Leann Howe

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Research Methodology

Genomics, genetics, bioinformatics

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Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Targeting of histone acetylation to transcribed chromatin (2018)

The enrichment of histone acetylation within transcribed chromatin was first observed in the 1960s, and how specific histones are acetylated has been a central question of chromatin biology ever since. One mechanism for specificity is through the targeted recruitment of histone acetyltransferases (HATs) to transcribed chromatin, and we first focused on recruitment of the NuA3 HAT complex in S. cerevisiae. NuA3 is known to bind to cotranscriptional histone methylation through two domains: the PHD finger in Yng1 and the PWWP domain in Pdp3, which in vitro bind to H3K4 and H3K36 methylation, respectively. While the in vitro binding has been well characterized, the relative in vivo contributions of these histone methylation marks in targeting NuA3 is unknown. Here, through genome-wide colocalization and mutational interrogation, we demonstrate that the PHD finger of Yng1 and the PWWP domain of Pdp3 independently target NuA3 to H3K4 and H3K36 methylated chromatin, respectively. Interestingly however, the simple presence of NuA3 is insufficient to ensure the acetylation of associated nucleosomes, suggesting a secondary level of regulation that does not involve control of HAT-nucleosome interactions.Next we studied targeting of histone acetylation itself, focusing on the causality of the relationship between histone acetylation and RNAPII transcription. Through genome-wide analysis of mammalian cell culture and budding yeast, we reveal that the preponderance of histone acetylation is tightly linked with RNAPII occupancy, and, in S. cerevisiae, chemically or genetically altering RNAPII localization results in a corresponding change in histone acetylation. These findings show that histone acetylation is primarily targeted through RNAPII as a consequence of transcription. Importantly, several lines of evidence suggest that RNAPII does not promote acetylation by simple HAT targeting. First, we show that HAT occupancy is a poor predictor of histone acetylation. Second, NuA4 recruitment to upstream activation sequences of either Taf1 (TFIID) enriched or depleted promoters does not result in acetylation in the absence of transcription. Collectively, these data suggest that the activity of HATs is regulated post-recruitment by a mechanism that is dependent on RNAPII.

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Chromatin-Targeting Domains and the Divergence of Histone H3 (2016)

NuA3 is one of the major histone H3 HATs in yeast, as its catalytic subunit, Sas3, is responsible for acetylation of K14 and 23. The only characterized chromatin-targeting domain within the HAT is the PHD finger of Yng1, which associates with H3K4me3 and directs NuA3 to the 5’ ends of genes. We examined the genome-wide localization of Sas3, and found that it strongly correlates with H3K36me3. We demonstrated that recruitment of NuA3 to chromatin is dependent on methylation of both H3K4 and K36, and have implicated a novel member of the NuA3 complex – Pdp3 – as being responsible for this interaction. This likely occurs through its PWWP domain, which is a known H3K36me3-interactor in other proteins. In combination with the PHD finger of Yng1, this provides a mechanism by which NuA3 is recruited across the entirety of transcribed genes. In addition to its PHD finger and PWWP domain, NuA3 also contains the YEATS domain of Taf14. This is a conserved eukaryotic domain of unknown function present exclusively in transcription-related complexes. Although evidence exists suggesting that YEATS domains in other proteins interact directly with histones, its role in the NuA3 complex has remained elusive. We confirmed that the YEATS domain functions in chromatin-targeting of NuA3, and that it interacts directly with H3K9, 18, and 27 acetylated peptides. Finally we showed that NuA3 recruitment is dependent on Gcn5. This work describes a novel mechanism by which acetylation by one HAT targets further acetylation by another, and provides an additional mechanism for recruitment of NuA3.Finally, we explored the functional divergence of residues within histone H3 in yeast and humans. We showed that, while amino acids that define histone H3.3 are dispensable for yeast growth, substitution of residues within the histone H3 α3 helix with their human counterparts resulted in a severe growth defect. Furthermore, these mutations resulted in altered nucleosome positioning, both in vivo and in vitro, which was accompanied by an increased preference for nucleosome positioning sequences. Taken together, this suggests that divergent residues within the histone H3 α3 helix play differing roles in chromatin regulation between yeast and metazoans.

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"Open" Chromatin: Histone Acetylation, Linker Histones, and Histone Variants (2013)

DNA in the eukaryotic cell is packaged into a structure called chromatin. Chromatin is a dynamic structure that regulates access to DNA in response to environmental stimuli. Two widely conserved mechanisms that influence chromatin structure are the addition of post-translational modifications (PTMs) to histones and other chromatin-associated proteins, and the replacement of canonical histones with histone variants.Histone acetylation is catalyzed by histone acetyltransferases (HATs). HATs are comprised of a catalytic subunit, and associated proteins. Genetic analysis of the yeast HATs has shown that the combined deletion of the two HAT genes, GCN5 and SAS3, results in an inviable strain of yeast. In this thesis, I show that the inviability of the gcn5Δsas3Δ mutant is due to a combined failure to acetylate both histone H3 and the chromatin-remodeler protein Rsc4. Further, I show that acetylation of Rsc4 is catalyzed by Gcn5 in a HAT complex-independent manner.The linker histone, H1, is associated with higher-order chromatin structure; it has been shown that removal of H1 is required to allow access to DNA. In this work, I show that deletion of the linker histone rescues the growth of a conditional gcn5Δ sas3Δ mutant expressing a temperature-sensitive version of Sas3. Further, I present the incorporation of the histone variant Htz1 as an additional mechanism for mobilizing the linker histone away from the +1 nucleosome. I, also, provide data that corroborates evidence suggesting that the yeast linker histone binds a single nucleosome.Another histone variant found in many eukaryotes is histone H3.3, which is primarily incorporated into transcriptionally active regions in chromatin. In this dissertation, we created a series of human-yeast histone hybrids and tested their ability to rescue yeast lacking both endogenous copies of histone H3. Our data shows that the two human histone H3 variants, H3.1 and H3.3, are functionally interchangeable for growth in most nutrient conditions, confirming that the four amino acids that are different between H3.1 and H3.3 are not necessary to create transcriptionally permissive chromatin. Finally, we present evidence that three yeast H3 C-terminal domain amino acids play an important role in regulating the interactions of yeast H3.

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Regulation of chromatin by FACT and NuA3 (2013)

The presence of nucleosomes over vast regions ofgenome negatively influencestranscription creating a need for temporal and structural regulation of chromatin. Thedefaulttranscription-repressive state can be countered by addition of post-translationalmodifications by chromatin modifiersor chromatin alterationbyhistone chaperones.Chaperones alterchromatin structurebeforetheRNAPIIpassageand restore itafterwards. Howmodifiers and chaperones function within chromatin is an area ofintense research.Herewe show how two complexes, the yeastFACTand NuA3contribute tochromatinfunction.Yeast Facilitates Chromatin Transactions (yFACT) is a histone chaperone thatmaintains chromatin structure.The model of yFACT functionin vivo is asubject of muchdebate. Weprovide evidencethat yFACT acts by stably binding and alteringnucleosomes. We alsopresenttheEM structure of yFACT associated withnucleosomes.We find that yFACT-associated nucleosomes are hyper-acetylated and show evidencefor it being an effect of a direct interaction between yFACT and NuA3.At the same time, acetylation ofthe H3K56 residue bythe histone acetyltransferaseRtt109, acts to recruit yFACT to chromatin through a nucleosome-dependent mechanism.To determinethe distribution of yFACT-associated nucleosomesweconstructeda map of yFACT-nucleosomelocalization atsingle-nucleosomeresolution. We showthat while yFACT-bound nucleosomes are distributed thought thegenomethey are alsopositionedoverthecanonical Nucleosome Depleted Regions (NDR).The yFACT-boundnucleosomesare positionedaround TATA-elements andNhp6-target sequencesgenome-wide.Deletionof NHP6A/Bleads toloss ofchromatinat these loci. Our worksuggeststhe first ever sequence-dependent mechanism of histone chaperoneactioninSaccharomyces cerevisiae.We also examined NuA3 recruitment to chromatin andshowedthat Yng1, asubunit of NuA3 with a known affinity for H3K4me3 is a bivalent protein.Whileaspreviously shown,the C-terminal PHD finer of Yng1 binds to H3K4me3, the N-terminusof Yng1 canalsobind to unmodified chromatin.Although these motifscan bindindependently, together they increase the apparent association of Yng1 withchromatin. Yng1 binding to chromatin is regulated by the HDA1 complex.

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Investiagation into the role and regulation of histone lysine methylation in the yeast Saccharomyces cerevisiae (2012)

No abstract available.

Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Investigating the effects of expressing MECP2 in Saccharomyces cerevisiae (2023)

Methyl-CpG-binding protein 2 (MECP2) plays a key role in brain homeostasis. MECP2 function is sensitive to changes in its concentration, as both overexpression and reduction of MECP2 result in neurodevelopmental disorders in humans and mice. MEPC2 was originally discovered based on its ability to bind methylated-CpG DNA to repress gene expression. Later research suggested that MECP2 may also interact with unmethylated DNA. While there have been in vitro studies supporting this idea, there were few in vivo studies. In this thesis, we demonstrate that expression of the human MECP2 in an organism lacking DNA methylation, the budding yeast Saccharomyces cerevisiae, disrupts normal yeast growth, by inhibiting passage through S phase. Further investigation revealed that MECP2-expressing yeast had defects in their cell cycle, which may be the cause of the growth defect. We found that the phenotype of yeast expressing MECP2 with disease-associated mutations reflects disease severity in humans. Using single-cell techniques such as flow cytometry and fluorescence microscopy, we confirmed the expression of MECP2 in yeast and its nuclear localization. Disease-associated mutations that reversed the growth defect either impair nuclear localization or, as previously reported, disrupt MECP2 binding to DNA. In addition, we found differences between the two MECP2 isoforms in terms of protein stability, and impact on the cell cycle and viability. This suggests that the isoforms have different functions and potentially, differing interacting partners within cells. Collectively, our findings confirmed the feasibility of using yeast as a model organism to study MECP2 function in vivo. Moreover, using this system, we are able to rapidly screen for phenotypes of MECP2-disease-associated mutations that reflect disease severity in humans.

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Investigation of cryptic transcription suppressing factors using a FLO8:sfGFP reporter (2023)

Chromatin disruption caused by polymerase passage during transcription or DNA replication has the potential to lead to cryptic transcription originating within aberrant initiation sites across the genome. We developed a sfGFP fluorescence reporter to track cryptic transcription initiating from an intragenic cryptic promoter within the FLO8 coding region and confirmed reporter utility in a spt16-197 mutant strain. Using this reporter, our results showed that the proteins, Spt10, Spt21, Cac1, and Asf1 are required for chromatin stability during DNA replication, while Spt2 plays a replication-independent role in maintaining chromatin. We also showed that blocking DNA replication only partially suppresses cryptic transcription in Spt5, Spt6, or Spt16-depleted cells, indicative of these factors functioning in a process independent of replication. Taken together, our results demonstrate that restoration of nucleosome stability following disruption is facilitated by a number of collaborating factors functioning in various pathways.

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Histone acetylation, not stoichiometry, regulates linker histone binding in Saccharomyces cerevisiae (2019)

Linker histones play a fundamental role in shaping chromatin structure, but how their interaction with chromatin is regulated is not well understood. A combination of genetic and genomic approaches were used to explore the regulation of linker histone binding in the yeast, Saccharomyces cerevisiae. We found that increased expression of Hho1, the yeast linker histone, resulted in a severe growth defect, despite only subtle changes in chromatin structure. Further, this growth defect was rescued by mutations that increase histone acetylation. Consistent with this, genome-wide analysis of linker histone occupancy revealed an inverse correlation with histone tail acetylation in both yeast and mouse embryonic stem cells. Collectively, these results suggest that histone acetylation negatively regulates linker histone binding in S. cerevisiae and other organisms and provides important insight into how chromatin structure is regulated and maintained to both facilitate and repress transcription.

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Investigating histone methylation in yeast : regulation of H3K4me3, and the role of the methyl-histone binding domains of Isw1b (2010)

Chromatin structure is regulated in part by the post-translational modification of histones. Histone methylation is highly conserved amongst eukaryotes, and is arguably one of the best characterized indicators of whether a gene is repressed or active. There are several unique states of histone methylation, each capable of specific downstream effects through the recruitment of highly specific methyl-histone binding domains and their associated chromatin-altering protein complexes. Histone H3 lysine 4 tri-methylation (H3K4me3) is a well known mark of actively transcribed genes, and co-localizes with histone H3 lysine 14 acetylation (H3K14ac), another mark of actively transcribed genes. The discovery that H3K4me3 is lost when H3K14 is substituted with another residue, led to the possibility of cross-talk between H3K4me3 and H3K14ac. The first part of this thesis demonstrates that H3K4me3 is indeed dependent on H3K14ac. Furthermore, we go on to show for the first time, that H3K14ac protects H3K4me3 from demethylation by the histone demethylase Jhd2.Though the mechanisms by which methyl-histone binding domains recognize methylated chromatin have been well studied, the specific physiological roles of the numerous methyl-histone binding domains have yet to be investigated. Isw1 is a highly conserved catalytic subunit of several ATP-dependent chromatin-modifying complexes. One of these complexes, Isw1b, has two putative methyl-histone binding domains, the PHD finger of Ioc2, and the PWWP domain of Ioc4. The second part of this thesis investigates the role that these domains play in the localization of the Isw1b complex to a specific region of the genome. Though we were unable to demonstrate a role for the PHD finger of Ioc2, we did demonstrate that the PWWP domain of Ioc4 is involved in chromatin localization. Additionally we found that Ioc2’s ability to bind chromatin is negatively affected by association with Ioc4 in the Isw1b complex, though the significance of this finding has yet to be determined.

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