Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

Mixed Lineage Leukemia protein (MLL) is required for proper embryonic development, and hematopoiesis. It is a SET domain containing histone methyl transferase that trimethylates histone H3 on lysine 4 (H3K4Me3), a histone modification that correlates with active transcription. The 3rd PHD finger of MLL binds to H3K4me3. Thus MLL is a "writer" with an embedded "reader" for H3K4Me3. Cyp33 is another known ligand of MLL PHD3. Over expression of Cyp33 results in transcriptional repression of MLL target genes.

The aim of this study is to determine the biological function of MLL PHD3 binding to H3K4Me3 or Cyp33. Cyp33 binding to MLL PHD3 reduces the binding affinity of the MLL PHD3 for H3K4Me3 by 5.7 fold in vitro. To know if this is true in a cellular system, we sought to study the effect of excess of Cyp33 on H3K4 trimethylation and on transcription of MLL target genes. Cyp33 over expression resulted in a decrease in H3K4Me3 and increase in recruitment of JARID1A and JARID1B, the H3K4 specific demethylases, at the MLL target gene promoters.

To determine the transcriptional outcome of the MLL PHD3 binding to H3K4Me3 or Cyp33, we abrogated either H3K4Me3 binding or Cyp33 binding to the MLL PHD3 by point mutations in the MLL-N coding sequence and assessed the effect of expressing the mutant proteins on MLL target gene expression. Abrogation of H3K4Me3 binding to MLL PHD3 resulted in decrease in transcription of MLL target genes and decrease in H3K4Me3 marks when compared to the MLL-N wt. Also, abrogation of MLL PHD3 binding to H3K4Me3 resulted in decreased expansion of mouse hematopoietic progenitors in a methyl cellulose colony formation assay when compared to wt MLL-N. This indicates that the MLL PHD3 binding to H3K4Me3 contributes to histone H3K4 trimethylation, for the transcriptional activation of MLL target genes and for the expansion of hematopoietic progenitors. Taken together, we have identified a novel link between Cyp33 and JARID1 histone demethylases in the transcription regulation of MLL target genes; and the transcriptional outcome of MLL target genes is influenced by Cyp33 and H3K4Me3, the ligands of MLL PHD3.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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