Marcie H. Warner

Regulation of histone modification and cryptic transcription by the Bur1 and Paf1 complexes

The Bur1–Bur2 and Paf1 complexes function during transcription elongation and affect histone modifications. Here we describe new roles for Bur1–Bur2 and the Paf1 complex. We find that histone H3 K36 tri‐methylation requires specific components of the Paf1 complex and that K36 tri‐methylation is more strongly affected at the 5′ ends of genes in paf1Δ and bur2Δ strains in parallel with increased acetylation of histones H3 and H4. Interestingly, the 5′ increase in histone acetylation is independent of K36 methylation, and therefore is mechanistically distinct from the methylation‐driven deacetylation that occurs at the 3′ ends of genes. Finally, Bur1–Bur2 and the Paf1 complex have a second methylation‐independent function, since bur2Δ set2Δ and paf1Δ set2Δ double mutants display enhanced histone acetylation at the 3′ ends of genes and increased cryptic transcription initiation. These findings identify new functions for the Paf1 and Bur1–Bur2 complexes, provide evidence that histone modifications at the 5′ and 3′ ends of coding regions are regulated by distinct mechanisms, and reveal that the Bur1–Bur2 and Paf1 complexes repress cryptic transcription through a Set2‐independent pathway.

Rtf1 is a multifunctional component of the Paf1 complex that regulates gene expression by directing cotranscriptional histone modification.

ABSTRACT Numerous transcription accessory proteins cause alterations in chromatin structure that promote the progression of RNA polymerase II (Pol II) along open reading frames (ORFs). The Saccharomyces cerevisiae Paf1 complex colocalizes with actively transcribing Pol II and orchestrates modifications to the chromatin template during transcription elongation. To better understand the function of the Rtf1 subunit of the Paf1 complex, we created a series of sequential deletions along the length of the protein. Genetic and biochemical assays were performed on these mutants to identify residues required for the various activities of Rtf1. Our results establish that discrete nonoverlapping segments of Rtf1 are necessary for interaction with the ATP-dependent chromatin-remodeling protein Chd1, promoting covalent modification of histones H2B and H3, recruitment to active ORFs, and association with other Paf1 complex subunits. We observed transcription-related defects when regions of Rtf1 that mediate histone modification or association with active genes were deleted, but disruption of the physical association between Rtf1 and other Paf1 complex subunits caused only subtle mutant phenotypes. Together, our results indicate that Rtf1 influences transcription and chromatin structure through several independent functional domains and that Rtf1 may function independently of its association with other members of the Paf1 complex.

Small region of Rtf1 protein can substitute for complete Paf1 complex in facilitating global histone H2B ubiquitylation in yeast

Histone modifications regulate transcription by RNA polymerase II and maintain a balance between active and repressed chromatin states. The conserved Paf1 complex (Paf1C) promotes specific histone modifications during transcription elongation, but the mechanisms by which it facilitates these marks are undefined. We previously identified a 90-amino acid region within the Rtf1 subunit of Paf1C that is necessary for Paf1C-dependent histone modifications in Saccharomyces cerevisiae. Here we show that this histone modification domain (HMD), when expressed as the only source of Rtf1, can promote H3 K4 and K79 methylation and H2B K123 ubiquitylation in yeast. The HMD can restore histone modifications in rtf1Δ cells whether or not it is directed to DNA by a fusion to a DNA binding domain. The HMD can facilitate histone modifications independently of other Paf1C subunits and does not bypass the requirement for Rad6–Bre1. The isolated HMD localizes to chromatin, and this interaction requires residues important for histone modification. When expressed outside the context of full-length Rtf1, the HMD associates with and causes Paf1C-dependent histone modifications to appear at transcriptionally inactive loci, suggesting that its function has become deregulated. Finally, the Rtf1 HMDs from other species can function in yeast. Our findings suggest a direct and conserved role for Paf1C in coupling histone modifications to transcription elongation.

Identification of a Role for Histone H2B Ubiquitylation in Noncoding RNA 3′-End Formation Through Mutational Analysis of Rtf1 in Saccharomyces cerevisiae

The conserved eukaryotic Paf1 complex regulates RNA synthesis by RNA polymerase II at multiple levels, including transcript elongation, transcript termination, and chromatin modifications. To better understand the contributions of the Paf1 complex to transcriptional regulation, we generated mutations that alter conserved residues within the Rtf1 subunit of the Saccharomyces cerevisiae Paf1 complex. Importantly, single amino acid substitutions within a region of Rtf1 that is conserved from yeast to humans, which we termed the histone modification domain, resulted in the loss of histone H2B ubiquitylation and impaired histone H3 methylation. Phenotypic analysis of these mutations revealed additional defects in telomeric silencing, transcription elongation, and prevention of cryptic initiation. We also demonstrated that amino acid substitutions within the Rtf1 histone modification domain disrupt 3′-end formation of snoRNA transcripts and identify a previously uncharacterized regulatory role for the histone H2B K123 ubiquitylation mark in this process. Cumulatively, our results reveal functionally important residues in Rtf1, better define the roles of Rtf1 in transcription and histone modification, and provide strong genetic support for the participation of histone modification marks in the termination of noncoding RNAs.

Genome Sequences of Gordonia Phages Bowser and Schwabeltier

ABSTRACT Gordonia phages Bowser and Schwabeltier are newly isolated phages infecting Gordonia terrae 3612. Bowser and Schwabeltier have similar siphoviral morphologies and their genomes are related to each other, but not to other phages. Their lysis cassettes are atypically situated among virion tail genes, and Bowser encodes two tyrosine integrases.

Genome Sequence of a Newly Isolated Mycobacteriophage, ShedlockHolmes.

ABSTRACT Mycobacteriophage ShedlockHolmes is a newly isolated phage infecting Mycobacterium smegmatis mc2155. It has a 61,081-bp genome containing 99 predicted protein-coding genes and one tRNA gene. ShedlockHolmes is closely related to mycobacteriophages Pixie, Keshu, and MacnCheese and is a new member of subcluster K3.

Genome Sequences of Gordonia Bacteriophages Obliviate, UmaThurman, and Guacamole

ABSTRACT We describe three newly isolated phages—Obliviate, UmaThurman, and Guacamole—that infect Gordonia terrae 3612. The three genomes are related to one another but are not closely related to other previously sequenced phages or prophages. The three phages are predicted to use integration-dependent immunity systems as described in several mycobacteriophages.

Genome Sequence of Gordonia Bacteriophage Lucky10

ABSTRACT Lucky10 is a newly isolated phage of Gordonia terrae 3612 that was recovered from a soil sample in Pittsburgh, PA. Lucky10 has siphoviral morphology and a double-stranded DNA (dsDNA) genome of 42,979 bp, with 70 predicted protein-coding genes. Lucky10 shows little similarity to previously reported Gordonia phages.

Genome Sequences of Gordonia Phages Hotorobo, Woes, and Monty

ABSTRACT Hotorobo, Woes, and Monty are newly isolated bacteriophages of Gordonia terrae 3612. The three phages are related, and their genomes are similarly sized (76,972 bp, 73,752 bp, and 75,680 bp for Hotorobo, Woes, and Monty, respectively) and organized. They have extremely long tails and among the longest tape measure protein genes described to date.

Genome Sequences of Gordonia Phages BaxterFox, Kita, Nymphadora, and Yeezy

ABSTRACT Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%.