Zbornik - Društvo genetičara Srbije

46 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE II-Uvo-3
Subotica, 30. novembar - 4. decembar 2004.
IDENTIFIKACIJA KONTROLNIH ELEMENATA UKLJUÈENIH U
REGULACIJU EKSPRESIJE SOX3 GENA RETINOIÈNOM KISELINOM
Marija Mojsin, Nataša Kovaèeviæ-Grujièiæ, A. Krstiæ i Milena Stevanoviæ
Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd
SOX geni pripadaju familiji gena koji pokazuju homologiju sa SRY genom (sex-determining
gene) sisara. Ova homologija odnosi se na region oznaèen kao HMG domen koji
je odgovoran za vezivanje SOX proteina za specifiènu DNK sekvencu u kontrolnim
regionima ciljnih gena.
NT2/D1 je permanentna æelijska linija humanog teratokarcinoma. Ove æelije su na stadijumu
opredeljenih neuralnih progenitora, a retinoièna kiselina indukuje njihovu diferencijacija u
neurone. Ova karakteristika èini NT2/D1 æelije jedinstvenim model sistemom za prouèavanje
humanih gena ukljuèenih u procese diferencijacije nervnog sistema.
Ovaj model sistem je korišæen za prouèavanje transkripcione regulacije SOX3 gena i
njegove ekspresije tokom neuralne diferencijacije. Pokazano je da u ranim fazama
neuralne diferencijacije NT2/D1 æelija, indukovanih retinoiènom kiselinom, dolazi do
aktivacije SOX3 gena. Naš cilj bio je da identifikujemo kontrolne elemente odgovorne za
vezivanje transkripcionih faktora i elemente preko kojih retinoièna kiselina indukuje
aktivaciju SOX3 gena. Korišæenjem nuklearnih ekstrakata izolovanih iz neindukovanih i
indukovanih NT2/D1 æelija uraðene su metoda otisaka proteina na DNK i metoda usporene
pokretljivosti u gelu. Rezultati ovih eksperimenata ukazuju na postojanje razlika u
formiranju kompleksa DNK-protein u neindukovanim i indukovanim NT2/D1 æelijama, i
omoguæavaju identifikaciju kontrolnih elemenata odgovornih za aktivaciju transkripcije
SOX3 gena retinoiènom kiselinom.
IDENTIFICATION OF CONTROL ELEMENTS INVOLVED IN
REGULATION OF SOX3 GENE EXPRESSION BY RETINOIC ACID
SOX genes comprise a family of genes related to the mammalian sex-determining gene
SRY in the region that encodes the HMG-box domain responsible for the sequence-specific
DNA-binding activity. Spatial and temporal expression of SOX genes suggests they
play roles in the regulation of development and differentiation. The developing brain is
the major site of expression of many SOX genes. During mammalian embryogenesis,
SOX3 gene is predominantly expressed in developing nervous system.
NT2/D1 is human teratocarcinoma cell line that resembles human committed neuronal
progenitor cells that can be induced to differentiate into neurons by retinoic acid. Thus,
retinoic acid-induced differentiation of NT2/D1 providing a unique in vitro model for
studying human genes that promote and regulate neural differentiation.
We used this model to study transcriptional regulation of SOX3 gene expression during
neuronal differentiation of NT2/D1 cells. It was shown that retinoic acid-induced differentiation
of NT2/D1 cells is marked by activation of SOX3 gene during early phases of
induction. In order to determine regions recognized by transcription factors and to identify
control elements responsible for retinoic acid induction of SOX3 gene we performed
in vitro DNaseI footprinting analysis and gel mobility shift assays with nuclear extracts
from uninduced and induced NT2/D1 cells. These experiments showed significant difference
between protein-DNA complexes in undifferentiated and differentiated NT2/D1
cells allowing us to identify control regions that mediate a stimulatory effect of retinoic
acid on SOX3 gene expression.