1. Elucidation of mechanisms that coodinate genome expression
We have been investigating the mechanisms that coordinate genome
expression, especially those that control the process of transcription
elongation in our cells. Some time ago, we focused on low molecular weight
transcriptional inhibitors such as DRB and Flavopiridol, which are also
known as potential drugs for cancers and AIDS, and initiated the study
on the mechanism of action of these inhibitors. We have shown that these
inhibitors block mRNA synthesis by RNA polymerase II (RNAPII) by inducing
its “promoter-proximal pausing” (reviewed in Yamaguchi et al. Genes Cells 1998). We identified protein
factors, termed DSIF and NELF, that induce transcriptional pausing (Wada et al.
Genes Dev. 1998; Yamaguchi et al. Cell 1999). DSIF
and NELF bind to RNAPIIa, the RNAPII molecule carrying the unphosphorylated
C-terminal domain (CTD) (Wada et al. Genes Dev. 1998; Yamaguchi et
al. Cell 1999) and, through the direct interactions, interfere
with mRNA synthesis (Yamaguchi et al. J. Biol. Chem. 1999;
Yamaguchi et al. Mol.Cell. Biol. 2002). The protein kinase P-TEFb
converts RNAPIIa into RNAPIIo, the phosphorylated form of RNAPII, by
phosphorylating the RNAPII CTD and thereby facilitates the release of RNAPII
pausing and the restart of mRNA synthesis (Wada et al. EMBO J.
1998). Nevertheless, P-TEFb does not suffice for the reversal of pausing, but
the FACT protein and the helicase activity of the general transcription factor
TFIIH are additionally required (Wada et al. Mol. Cell 2000).
Following the release of RNAPII pausing, the protein factor called hSpt6 is
recruited to the RNAPII elongation complex and enhances mRNA synthesis (Endou et
al. Mol. Cell. Biol. 2004). Moreover, we reported recently that
P-TEFb phosphorylates not only RNAPII but also DSIF, and that the latter
phosphorylation leads to the activation of RNAPII elongation activity and to the
enhanced synthesis of long mRNA (Yamada et al. Mol. Cell 2006).
Thus, the unphosphorylated form of DSIF cooperates with NELF to act as a brake
for mRNA synthesis, whereas the phosphorylated form of DSIF cooperates with
other unidentified proteins to act as an accelerator. The DSIF sequence
phosphorylated by P-TEFb contains a repetitive amino acid motif, termed the
C-terminal region (CTR), that is similar to the RNAPII CTD.
A number
of interesting data have been obtained as to the biological significance of
promoter-proximal pausing. First, it plays a role during early development.
Zebrafish carrying a point mutation that destroys DSIF brake activity shows
abnormal differentiation of neural cells, having a reduced number of
dopamine-containing neurons and an excess of serotonin-containing neurons (Guo
et al. Nature 2000). A similar mutation of DSIF in fruitfly leads
to defective formation of body segments (Jennings et al. Curr.
Biol. 2004). Second, promoter-proximal pausing controls many of the
genes rapidly induced by external stimuli, such as heat shock-responsive genes,
inflammatory cytokine-responsive genes, sex hormone-responsive genes, and growth
stimuli-responsive genes (Wu et al. Genes Dev. 2003; Ainbinder et
al. Mol. Cell. Biol. 2004; Aiyar et al. Genes Dev.
2004; Yamada et al. Mol. Cell 2006). Third, promoter-proximal
pausing is involved in the growth of some pathogenic viruses. During the latent
infection of human immunodeficiency virus (HIV), the causative agent of AIDS,
RNAPII pauses at the HIV promoter-proximal region through the action of DSIF and
NELF (reviewed in Yamaguchi et al. Microbes Infect. 2002). When
the HIV Tat protein is expressed, Tat recruits P-TEFb to the HIV promoter
region, induces phosphorylation of the CTD of paused RNAPII, and thereby
facilitates the restart of mRNA synthesis. Hepatitis delta virus (HDV) encodes a
protein called hepatitis delta antigen, which we found to interact with RNAPII.
Through the interaction, hepatitis delta antigen prevents NELF from associating
with RNAPII and strongly enhances the rate of mRNA synthesis (Yamaguchi et al.
Science 2001). Such a viral protein that directly interacts with
RNAPII and regulates the process of transcription elongation is
unprecedented.
Currently we are studying various issues such as novel
protein factors inducing or suppressing promoter-proximal pausing, the
biological significance of dynamically controlling the rate of mRNA synthesis,
the relationship between the rate of mRNA synthesis and its stability, and the
relationship between transcription elongation and mRNA processing, at a
molecular level. We are also conducting an investigation using model organisms
such as zebrafish and mice, aiming at comprehensively understanding
transcription elongation control in higher eukaryotes.
We have
pioneered the research area over the last decade. Our research is highly
appreciated internationally. We have concisely reviewed recent developments in
this research area in the book “Mastering the world of transcription” (eds. H.
Handa, T. Wada, and Y. Yamaguchi, Yodosha, Tokyo, Japan, 2005).