We have demostrated that Myo6 is present in the nucleus and is involved in transcription (1).
The nuclear localization of Myo6 depends on a bipartite NLS, located at the C-terminus of the
protein and is enhanced following stimulation of a transcriptionally inducible cell system
(HepG2). Nuclear Myo6 associates with CDK9 and modulates the phosphorylation of RNAP II
at serine 2 of the CTD, affecting transcriptional elongation. This process occurs at active
transcription factories that, in HepG2 cells, co-exist with transcriptionally inactive, �poised�
factories, associated with RNAP II phosphorylated at serine 5 of the CTD (2).
Thus Myo6 may play a role in establishing a functional nuclear architecture.
To investigate the molecular details of this hypothesis we plan to perform high resolution
chromatin immunoprecipitation experiments (3) with specific antibodies against Myo6, CDK9,
CDK7 and both of the phosphorylated forms of RNAP II, followed by sequencing of all the
recovered DNA fragments. By using material from unstimulated/stimulated HepG2 cells we
plan to build a genome-wide map of the co-localizing proteins and detect relevant changes in
the absence/presence of a stimulus. This approach will be complemented with an immuno
FISH approach, aimed at: i) establishing which genes occupy the same (poised or active)
factory (and, possibly, how many of them); ii) determining if genes activated by a stimulus
relocate to different factories or not.
We also plan to broaden the investigation by performing proteomics on nuclear Myo6 using
the SILAC approach to identify partners of the protein that are involved both in transcription
and in the mobilization of nucleic acids, thus contributing to shape the functional nuclear
landscape.
(1) Vreugde, S. et al. (2006) Mol Cell 23, 749-755.
(2) Ferrai, C. et al. (2010) PLoS Biology 8(1): e1000270
(3) Ferrai, C. et al. (2007) J Biol Chem. 282: 12537-12546.

Unconventional myosin VI (Myo6) mRNA and protein are overexpressed in prostate cancer
tissues and cell lines [1]. Depletion of Myo6 in the castration-resistant, prostate
adenocarcinoma PC3 cell line by transient siRNA transfection dramatically reduces their
mobility, invasive potential, ability to grow in an achorage-independent manner and
tumorigenic potential when xenografted in NOD-SCID mice (only 70% of animals develop
very slow-growing tumors). When they are palpable, however, tumors from control and Myo6-
depleted cells express comparable levels of the protein, indicating that the growth rate of the
latter is not rescued.
Althouth the results indicate that Myo6 plays a role in the maintenance of the tumorigenic
phenotype, it is not clear which mechanism (possibly epigenetic in nature) is altered by its
depletion. It is particularly important to approach this question, since Myo6 appears as a
suitable target for androgen-independent tumors, for which, to date, there is no therapy.
We plan to tackle this problem by high throughput techniques. In the first place we must
establish stable prostate cancer cell lines transduced with a viral vector to conditionally
perform RNA interference on Myo6. These cell lines will allow to investigate the transcriptome
of Myo6-depleted (vs. control) cells, showing which genes are up-/downregulated by the lack
of the protein. The same lines will allow studying the protein complexes with which Myo6 is
associated (Myo6 proteome by SILAC). We plan to perform this investigation both at the
cytoplasmic level, where Myo6 is known to play a role [2], and at the nuclear level, since the
effect of Myo6 depletion in PC3 cells appears to be epigenetic. The transcriptomic and
proteomic results will be validated by quantitative-PCR and co-immunoprecipitation
experiments, respectively.
1. Dunn T.A. et al. (2006) Am J Pathol 169: 1843-1854.
2. Sweeney H.L., Houdusse, A. (2007) Curr Opin Cell Biol 19: 57-66