnlng condnions (Fig. 24). Oir€ct compaison ol CAT assay resu Is
with rcporter gen6 consLucts contai^ing the naural hi$one H4 promorer and
rh€ wildrype TM.3 p.omoter ar6 nor srarisrics ly difisr€nt (dala nol shown).
Upon transienl translectio. in prolileralirg Heta 53 c€lls, mutanl MC-7/CAT
dlsplays up to 10 lold reduc€d promoter adivily relatiw to lhe wildlypo
conslrucl TM.S/CAT (Fig 28). Thls !h6 r€cognirion morls n Site ll which
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