Characterization of repression capability of Sin3-associated protein 30-like and its alternatively spliced isoforms
KORKEAMÄKI, HANNA (2008)
KORKEAMÄKI, HANNA
2008
Biokemia - Biochemistry
Lääketieteellinen tiedekunta - Faculty of Medicine
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Hyväksymispäivämäärä
2008-05-28
Julkaisun pysyvä osoite on
https://urn.fi/urn:nbn:fi:uta-1-18363
https://urn.fi/urn:nbn:fi:uta-1-18363
Tiivistelmä
Background and Aims: Within the nucleus of eukaryotic cell, DNA is packaged with histone proteins to from nucleosomes that arrange further to form higher-order packaged structures. This three dimensional structure forms a physical barrier between DNA and proteins involved in transcription initiation. However, extent of chromatin packaging can be affected by post-translational covalent modifications to lysine residues of N-terminal histone tails. Acetylated state correlates with gene activation while deacetylation often associates with gene silencing. The reaction is reversible and catalysed by two groups of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Sin3 corepressor complex is a well known multiprotein assembly involved in repression of transcription in eukaryotic cells. Sin3-associated protein 30-like (SAP30L) has been proven to be a member of this complex. Several SAP30L isoforms derived from alternative pre-mRNA splicing exist. The aim of this research was to study the repression capability of SAP30L and its isoforms derived from alternative pre-mRNA splicing.
Methods: Repression capability was studied by transient transfection of expression vectors for target protein and luciferase reporter gene into immortalized human cell line. Fire fly luciferase enzyme activity was measured from cell lysates to determine transcription activity. Measurements were carried out for truncated deletion mutants and isoforms arising from alternative pre-mRNA splicing in addition to wild type. For splice isoforms also Sin3A interaction, associating HDAC activity and subcellular localization were studied.
Results: SAP30L was found capable of silencing gene transcription in HDAC-dependent manner. Repression activity correlates with Sin3A interaction and is dependent on intact C-terminus of the protein. Isoforms were also capable of transcriptional repression with the exception of naturally occurring C-terminal deletion form ΔEx3,4.
Conclusions: SAP30L is capable of association with functional Sin3A corepressor complex and inflicting gene repression through associating HDAC activity. The isoform ΔEx3,4 differs from other naturally occurring forms as it cannot interact with Sin3A and does not localize to the nucleolus. Functional significance of this isoform remains to be elucidated.
Asiasanat: SAP30L, histone deacetylation, repression, splicing
Methods: Repression capability was studied by transient transfection of expression vectors for target protein and luciferase reporter gene into immortalized human cell line. Fire fly luciferase enzyme activity was measured from cell lysates to determine transcription activity. Measurements were carried out for truncated deletion mutants and isoforms arising from alternative pre-mRNA splicing in addition to wild type. For splice isoforms also Sin3A interaction, associating HDAC activity and subcellular localization were studied.
Results: SAP30L was found capable of silencing gene transcription in HDAC-dependent manner. Repression activity correlates with Sin3A interaction and is dependent on intact C-terminus of the protein. Isoforms were also capable of transcriptional repression with the exception of naturally occurring C-terminal deletion form ΔEx3,4.
Conclusions: SAP30L is capable of association with functional Sin3A corepressor complex and inflicting gene repression through associating HDAC activity. The isoform ΔEx3,4 differs from other naturally occurring forms as it cannot interact with Sin3A and does not localize to the nucleolus. Functional significance of this isoform remains to be elucidated.
Asiasanat: SAP30L, histone deacetylation, repression, splicing