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The primary goal of this work was to characterise the histone methyltransferase activity
of PRMT6. Despite the fact that PRMT6 was found to be mainly nuclear localised and
that several PRMT members exhibit activity towards histones, the histone-methylation
by PRMT6 has not been investigated so far.
In this work PRMT6 was found to methylate histones H3, H2A and H4 in vitro. Free
histones were substrates of PRMT6, whereas nucleosomes could not be methylated by
PRMT6 in vitro. The methylation sites were localized in the N-termini of histones and
identified as R2 in histone H3 and R3 in histones H4 and H2A, respectively. The
characterisation of the methylation mechanism resulted in a catalytic preference of
PRMT6 for monomethylated arginines. This indicates that PRMT6 is mainly
responsible for the dimethylation of arginines. Considering the methylation of R2 in
histone H3 other histone modifications were found to exhibit an influence on the in
vitro activity of PRMT6. While methylation of K4 and K9 in H3 inhibited PRMT6
activity, methylation of K27 increased PRMT6 activity.
Subsequently, the investigation of H3R2 dimethylation function in the context of the
histone code was the next goal of this work. This should be achieved by identifying
potential binding partners (effector proteins) for this modification. Peptide pulldowns
with synthetically modified histone H3-pepides were used to search for differentially
interacting proteins. This technique did not result in the identification of specific
interaction partners for H3R2 dimethylation. A supposed functional relationship
between PRMT6 and the PRC2-complex as well as between their corresponding
modifications R2 and K27 could not be confirmed using peptide pulldowns.
Recent studies considering the genome-wide localisation of R2 di- and the K4
trimethylation suggested a negative cross-talk between these histone marks.
Additionally, PRMT6 overexpression experiments demonstrated a repressive effect of
PRMT6 on the expression of distinct HoxA genes and c-myc targets. The regulation of
c-myc gene expression by the Wnt-pathway was used to further characterise the
regulation mechanism of PRMT6. RNAi-mediated PRMT6-knockdown exhibited no
effect of PRMT6 on the gene expression of c-myc in this context.
Alternatively, the negative cross-talk between R2Me2 and K4Me3 should be described
on the mechanistic level. Therefore, the in vitro influence of R2 dimethylation on K4
trimethyltransferase MLL activity as well as on histone binding affinity of MLL-complex subunit WDR5 was investigated. The R2 dimethylation exerted a strong
inhibitory effect on MLL activity and on WDR5 binding to the histone H3-tail. The
negative effect of R2 methylation on K4 methylation could be verified in vivo using
chromatin immunoprecipitation on the HoxA2 promoter in PRMT6 overexpressing
cells. Exogenous PRMT6 levels resulted in increased PRMT6 and R2 dimethylation
levels at the promoter and decreased K4 trimethylation as well as reduced recruitment
of MLL and WDR5. These promoter events were described in a model of neuronal cell
differentiation, which confirmed the model for the repressive function of PRMT6-
mediated R2 dimethylation in gene regulation.
Together, the data from the present work identify PRMT6 as a novel histone
methyltransferase, which dimethylates R2 in histone H3. Furthermore, the specific gene
regulation mechanism for PRMT6 was characterised in the context of the histone code,
where H3R2Me2 regulates H3K4Me3-mediated expression of certain genes.