Phosphorylation of Histone H1 by P-TEFb is a Necessary Step in Skeletal Muscle Differentiation

Transcription of many genes by RNA polymerase II (Pol II) is controlled at the step of mRNA elongation. The transcription elongation factor positive transcription elongation factor b (P-TEFb) participates in the activation of expression of many genes transcribed by Pol II. Comprised of the cyclin-dependent kinase CDK9 and Cyclin T1, P-TEFb phosphorylates the heptapeptide repeat of the largest subunit of RNA Pol II, increasing its processivity during elongation (Kim and Sharp, 2001; Peterlin and Price, 2006; Kohoutek, 2009). To enhance elongation, P-TEFb also phosphorylates other substrates such as the negative elongation factors DRB sensitivity-inducing factor and NELF (Ping and Rana, 2001). Our prior work has shown that P-TEFb also phosphorylates histone H1 to activate HIV-1 and immediate-early transcription (O’Brien et al., 2010). When phosphorylated by P-TEFb, H1 dissociates from DNA, allowing factors to access chromatin and Pol II to progress. We sought to know whether H1 phosphorylation is a requisite for transcription of other P-TEFb-regulated systems. As a model system for differentiation regulated by P-TEFb, we induced skeletal muscle differentiation in the myoblast cell line C2C12 and examined the activity of P-TEFb. P-TEFb is indispensable for the transcription of HIV-1 genes as well as a number of cellular genes such as c-fos, c-myc, and the heat shock genes (Mancebo et al., 1997; Garber et al., 1998; Lis et al., 2000; Eissenberg et al., 2007). P-TEFb activity is also necessary for the differentiation of some tissues such as skeletal and cardiac muscle (Simone et al., 2002; Giacinti et al., 2006). In skeletal muscle, P-TEFb is recruited to differentiation-specific promoters by the major muscle transcription factors MyoD and MEF2 (Simone et al., 2002; De Falco and De Luca, 2006; Nojima et al., 2008). MyoD and the MEF2 family of transcription factors are responsible for the early expression of other muscle-specific transcription factors and expression of structural genes, respectively (Bergstrom et al., 2002; Blais et al., 2005). Because we found that P-TEFb phosphorylation of histone H1 is necessary for the transcription of HIV-1 genes and the cellular immediate early genes c-fos and hsp70 (O’Brien et al., 2010).We hypothesized that P-TEFb could also modulate myogenic gene expression in C2C12 cells by phosphorylating H1. In particular, we chose to examine the early muscle marker gene and transcription factor myogenin, and the later-expressing markers muscle creatine kinase (MCK) and myosin heavy chain (MyHC), both essential to muscle contractile function. Using P-TEFb inhibition and H1 phosphorylation site mutants, qPCR, ChIP, and MyHC immunofluorescence, we show that P-TEFb phosphorylation of H1 is an important step in myogenic differentiation.