Investigating the role of the mesoderm induction early response (MIER) family members as transcriptional co-repressors

Derwish, Roya (2019) Investigating the role of the mesoderm induction early response (MIER) family members as transcriptional co-repressors. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Large coregulator complexes are recruited to specific gene loci to modulate chromatin structure by altering epigenetic marks on DNA and histones to control gene expression. Mesoderm induction early response 1 (MIER1) is a nuclear protein known to function in transcriptional repression through its ability to recruit histone deacetylase 1 (HDAC1) and 2. The MIER family consists of three related genes encoding proteins containing ELM2-SANT functional domains. MIER1 is the prototypical member, well characterized in our lab but little is known about MIER2 or MIER3 function and there is no data characterizing these two proteins. In my thesis, I have begun to characterize MIER2 and MIER3 proteins and to compare them to MIER1. I investigate their subcellular localization, their potential association with each other, their interaction with HDAC1 and 2 and chromodomain Y-like (CDYL), the activity of associated deacetylases and key residues for HDAC and CDYL recruitment. Immunostaining followed by confocal microscopy analysis revealed that, while MIER2 and MIER3 are mainly nuclear proteins, a substantial proportion (32%) of MIER2 is localized in the cytoplasm. Co-immunoprecipitation (co-IP) experiments demonstrated that the MIER proteins do not form dimers, neither homodimers nor heterodimers with either of the other two family members. Our data also showed that MIER2, but not MIER3, can recruit HDAC1 and 2. Co-IP experiments showed that MIER1 and MIER2, but not MIER3, interact with CDYL through the ELM2-SANT domains. Both MIER1 and MIER2 augment interaction between CDYL and HDACs. Finally, ChIP-Seq analysis revealed that each MIER member has unique targets and that they share target genes. In addition, consensus DNA sequences for MIER protein occupancy are nearly identical to binding motif for the transcriptional repressor RE-1 Silencing Transcription Factor (REST). REST is known to regulate expression of neural genes by recruiting corepressor complexes. Co-IP experiments demonstrated that MIER1 and MIER2, but not MIER3, interact with REST. Suppression of MIER1 or MIER2 expression in P19 embryonal carcinoma cells results in neuronal differentiation. Observations made in this report suggest that MIER1 and MIER2 play an important role in the repression of neuronal genes by the REST complex. Overall, I am the first to characterize MIER2 and MIER3. The results presented in this thesis show that MIER2 is similar to MIER1 in that it recruits some of the same epigenetic regulators and both proteins are enriched on REST target genes. In contrast, I showed MIER3 to be distinct: despite a high degree of amino acid similarity between MIER3 and MIER1/2, it did not interact with any of the MIER1/2 recruited regulators and is enriched on FOXA1 target genes.

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