Gene-Specific H1 Eviction through a Transcriptional Activator→p300→NAP1→H1 Pathway.

Linker histone H1 has been correlated with transcriptional inhibition, but the mechanistic basis of the inhibition and its reversal during gene activation has remained enigmatic. We report that H1-compacted chromatin, reconstituted in vitro, blocks transcription by abrogating core histone modifications by p300 but not activator and p300 binding. Transcription from H1-bound chromatin is elicited by the H1 chaperone NAP1, which is recruited in a gene-specific manner through direct interactions with activator-bound p300 that facilitate core histone acetylation (by p300) and concomitant eviction of H1 and H2A-H2B.

The human PAF1 complex acts in chromatin transcription elongation both independently and cooperatively with SII/TFIIS.

Genetic and cell-based studies have implicated the PAF1 complex (PAF1C) in transcription-associated events, but there has been no evidence showing a direct role in facilitating transcription of a natural chromatin template. Here, we demonstrate an intrinsic ability of human PAF1C (hPAF1C) to facilitate activator (p53)- and histone acetyltransferase (p300)-dependent transcription elongation from a recombinant chromatin template in a biochemically defined RNA polymerase II transcription system. This represents a PAF1C function distinct from its established role in histone ubiquitylation and methylation.

RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells.

H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (1) that hRAD6 serves as the cognate E2-conjugating enzyme; (2) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120; (3) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery; (4) that H2B ubiquitylation per se does not affect the level of hPAF-, SII-, and p300-dependent transcription and likely functions downstream; and (5) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and trimethylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.

Transcription of in vitro assembled chromatin templates in a highly purified RNA polymerase II system.

In mammalian cells RNA polymerase II efficiently transcribes nucleosome-packaged DNA. In this regard, a fundamental question concerns the nature and mechanism of action of the accessory factors that are necessary and sufficient for, or enhance, transcription through nucleosomal arrays by RNA polymerase II. Here we describe a highly purified system that allows for efficient activator-dependent transcription by RNA polymerase II from the promoter through several contiguous nucleosomes on defined chromatin templates.