Characterisation and Functional Studies of the DNA Cytosine-5-Methyltransferase 3-like Gene

Abstract

DNMT3L-GEENIN KARAKTERISOINTI JA TOIMINTA

Epigenetiikka tutkii DNA:n emäsjärjestyksestä riippumattomia geenin toimintaan vaikuttavia perinnöllisiä muutoksia. DNA:n metylaatio on epigeneettinen säätelymekanismi, joka vaikuttaa geenien ilmentymiseen, alkiokehitykseen ja syövän syntyyn. Nisäkässoluissa DNA:ta metyloivat neljä erilaista DNA-sytosiini-5-metyylitransferaasi-entsyymiä, DNMT1, DNMT2, DNMT3A ja DNMT3B.

Tutkimuksessa tunnistettiin DNMT3-geeniperheeseen kuuluva uusi geeni, DNMT3L (DNA cytosine-5-methyltransferase 3-like) ja selvitettiin Dnmt3L-geenin säätelyyn vaikuttavat promoottorirakenteet. Lisäksi osoitettiin, että DNMT3L estää muiden geenien ilmentymistä sitoutumalla transkription säätelyssä toimivaan histonideasetylaasi 1­proteiniin.

Tutkimustulokset ja niiden pohjalta muualla tehdyt jatkotutkimukset ovat auttaneet ymmärtämään epigeneettisen säätelyn toimintamekanismeja.

DNA methylation is important in the regulation of gene expression, embryonic development, genomic imprinting and X chromosome inactivation. Abnormal methylation has been associated with carcinogenesis. So far, four separate genes, DNMT1, DNMT2, DNMT3A and DNMT3B encoding DNA cytosine-5-methyltransferases have been cloned from mammalian cells. DNMT1 is a maintenance methyltransferase, copying existing methylation patterns into newly replicated DNA strand. Dnmt3a and Dnmt3b are required for genome-wide de novo methylation and are essential for mammalian development. Gene expression is also regulated through modification of histone proteins that are essential for folding and packing of DNA inside the cells. Usually, histones are modified by acetylation, methylation and phosphorylation. In this thesis we have identified a novel member of the DNMT3 gene family, DNA cytosine-5-methyltransferase 3-like gene (DNMT3L). DNMT3L protein is similar to DNMT3A/Dnmt3a and DNMT3B/Dnmt3b, sharing the greatest similarity with other DNMT3 family members at the zinc finger region in the N-terminal part of the DNMT3L/Dnmt3L. In order to perform functional studies, the mouse Dnmt3L gene was also identified. Human and mouse genes showed high identity with each other and were both strongly expressed in testis and to a lesser extent also in ovary, thymus and fetal tissues. To study the regulation of Dnmt3L, we isolated the Dnmt3L promoter region and identified a minimal promoter area and regulatory elements in it. We demonstrated that Dnmt3L was regulated through Sp1/Sp3 transcription factors and epigenetic chromatin modifications, DNA methylation and histone deacetylation. Since Dnmt3a and Dnmt3b function as transcriptional repressors using zinc finger-domain to interact with histone deacetylase, HDAC1, we tested the repressional capacity of DNMT3L. Our data indicated that DNMT3L represses transcription by binding directly to HDAC1 protein and demonstrated the PHD-like zinc finger as a main repressional domain of DNMT3L.