The elongation phase of transcription is a point of regulation for many genes transcribed by RNAPII in eukaryotes, and control of elongation is critical for coupling of transcription to downstream steps in gene expression 
. Whereas the regulation of transcription at the initiation step has been studied extensively, many of the mechanisms governing elongation in vivo remain to be elucidated. Transcription is accompanied by post-translational modification of nucleosomal histones in a highly conserved pattern, stereotypical features of which include methylation of histone H3 Lys4 (H3K4me) and acetylation of histones H3 and H4 at 5′ ends, methylation of histone H3 Lys36 (H3K36me) towards 3′ ends, and mono-ubiquitylation of histone H2B at a conserved site in the carboxyl-terminus (H2Bub1) throughout coding regions of genes 
. Conservation of this pattern suggests an important role in coordinating gene expression, but the precise functions of individual modifications in elongation control are poorly understood.
H2Bub1, which appears to play a central role in the interplay between chromatin and the RNAPII elongation complex, is catalyzed in budding yeast by the ubiquitin-conjugating enzyme Rad6 and the E3 ubiquitin ligase Bre1 
. Formation of H2Bub1 on transcribed chromatin also requires PAF, a conserved complex with multiple functions during elongation 
. H2Bub1 is required for co-transcriptional generation of H3K4me by the methyltransferase Set1 in yeast, and contributes to global H3K4me levels in metazoans 
. In vitro, H2Bub1 directly stimulates activity of Set1 towards a reconstituted chromatin substrate 
H2Bub1 also acts independently of histone methylation; our work in the fission yeast S. pombe
revealed that the Set1-independent pathway is important for normal cell growth and morphology, and for elongation by RNAPII at select target genes 
. Similar findings have now been reported in S. cerevisiae
and mammalian cells 
, but the mechanism of methylation-independent effects of H2Bub1 is unknown.
Co-transcriptional histone modifications are regulated by a conserved subset of cyclin-dependent kinases (CDKs) associated with the transcription machinery 
. In metazoans these include Cdk7, a component of the initiation factor TFIIH, and Cdk9, catalytic subunit of positive transcription elongation factor b (P-TEFb). Cdk7, Cdk9 and their yeast orthologs phosphorylate multiple proteins important for elongation, including the Rpb1 subunit of RNAPII, at Ser2, Ser5 and Ser7 positions within the repeated YSPTSPS motif of its carboxyl-terminal domain (CTD) 
; and the Spt5 subunit of a conserved elongation factor, known in metazoans as DRB-sensitivity inducing factor (DSIF) 
. Those phosphorylations control recruitment of pre-mRNA-processing and chromatin-modifying enzymes 
.In metazoans, moreover, Cdk9 activity overcomes promoter-proximal pausing imposed by unphosphorylated DSIF and a negative elongation factor (NELF) 
. Pausing is thought to act both as a quality control over gene expression, by facilitating recruitment of mRNA-processing factors 
; and as a rate-limiting determinant of expression for subsets of stringently regulated genes 
P-TEFb also regulates histone modification. In mammalian cells, levels of H2Bub1, H3K4me, and H3K36me decrease after depletion of Cdk9 by RNA interference (RNAi), or treatment with flavopiridol, an inhibitor of Cdk9 and related kinases 
. Bur1, the essential ortholog of Cdk9 in budding yeast, is required for co-transcriptional H3K36me and consequent action of the Rpd3S histone deacetylase complex to prevent transcription initiation within coding regions 
. Similarly, co-transcriptional H2Bub1 depends on Bur1 
and the carboxyl-terminal region of Spt5, which contains sites phosphorylated by Bur1 
. Budding yeast contains another, non-essential CDK, Ctk1, which is the major Rpb1-Ser2 kinase in vivo and is required for H3K36me 
. Although Ctk1 was proposed to be a yeast-specific P-TEFb paralog 
, putative orthologs of Ctk1 have recently been identified in human and Drosophila
, suggesting faithful conservation of the entire CTD kinase network.
CDKs are themselves regulated by histone modification. In mammals, P-TEFb binds the bromodomain protein Brd4, whose recruitment to promoter-proximal sites is stimulated by acetylation of histone H4 
. Phosphorylation of histone H3 also favors recruitment of P-TEFb 
. On the other hand, certain histone marks work in opposition to specific CDKs during elongation. For example, in budding yeast, lethality of a BUR1
deletion is suppressed by deletion of the H3K36 methyltransferase Set2 
; and H2B de-ubiquitylation by Ubp8—a component of the SAGA complex—is required for Ctk1 recruitment and Ser2 phosphorylation at some genes 
In fission yeast, the P-TEFb ortholog is the essential Cdk9/Pch1 complex. In vitro, Cdk9 phosphorylates the Rpb1 CTD at both Ser5 and Ser2 
; inhibition of Cdk9 in vivo reduced the apparent stoichiometry of Rpb1 phosphorylation but did not cause selective loss of either Ser5 or Ser2 signals 
. Cdk9 also phosphorylates the CTD of S. pombe
mutations that prevent this phosphorylation cause slow growth, defects in transcript elongation and, when combined with partial truncations of the Rpb1 CTD, synthetic lethality 
. Genetic epistasis suggests, moreover, that the Spt5 CTD contains an exclusive, albeit nonessential, target of Cdk9 in vivo 
. The potential roles of CDKs and their substrates in regulating histone modifications in fission yeast have not been explored.
Here we uncover a mutual dependence of H2Bub1 and P-TEFb function in S. pombe: mutations that impair phosphorylation of Spt5 by Cdk9 diminish levels of H2Bub1 and, reciprocally, mutants unable to generate H2Bub1 have reductions in chromatin-associated Cdk9 and phosphorylated Spt5. Ablation of the preferred phosphoacceptor site in Spt5 combined with deletion of set1+ phenocopies morphological defects of htb1-K119R mutants, suggesting that Cdk9 and Set1 govern separate, partially redundant pathways downstream of H2Bub1. Despite the dependencies between H2Bub1 and Spt5 phosphorylation, mutations that impair Cdk9 function suppress abnormal cell morphologies and reverse an RNAPII distribution defect, both due to loss of H2Bub1. Conversely, growth of htb1-K119R cells is resistant to selective Cdk9 inhibition, relative to that of htb1+ cells. Taken together, biochemical and genetic results suggest that P-TEFb and H2Bub1 oppose one another to regulate RNAPII elongation, but that proper balance between the two is ensured via a positive feedback loop, in which phosphorylation by Cdk9 stimulates H2Bub1 and vice versa.