Date of Award

2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biochemistry Program

Abstract

MLL or Mixed Lineage Leukemia gene is clinically known for its involvement in genetic translocation with more than 70 different partners identified each giving rise to a highly leukemogenic fusion protein. With a poor prognosis of MLL related leukemia, an investigation into its role during hematopoiesis has been a very active field of research. In order to design strategies to combat the MLL leukemia, it becomes essential to delineate the molecular mechanisms behind the function of MLL wild type protein. Wild type MLL is a transcriptional maintenance protein from the Trithorax Group (TrxG) that resides in complex with other chromatin regulators to maintain the downstream target genes, in particular the HOX genes, in their active state of transcription. A nuclear cyclophilin called CYP33 interacts with the third Plant Homeo Domain (PHD) of MLL via its N terminal RNA Recognition Motif (RRM) such that its overexpression switches the function of transcriptional activator protein MLL towards transcriptional repressor. This repression is mediated in part by an enhanced recruitment of histone deacetylase 1 to MLL and a subsequent decrease in the histone H3 acetylation at the MLL target gene promoters, including the HOXC8 promoter. CYP33 RRM binds Poly A and Poly U RNA in addition to MLL. A regulatory region of MLL target gene HOXC8 is transcribed into a non-coding transcript, which we call NC4 in this study. We hypothesize that the regulation of HOXC8 via MLL may involve NC4 function such that its expression would disrupt CYP33 and MLL interaction and consequent repression at the gene promoter. In order to address this, first a more conserved region between HOXC8 and HOXC6 was analyzed for transcription into non-coding RNA based on the presence of ESTs and in silico analysis followed by the monitoring of transcription in human and mouse cell lines as well as in mouse embryos. After comparing the expression kinetics of NC4 and HOXC8 during in vitro differentiation of mouse embryonic stem (mES) cells, NC4 transcription was found to precede and exceed that of the HOXC8 during the mES differentiation implying its early role during differentiation. In order to determine if CYP33 binds a specific RNA sequence, an in vitro selection and enrichment technique was performed that identified a YAAUNY consensus RNA sequence preferentially bound by CYP33. An NMR based collaborative study revealed an overlap between both MLLPHD3 and core AAU RNA sequence binding surfaces on CYP33 RRM that also suggested an exclusive binding by MLLPHD3 and RNA. Furthermore, CYP33 preferred YAAUNY motifs were found to be present at a greater density in the 3' region of NC4 suggesting its possible role in HOXC8 regulation by MLL and CYP33. This possibility was first tested using in vitro competition assays based on Forster Resonance Energy Transfer method of studying molecular interactions. YAAUNY containing RNA sequences (both synthetic as well as endogenous NC4 3' region) could disrupt the CYP33-MLLPHD3 interaction by 40.66% and 35.78% respectively. Following this, MSA cells that lack endogenous expressions of both HOXC8 and NC4 and show the binding of MLL and CYP33 at their promoters, expressed the HOXC8 gene after ectopic expression of a NC4 transcript from a transfected plasmid.

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