The timing of gene transcription during development and differentiation of tissues needs to be tightly controlled. To prevent the aberrant growth of different tissue types, gene expression must be activated specifically. Because of its role in elongation of transcripts with poised polymerases, we wanted to explore the role P-TEFb plays in the context of muscle development and regeneration.
We found that P-TEFb inhibition, both by flavopiridol and CDK9 siRNA, decreased both early and late transcription using three muscle marker genes in C2C12 cells. After observing the dependence of muscle gene transcription on active P-TEFb, we wanted to examine the effects of P-TEFb inhibition on cell differentiation and morphology change. Flavopiridol treatment and CDK9 depletion not only inhibited the expression of MyHC in immunofluorescence experiments, but also prevented the formation of elongated, organized myotubes.
In our previous work, we characterized the phosphorylation of histone H1 by P-TEFb (O’Brien et al., 2010
). We therefore examined H1 phosphorylation in C2C12 cells. In differentiating C2C12 cells, H1 phosphorylation is induced, but P-TEFb inhibition prevents this phosphorylation. These data suggest that, along with activating muscle gene transcription, P-TEFb also directs H1 phosphorylation during differentiation.
Previously, we characterized a specific site on histone H1.1 that is phosphorylated by P-TEFb. To further examine the role of H1 phosphorylation in muscle gene transcription, we expressed GFP-WT, T152A, S183A, T152A S183A, and S183E H1.1 in C2C12 cells. S183A and T152A S183A GFP-H1.1 expression prevented the induction of myogenin, MCK, and MyHC when cells were placed in differentiation media. Thus, preventing H1 phosphorylation by P-TEFb at S183 specifically inhibited transcription. H1 ChIP experiments also showed that H1 is released from these genes during differentiation, while flavopiridol treatment inhibits this release. Like our previous work, which studied H1 phosphorylation at the c-fos
genes and the HIV-1 LTR (O’Brien et al., 2010
), we find that H1 needs to be phosphorylated at P-TEFb-specific sites and to dissociate from DNA in order for muscle-specific transcription to proceed. H1 phosphorylation is therefore critical to the skeletal muscle differentiation program.
Our data provide an additional role for P-TEFb in differentiation. Other groups had previously characterized P-TEFb as a necessary factor for muscle differentiation (Simone et al., 2002
; Giacinti et al., 2006
; Giacinti et al., 2008
). Original studies in C2C12 showed that over-expression of dominant negative CDK9 prevents myotube formation (Simone et al., 2002
). Similarly, we report that inhibition of CDK9 by flavopiridol and depletion by RNAi result in the same phenotype as depletion of myogenin. We additionally show that P-TEFb inhibition prevents the expression of genes specific to myotube formation and contractile function, and that H1 phosphorylation is required for the transcription of these genes.
The major muscle regulatory factor MyoD interacts with and recruits P-TEFb to the promoters of muscle-specific genes (Simone et al., 2002
; Giacinti et al., 2006
). P-TEFb also interacts with the MEF2 family of transcription factors, providing additional activation of muscle structural genes (Nojima et al., 2008
). Once these factors recruit P-TEFb to promoters, CDK9 is then able to phosphorylate RNA Pol II and the NELFs and promote transcription elongation. Our data suggest that H1 also needs to be phosphorylated to promote the transcription of these genes. MyoD also binds the histone acetyltransferases p300 and pCAF, and their activity in turn recruits the SWI/SNF complex to remodel chromatin (Puri et al., 1997
; Sartorelli et al., 1999
; Giacinti et al., 2008
). Although the timing of events is unclear, it is possible that phosphorylation of H1 by P-TEFb can result in increased accessibility for these acetyltransferases and chromatin modifiers to nucleosomes. This activity would facilitate Pol II transcription through chromatin.
P-TEFb has also been implicated in muscle regeneration. In experiments using skeletal muscle tissue from mice, CDK9 mRNA and protein expression is induced in satellite cells when muscle is damaged (Giacinti et al., 2008
). While we have not examined muscle regeneration in this study, we would hypothesize that H1 phosphorylation is induced during regeneration after damage as well as initial tissue differentiation.
As a chromatin structural protein, histone H1 has proven essential for proper development in mammals. As pluripotent cells differentiate, the total amount of H1 in cells increases (Fan et al., 2003
; Fan et al., 2005
). Likewise, higher order packing of chromatin occurs, determining the accessibility of genes for transcription. Temporary changes in H1 binding would be beneficial for timing the expression of specific genes. P-TEFB is emerging as an important factor in differentiation-specific transcription not only in myotube precursors but also in pluripotent and embryonic stem cells (Kohoutek et al., 2009
; Kaichi et al., in press
). Our work suggests that H1 is one substrate by which P-TEFb acts to trigger differentiation.