THE STUDY OF THE EFFECTS OF A CHANGE IN THE EXPRESSION OF MIXED LINEAGE LEUKEMIA 5 ON TRANSCRIPTION REGULATION

Abstract

Mixed Lineage Leukaemia 5 (MLL5) is a mammalian Trithorax group (TrxG) gene located at chromosome band 7q22, a frequently deleted region in myeloid malignancies. MLL5 was discovered and subsequently cloned in year 2002. Currently, there are a total of fifteen publications dedicated to MLL5 MLL5 is identified as a nuclear protein and either over-expression or depletion of MLL5 resulted in dual-phase cell cycle arrest. In interphase cells, MLL5 exhibits distinct irregular, punctate intra-nuclear speckles but with uncharacterized biological functions. Intrigued by the complexities of nuclear speckles, which are dynamic structures enriched with a reservoir of factors that participate in transcription and pre-mRNA processing, we attempted to unravel the biological functions of MLL5 within the nuclear speckles. To begin with, we examined the co-staining pattern of MLL5 with several well-characterized proteins that were known to display nuclear speckle pattern by immunofluorescence staining. Interestingly, we found that MLL5 nuclear speckles exhibited extensive co-localization with the spliceosome protein SC35 which has recently been reported to be involved in the bi-directional coupling of transcription and splicing. Given the fact that alterations in MLL5 level through ectopic over-expression or siRNA-mediated knockdown resulted in the enlargement and aggregation of nuclear speckles, a phenotype that indicated a defect in co-transcriptional splicing process, we therefore speculate a novel biological role of MLL5 involving in the transcription and splicing processes. We tested this hypothesis by examining if MLL5 is sensitive to transcription inhibitors and whether MLL5 is associated with RNA Polymerase II (RNAPII) transcription machinery. Results showed that MLL5 not only physically interacted with RNAPII but also affected the progression of RNAPII along the DNA template as MLL5 depletion resulted in chromatin compaction and affected the subunits of chromatin remodelling proteins. In addition, histone signatures signifying transcription activation, namely H3K4 tri-methylation and H4 acetylation, were largely reduced in MLL5-kockdown cells. Splicing activity was also reduced as a result of a disruption in the transcription process. Taken together, our findings suggest that MLL5 participates in transcription regulation, which consequently affects gene regulation and cell-cycle progression.