Background: It remains unknown how Dot1 or the Dot1 complex specifically targets the transcribed regions. Results: A functional interaction between hDOT1L and RNAPII targets hDOT1L and subsequent H3K79 methylations to active genes. Conclusion: hDOT1L interacts with phosphorylated CTD of RNAPII. Significance: This represents novel mechanistic insight into the understanding of targeting and propagation of hDOT1L along gene transcription.

Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1⅐Cullin 1⅐F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein.Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206-215 and 216-225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys 48 and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe Ubiquitination is a complex protein modification reaction that produces a covalent isopeptide bond linking the ubiquitin Gly 76 carboxyl end to the ⑀-amino group of a lysine residue within a substrate protein (1). One of the hallmarks of this modification is that substrates are frequently attached with polyubiquitin chains, formed by isopeptide linkage joining the ubiquitin Gly 76 carboxyl end and one of its seven internal lysine residues. Three enzymatic activities typically participate in the ubiquitination reaction, including a ubiquitin-activating enzyme (E1) that forms an E1-Sϳubiquitin thiol ester complex in an ATP hydrolysis-dependent manner, a ubiquitin-conjugating enzyme (E2) capable of tethering a ubiquitin in thiol ester form that is transferred from E1, and a ubiquitin-ligase (E3). RING finger domain-containing E3s orchestrate the transfer of ubiquitin to a substrate by recruiting both the target protein and E2-Sϳubiquitin thiol ester and by helping position the attacking substrate lysine and the E2-bound donor ubiquitin optimally for conjugation (2). SCF (Skp1⅐Cullin⅐F-box) is a four-subunit RING E3 ligase complex with CUL1 (cullin 1) as scaffold that at its N terminus tethers the Skp1⅐F-box protein complex to target a substrate and utilizes its C terminus to dock the ROC1/Rbx1/Hrt1 RING finger subunit for recruiting an E2 ubiquitin-conjugating enzyme (3-9). SCF-mediated polyubiquitination is activated by conjugation of Nedd8, a ubiquitin-like protein, to human CUL1 at lysine 720 (10, 11), most likely due to Nedd8-imposed conformational changes in CUL1 (12), thereby relieving autoinhibitory interactions between the extreme C terminus of CUL1 and ROC1 (13).Accumulating evidence demonstrates a role for the Cdc34 E2 in SCF-dependent ubiquitination. Yeast Cdc34 (Ubc3) is essential for viability and is required for SCF Cdc4 -dependent degradation of the cyclin-dependent kinase inhibitor Sic1 (see Refs. 3 and 4 and references therein). This regulation has been recapitulated by in vitro experiments that reconstitute the SCF Cdcc4 -dependent and Cdc34-catalyzed ubiquitination of Sic1 and the degradation of the modified substrate by the 26 S proteasome (3,4,14). Human Cdc34 (hCdc34) 5 is highly conserved as it is

Dot1 (disruptor of telomeric silencing-1, DOT1L in humans) is the only known enzyme responsible for histone H3 lysine 79 methylation (H3K79me) and is evolutionarily conserved in most eukaryotes. Yeast Dot1p lacks a SET domain and does not methylate free histones and thus may have different actions with respect to other histone methyltransferases. Here we show that Dot1p displays histone chaperone activity and regulates nucleosome dynamics via histone exchange in yeast. We show that a methylation-independent function of Dot1p is required for the cryptic transcription within transcribed regions seen following disruption of the Set2–Rpd3S pathway. Dot1p can assemble core histones to nucleosomes and facilitate ATP-dependent chromatin-remodeling activity through its nucleosome-binding domain, in vitro. Global analysis indicates that Dot1p appears to be particularly important for histone exchange and chromatin accessibility on the transcribed regions of long-length genes. Our findings collectively suggest that Dot1p-mediated histone chaperone activity controls nucleosome dynamics in transcribed regions.

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