Zbornik - Društvo genetičara Srbije

52 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE II-Usm-5
Subotica, 30. novembar - 4. decembar 2004.
STRUKTURNA I FUNKCIONALNA ANALIZA PROMOTORA
HUMANOG SOX14 GENA
Jelena Ðuroviæ, Mina Seoviæ, Isidora Petroviæ i Milena Stevanoviæ
Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd
SOX14 je èlan SOX familije gena koji kodiraju transkripcione regulatore ukljuèene u
kontrolu procesa razviæa. Poznato je da su mutacije u SOX genima odgovorne za pojavu
genetièkih sindroma kod èoveka, što ukazuje na njihovu vanu ulogu u procesu razviæa. I
ako ekspresioni profil SOX gena ukazuje da je njihova ekspresija specifièna za tkivo i
stupanj razviæa, vrlo malo se zna o mehanizmima koji regulišu ekspresiju ovih gena.
U cilju prouèavanja molekularnih mehanizama koji regulišu transkripciju humanog
SOX14 gena, odreðen je start transkripcije metodom elongacije prajmera. Dobijena su
dva produkta elongacije prajmera duine 275 nukleotida i 194 nukleotida. Ovi rezultati
ukazuju da transkripcija otpoèinje od guanina na poziciji -251 bp u odnosu na start
kodon, mada postojanje starta transkripcije na poziciji -170 ne moe biti iskljuèeno.
Identifikacija regiona koji pokazuje bazalnu promotorsku aktivnost kao i proksimalni
enhenser, odreðeni su sposobnošæu konstrukata sa 5’ nekodirajuæim regionom SOX14
gena da indukuju ekspresiju CAT reporter gena. Ovim eksperimentima pokazano je da je
region izmeðu -470 i +201 odgovoran za bazalnu aktivnost SOX14 promotora.
NF-Y je transkripcioni faktor vaan za regulaciju transkripcije gena koji ne poseduju
TATA boks. U ovom radu pokazano je da se NF-Y vezuje za CCAAT motiv prisutan u
promotoru SOX14 gena. Mutacionom analizom pokazano je da je CCAAT motiv
funkcionalan i znaèajan za regulaciju transkripcije SOX14 gena.
STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE
HUMAN SOX14 PROMOTER
SOX14 is a member of SOX gene family of putative transcriptional regulators implicated
in the control of diverse developmental processes. Mutations in SOX genes are known to
be responsible for human genetic syndromes, demonstrating the critical role that they
play in normal embryonic development. Although SOX gene expression patterns suggest
tissue- and development stage-specific control of gene expression, little is known about
mechanisms responsible for expression of these genes.
In order to elucidate the molecular mechanisms controlling the expression of the human
SOX14 gene, we have determined the transcription start site using primer extension
method and carried out the structural and functional analysis of the regulatory region responsible
for its expression. Two distinct primer extension products were identified: the
single major product that migrates with a length of 275 nt and the minor product 194 nt in
size. This result indicates that the transcription of the SOX14 initiates at the single major
site at the guanine residue 251 bp upstream of the start codon, although the existence of a
minor transcription start site can not be excluded.
To identify the DNA regions responsible for the control of SOX14 gene transcription we
have analyzed the ability of truncated fragments from the 5’ flanking region of the
SOX14 gene to drive expression of CAT reporter gene. Functional mapping of the 5’ regulatory
region revealed that the sequence between -470 and +201 bp is essential for minimal
basal activity of the SOX14 promoter.
NF-Y is a transcriptional factor involved in transcription of TATA-less genes. We have
identified that NF-Y binds to the CCAAT box motif present in the regulatory region of
the SOX14 promoter. By mutation analysis we have shown that CCAAT box motif present
in the SOX14 promoter plays a functional role in the transcription of this gene.