| الوصف: |
Nature has evolved an additional level of genetic regulation by-passing direct changes in genetic code through the means of posttranslational modifications (PTMs) of nucleobases and histone proteins. Acetylation, methylation, phosphorylation, O-GlcNAcylation, ubiquitination, sumoylation, and ADP ribosylation are few common examples of various histone modifications. Identification of these modifications and subsequent access to homogeneously modified histone proteins are key for understanding the functional consequence of these PTMs. In this doctoral thesis, the role of PTMs of histone proteins in epigenetics was investigated with emphasis on understanding the role of O-GlcNAcylation in particular. In the second chapter, the functional consequence of O-GlcNAcylation at histone protein, H2B-Ser112 was explored. Homogeneously GlcNAcylated histones and nucleosomes were synthesized using protein chemical reactions. Mass Spectrometry (MS) based quantitative interaction proteomics revealed a direct interaction between GlcNAcylated nucleosomes and the Facilitates Chromatin Transcription (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes provides a molecular mechanism for FACT-driven transcriptional control. In the third chapter, the physical effect of O-GlcNAcylation on the nucleosome structure is described. Homogeneously GlcNAcylated histone protein, H2A-Thr101 was synthesized. The modified protein was used to reconstitute histone sub-complexes and nucleosomes. Various biophysical studies involving circular dichroism and native mass spectrometry revealed that H2A-T101 GlcNAcylation regulates the stability of the nucleosome structure, suggesting a role in transcriptional activation. In the fourth chapter, we discuss an interesting scenario where two PTMs - O-GlcNAcylation and phosphorylation - can compete for the same modification site of histone protein, H2B-Ser36. The resulting outcome is possibly a competitive antagonism or cross-talk, which can modulate the overall control of chromatin regulation. Using a "Tag-and-modify" approach, modified histone proteins bearing both modifications was synthesized, and was later used for nucleosome reconstitution. Quantitative interaction proteomics experiments with the modified nucleosome revealed key interacting protein partners for both the modifications. |