Characterization of the interactions between the H3K4 demethylase JARID1A and the SCL-nucleated oncogenic DNA binding complex

Abstract

Transcriptional regulation is of key importance to cellular processes such as development and differentiation, and it is dependent on the accessibility of DNA in the chromatin. Methylation and demethylation of histones alter properties of nucleosomes, thereby changing the accessibility of DNA. For many years, histone tail methylation was considered to be irreversible but in the last decade two families of histone demethylases were discovered, lysine specific demethylase 1 (LSD1) and Jumonji C (JMJC) demethylases. The work presented in this thesis focuses on the chromatin remodeling protein JARID1A, belonging to the JARID1 family of JMJC demethylases that demethylates lysine 4 (K4) on the tail of histone H3. Double and triple methylation of K4 on histone H3 are marks traditionally linked to transcriptionally active regions of DNA. Recently, a translocation fusing the third PHD domain (PHD3) of JARID1A to NUP98 (a common leukaemia fusion partner with transactivation activity) was identified in acute myeloid leukaemia patients (AML). Also, PHD3 was previously shown to mediate interactions between JARID1A and the second LIM domain of LMO2. LMO2 operates within the multiprotein DNA binding complex named "SCL complex" (including proteins SCL/TAL1, LDB1, E47, GATA1) that regulates gene expression at different stages of erythropoiesis. This thesis presents findings of an investigation into relations between JARID1A and the oncogenic multiprotein complex SCL-E47-LMO2-LDB1-GATA1. The study of the endogenous proteins from erythroid cells using techniques such as Co-immunoprecipitation, GST pull-down and Size-exclusion chromatography showed that JARID1A interacts with SCL and GATA1. These interactions were further characterized biochemically using analytical ultracentrifugation which revealed that the second PHD domain of JARID1A is involved in direct interaction with GATA1. Hence, JARID1A could play a role in regulation of erythropoiesis by modulating activities of transcription factors SCL and GATA1.</p