Rebecca J. Burgess

Histone chaperones in nucleosome assembly and human disease

Nucleosome assembly following DNA replication, DNA repair and gene transcription is critical for the maintenance of genome stability and epigenetic information. Nucleosomes are assembled by replication-coupled or replication-independent pathways with the aid of histone chaperone proteins. How these different nucleosome assembly pathways are regulated remains relatively unclear. Recent studies have provided insight into the mechanisms and the roles of histone chaperones in regulating nucleosome assembly. Alterations or mutations in factors involved in nucleosome assembly have also been implicated in cancer and other human diseases. This review highlights the recent progress and outlines future challenges in the field.

A Role for Gcn5 in Replication-Coupled Nucleosome Assembly

Acetylation of lysine residues at the H3 N terminus is proposed to function in replication-coupled (RC) nucleosome assembly, a process critical for the inheritance of epigenetic information and maintenance of genome stability. However, the role of H3 N-terminal lysine acetylation and the corresponding lysine acetyltransferase (KAT) in RC nucleosome assembly are not known. Here we show that Gcn5, a KAT that functions in transcription, works in parallel with Rtt109, the H3 lysine 56 KAT, to promote RC nucleosome assembly. Cells lacking both Gcn5 and Rtt109 are highly sensitive to DNA damaging agents. Moreover, cells lacking GCN5 or those that express N-terminal H3 mutants are compromised for deposition of new H3 onto replicating DNA and also show reduced binding of H3 to CAF-1, a histone chaperone involved in RC nucleosome assembly. These results demonstrate that Gcn5 regulates RC nucleosome assembly, in part, by promoting H3 association with CAF-1 via H3 acetylation.

All roads lead to chromatin: Multiple pathways for histone deposition

Chromatin, a complex of DNA and associated proteins, governs diverse processes including gene transcription, DNA replication and DNA repair. The fundamental unit of chromatin is the nucleosome, consisting of 147bp of DNA wound about 1.6 turns around a histone octamer of one (H3-H4)(2) tetramer and two H2A-H2B dimers. In order to form nucleosomes, (H3-H4)(2) tetramers are deposited first, followed by the rapid deposition of H2A-H2B. It is believed that the assembly of (H3-H4)(2) tetramers into nucleosomes is the rate-limiting step of nucleosome assembly. Moreover, assembly of H3-H4 into nucleosomes following DNA replication, DNA repair and gene transcription is likely to be a key step in the inheritance of epigenetic information and maintenance of genome integrity. In this review, we discuss how nucleosome assembly of H3-H4 is regulated by concerted actions of histone chaperones and modifications on newly synthesized H3 and H4. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The coordinated process of DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.

Thiopurine S-methyltransferase pharmacogenetics: autophagy as a mechanism for variant allozyme degradation

Objective Thiopurine S-methyltransferase (TPMT)*3A is degraded much more rapidly than is the ‘wild-type’ enzyme through a ubiquitin–proteasome-dependent process. It also forms aggresomes, suggesting a possible dynamic balance between degradation and aggregation. We set out to identify genes encoding proteins participating in these processes. Methods Green fluorescent protein tagged TPMT*3A was expressed in a Saccharomyces cerevisiae gene deletion library, and flow cytometry was used to screen for cells with high fluorescence intensity, indicating the loss of a gene essential for TPMT*3A degradation. Results Twenty-four yeast genes were identified in functional categories that included ubiquitin-dependent protein degradation, vesicle trafficking, and vacuolar degradation. The presence of genes encoding proteins involved in vesicular transport and vacuolar degradation suggested a possible role in TPMT*3A degradation for autophagy – a process not previously identified as a pharmacogenomic mechanism. In support of that hypothesis, TPMT*3A aggregates increased dramatically in mutants for vacuolar protease and autophagy-related genes. Furthermore, TPMT*3A expression in human cells induced autophagy, and small interfering RNA-mediated knockdown of ATG7, an autophagy-related human protein, enhanced TPMT*3A aggregation but not that of TPMT*3C or wild-type TPMT, indicating that autophagy contributes to TPMT*3A degradation in mammalian cells. We also demonstrated that UBE2G2, the human homologue of the E2 ubiquitin-conjugating enzyme identified during the yeast genetic screen, was involved in TPMT*3A degradation in human cells. Conclusion These results indicate that autophagy should be considered among mechanisms responsible for the effects of pharmacogenetically significant polymorphisms that alter encoded amino acids.

Human Histone Acetyltransferase 1 Protein Preferentially Acetylates H4 Histone Molecules in H3.1-H4 over H3.3-H4

Background: Hat1-RbAp46 acetylates H4 lysine 5 and 12 residues. Results: Hat1-RbAp46 acetylates H4 in H3.1-H4 complex more efficiently than that in H3.3-H4 complex. Conclusion: Hat1-RbAp46 differentially acetylates H4 in H3.1-H4 and H3.3-H4 and impacts nucleosome assembly of H3.1 and H3.3 differently. Significance: Determination of how Hat1-RbAp46 impacts nucleosome assembly of H3.1 and H3.3 differently will help understand how chromatin states are inherited during S phase of the cell cycle. In mammalian cells, canonical histone H3 (H3.1) and H3 variant (H3.3) differ by five amino acids and are assembled, along with histone H4, into nucleosomes via distinct nucleosome assembly pathways. H3.1-H4 molecules are assembled by histone chaperone CAF-1 in a replication-coupled process, whereas H3.3-H4 are assembled via HIRA in a replication-independent pathway. Newly synthesized histone H4 is acetylated at lysine 5 and 12 (H4K5,12) by histone acetyltransferase 1 (HAT1). However, it remains unclear whether HAT1 and H4K5,12ac differentially regulate these two nucleosome assembly processes. Here, we show that HAT1 binds and acetylates H4 in H3.1-H4 molecules preferentially over H4 in H3.3-H4. Depletion of Hat1, the catalytic subunit of HAT1 complex, results in reduced H3.1 occupancy at H3.1-enriched genes and reduced association of Importin 4 with H3.1, but not H3.3. Finally, depletion of Hat1 or CAF-1p150 leads to changes in expression of a H3.1-enriched gene. These results indicate that HAT1 differentially impacts nucleosome assembly of H3.1-H4 and H3.3-H4.

Histones, histone chaperones and nucleosome assembly

Chromatin structure governs a number of cellular processes including DNA replication, transcription, and DNA repair. During DNA replication, chromatin structure including the basic repeating unit of chromatin, the nucleosome, is temporarily disrupted, and then reformed immediately after the passage of the replication fork. This coordinated process of nucleosome assembly during DNA replication is termed replication—coupled nucleosome assembly. Disruption of this process can lead to genome instability, a hallmark of cancer cells. Therefore, addressing how replication-coupled nucleosome assembly is regulated has been of great interest. Here, we review the current status of this growing field of interest, highlighting recent advances in understanding the regulation of this important process by the dynamic interplay of histone chaperones and histone modifications.

Epigenetic changes in gliomas

Epigenetics are defined, in broad-terms, as alterations in gene expression without changes in DNA sequence. While histone modifications and DNA methylation are two classical means to regulate gene expression, miRNA has also recently been documented to govern gene expression in normal as well as cancer cells. In this review, we will first describe briefly histone modifications, DNA methylation and miRNAs and the functions of these epigenetic marks during different cellular processes involving DNA metabolism. We will then highlight some epigenetic changes in glioblastomas, a malignant form of brain tumor, and potential application of epigenetic means for diagnosis, prognosis, and treatment of gliomas. We expect that novel therapies will be developed to counter epigenetic changes in this deadly disease.

Arabidopsis Separase Functions beyond the Removal of Sister Chromatid Cohesion during Meiosis

Separase is a capase family protease that is required for the release of sister chromatid cohesion during meiosis and mitosis. Proteolytic cleavage of the α-kleisin subunit of the cohesin complex at the metaphase-to-anaphase transition is essential for the proper segregation of chromosomes. In addition to its highly conserved role in cleaving the α-kleisin subunit, separase appears to have acquired additional diverse activities in some organisms, including involvement in mitotic and meiotic anaphase spindle assembly and elongation, interphase spindle pole body positioning, and epithelial cell reorganization. Results from the characterization of Arabidopsis (Arabidopsis thaliana) separase (ESP) demonstrated that meiotic expression of ESP RNA interference blocked the proper removal of cohesin from chromosomes and resulted in the presence of a mixture of fragmented chromosomes and intact bivalents. The presence of large numbers of intact bivalents raised the possibility that separase may also have multiple roles in Arabidopsis. In this report, we show that meiotic expression of ESP RNA interference blocks the removal of cohesin during both meiosis I and II, results in alterations in nonhomologous centromere association, disrupts the radial microtubule system after telophase II, and affects the proper establishment of nuclear cytoplasmic domains, resulting in the formation of multinucleate microspores.

Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.