Characterization of DNA binding and oligomerization domains of stem cell transcription factor Sox9 and Sox5

• Main Author: Saravanan Vivekanandan
• Other Authors: Julien Lescar
• Format: Theses and Dissertations
• Language: English
• Published: 2013
• Subjects: DRNTU::Science::Biological sciences::Biochemistry; DRNTU::Science::Biological sciences::Biophysics
• Online Access: https://hdl.handle.net/10356/55052
• Institution: Nanyang Technological University

Sox (SRY-related HMG box) family of proteins is mammalian stem cell transcription factors, playing a pivotal role in regulation of numerous developmental processes. So far nearly 30 Sox proteins have been identified and categorized into eight subgroups of A to G, based on high sequence similarity of the conserved HMG box domain. Unlike other transcription factors, Sox proteins are unique as they recognize and bind the minor groove of DNA, inducing a strong DNA bend of 70-85°, an essential step in eukaryotic transcriptional regulation. Although the HMG domains of all Sox proteins are highly conserved and bind similar DNA elements of sequence (A/T) (A/T) CAA (A/T), they regulate a wide assortment of genes in diverse developmental processes. The functional specificity of Sox transcription factors depends on (i) subtle nucleotide variations in the DNA sequence; (ii) differential minor groove bend as a consequence of HMG domain mediated DNA interaction (iii) different co-factor recruitment through protein-protein interactions. In this regard, Sox9 (group SoxE), Sox5 and Sox6 (group SoxD), famously known as “Sox trio”, are ideal prototypes with conserved HMG domains and group specific domains for partner recruitment. Sox9 has a DNA dependent dimerization domain and Sox5 has “DNA independent” coiled-coil (CC) domain. The objective of the current study is to comprehend the underlying molecular mechanism of DNA recognition and transcriptional specificity of the Sox trio. Towards this end, the DNA binding HMG domain of Sox9 and Sox5; protein interacting domains of Sox9 (DNA dependent dimerization domain) and Sox5 (coiled-coil domain) were studied. In the case of protein-DNA interactions, the HMG domains of Sox9 and Sox5 were cloned, purified and their DNA binding abilities were determined by EMSA. Moreover, two novel Sox9 DNA binding motifs have been identified employing ultra-high-throughput DNA sequencing (ChIP-Seq) data. Functional validation of the novel motifs by EMSA and luciferase assay confirms Sox5 as downstream target of Sox9. Crystalisation of DNA bound Sox9/Sox5 HMG domains have yielded promising outcomes. Protein-protein mediated oligomerisation was analysed employing the DNA dependent dimerization domain of Sox9 and the “DNA independent” coiled- coil domain of Sox5, as model systems. The study involves the cloning, purification, crystallization, biochemical and biophysical characterization of the corresponding domains. The results presented indicate Sox9 to exclusively exist as dimers in the presence of DNA and contrastingly, Sox5 harboring intact CC domain tetramerizes, indicating a possibility of higher order homo/hetero oligomerisation. The current study is the first comprehensive biochemical validation of Sox protein-protein interacting domains, highlighting the role of Sox homo/hetero oligomers in dictating the transcriptional specificity of Sox transcription factors.