Catherine B. Millar

Genome-wide patterns of histone modifications in yeast

Post-translational histone modifications and histone variants generate complexity in chromatin to enable the many functions of the chromosome. Recent studies have mapped histone modifications across the Saccharomyces cerevisiae genome. These experiments describe how combinations of modified and unmodified states relate to each other and particularly to chromosomal landmarks that include heterochromatin, subtelomeric chromatin, centromeres, origins of replication, promoters and coding regions. Such patterns might be important for the regulation of heterochromatin-mediated silencing, chromosome segregation, DNA replication and gene expression.

Fas-associated death domain protein interacts with methyl-CpG binding domain protein 4: A potential link between genome surveillance and apoptosis

Fas-associated death domain protein (FADD) is an adaptor protein bridging death receptors with initiator caspases. Thus, its function and localization are assumed to be cytoplasmic, although the localization of endogenous FADD has not been reported. Surprisingly, the data presented here demonstrate that FADD is mainly nuclear in several adherent cell lines. Its accumulation in the nucleus and export to the cytoplasm required the phosphorylation site Ser-194, which was also required for its interaction with the nucleocytoplasmic shuttling protein exportin-5. Within the nucleus, FADD interacted with the methyl-CpG binding domain protein 4 (MBD4), which excises thymine from GT mismatches in methylated regions of chromatin. The MBD4-interacting mismatch repair factor MLH1 was also found in a complex with FADD. The FADD–MBD4 interaction involved the death effector domain of FADD and a region of MBD4 adjacent to the glycosylase domain. The FADD-binding region of MBD4 was downstream of a frameshift mutation that occurs in a significant fraction of human colorectal carcinomas. Consistent with the idea that MBD4 can signal to an apoptotic effector, MBD4 regulated DNA damage-, Fas ligand-, and cell detachment-induced apoptosis. The nuclear localization of FADD and its interaction with a genome surveillance/DNA repair protein that can regulate apoptosis suggests a novel function of FADD distinct from direct participation in death receptor signaling complexes.

Neurturin: An autocrine regulator of renal collecting duct development

Urine is produced in the kidney by excretory nephrons and is drained by a tree-like system of collecting ducts to the ureter. The collecting ducts develop by arborisation of an initially unbranched epithelial rudiment, the ureteric bud, which ramifies through the surrounding mesenchyme and induces the formation of nephrons by mesenchyme-to-epithelial transition. The question of how collecting duct morphogenesis is controlled is an important one, from the points of view of both basic developmental biology and congenital renal pathology (multi- and polycystic renal disease, and some forms of renal agenesis, arise from defective collecting duct development). We report that neurturin, a neurotrophin related to glial cell line-derived neurotrophic factor and expressed in the developing kidney, acts as a collecting duct morphogen in culture. Applied in culture medium, it promotes epithelial branching and can induced branch initiation that has otherwise been blocked by depleting cultured kidneys of their sulfated proteoglycans or by antibody treatments. Applied locally on agarose beads, neurturin induces supernumerary ureteric buds to emerge from the wolffian duct and causes nearby collecting duct branches to distend to an abnormally large diameter. Like its receptors, neurturin is expressed by the developing collecting ducts themselves, suggesting that it forms an autocrine morphoregulatory control loop. This is in marked contrast to previously identified morphogens such as glial cell line derived neurotrophic factor and hepatocyte growth factor, which act in a paracrine manner.

Organizing the genome with H2A histone variants.

Chromatin acts as an organizer and indexer of genomic DNA and is a highly dynamic and regulated structure with properties directly related to its constituent parts. Histone variants are abundant components of chromatin that replace canonical histones in a subset of nucleosomes, thereby altering nucleosomal characteristics. The present review focuses on the H2A variant histones, summarizing current knowledge of how H2A variants can introduce chemical and functional heterogeneity into chromatin, the positions that nucleosomes containing H2A variants occupy in eukaryotic genomes, and the regulation of these localization patterns.

A conserved function for the H2A.Z C-terminus

Background: The H2A variant H2A.Z is an important regulatory component of chromatin. Results: A novel form of human H2A.Z and mutants of yeast H2A.Z that share truncated C termini have altered properties. Conclusion: The C-terminal tail of histone H2A.Z is important for its stable association with nucleosomes. Significance: The strength of H2A.Zs association with nucleosomes is key to the biological functions of this histone variant. Histone H2A variants generate diversity in chromatin structure and functions, as nucleosomes containing variant H2A histones have altered physical, chemical, and biological properties. H2A.Z is an evolutionarily ancient and highly conserved H2A variant that regulates processes ranging from gene expression to the DNA damage response. Here we find that the unstructured portion of the C-terminal tail of H2A.Z is required for the normal functions of this histone variant in budding yeast. We have also identified a novel splice isoform of the human H2A.Z-2 gene that encodes a C-terminally truncated H2A.Z protein that is similar to the truncation mutants we identified in yeast. The short forms of H2A.Z in both yeast and human cells are more loosely associated with chromatin than the full-length proteins, indicating a conserved function for the H2A.Z C-terminal tail in regulating the association of H2A.Z with nucleosomes.

Histone variant Htz1 promotes histone H3 acetylation to enhance nucleotide excision repair in Htz1 nucleosomes

Nucleotide excision repair (NER) is critical for maintaining genome integrity. How chromatin dynamics are regulated to facilitate this process in chromatin is still under exploration. We show here that a histone H2A variant, Htz1 (H2A.Z), in nucleosomes has a positive function in promoting efficient NER in yeast. Htz1 inherently enhances the occupancy of the histone acetyltransferase Gcn5 on chromatin to promote histone H3 acetylation after UV irradiation. Consequently, this results in an increased binding of a NER protein, Rad14, to damaged DNA. Cells without Htz1 show increased UV sensitivity and defective removal of UV-induced DNA damage in the Htz1-bearing nucleosomes at the repressed MFA2 promoter, but not in the HMRa locus where Htz1 is normally absent. Thus, the effect of Htz1 on NER is specifically relevant to its presence in chromatin within a damaged region. The chromatin accessibility to micrococcal nuclease in the MFA2 promoter is unaffected by HTZ1 deletion. Acetylation on previously identified lysines of Htz1 plays little role in NER or cell survival after UV. In summary, we have identified a novel aspect of chromatin that regulates efficient NER, and we provide a model for how Htz1 influences NER in Htz1 nucleosomes.

Mutations in Non-Acid Patch Residues Disrupt H2A.Z's Association with Chromatin through Multiple Mechanisms

The incorporation of histone variants into nucleosomes is a critical mechanism for regulating essential DNA-templated processes and for establishing distinct chromatin architectures with specialised functions. H2A.Z is an evolutionarily conserved H2A variant that has diverse roles in transcriptional regulation, heterochromatin boundary definition, chromosome stability and DNA repair. The H2A.Z C-terminus diverges in sequence from canonical H2A and imparts unique functions to H2A.Z in the yeast S. cerevisiae. Although mediated in part through the acid patch-containing M6 region, many molecular determinants of this divergent structure-function relationship remain unclear. Here, by using an unbiased random mutagenesis screen of H2A.Z alleles, we identify point mutations in the C-terminus outside of the M6 region that disrupt the normal function of H2A.Z in response to cytotoxic stress. These functional defects correlate with reduced chromatin association, which we attribute to reduced physical stability within chromatin, but also to altered interactions with the SWR and INO80 chromatin remodeling complexes. Together with experimental data, computational modelling of these residue changes in the context of protein structure suggests the importance of C-terminal domain integrity and configuration for maintaining the level of H2A.Z in nucleosomes.

Stress-Activated Kinase MKK7 Governs Epigenetics of Cardiac Repolarization for Arrhythmia Prevention

Background: Ventricular arrhythmia is a leading cause of cardiac mortality. Most antiarrhythmics present paradoxical proarrhythmic side effects, culminating in a greater risk of sudden death. Methods: We describe a new regulatory mechanism linking mitogen-activated kinase kinase-7 deficiency with increased arrhythmia vulnerability in hypertrophied and failing hearts using mouse models harboring mitogen-activated kinase kinase-7 knockout or overexpression. The human relevance of this arrhythmogenic mechanism is evaluated in human-induced pluripotent stem cell–derived cardiomyocytes. Therapeutic potentials by targeting this mechanism are explored in the mouse models and human-induced pluripotent stem cell–derived cardiomyocytes. Results: Mechanistically, hypertrophic stress dampens expression and phosphorylation of mitogen-activated kinase kinase-7. Such mitogen-activated kinase kinase-7 deficiency leaves histone deacetylase-2 unphosphorylated and filamin-A accumulated in the nucleus to form a complex with Krüppel-like factor-4. This complex leads to Krüppel-like factor-4 disassociation from the promoter regions of multiple key potassium channel genes (Kv4.2, KChIP2, Kv1.5, ERG1, and Kir6.2) and reduction of their transcript levels. Consequent repolarization delays result in ventricular arrhythmias. Therapeutically, targeting the repressive function of the Krüppel-like factor-4/histone deacetylase-2/filamin-A complex with the histone deacetylase-2 inhibitor valproic acid restores K+ channel expression and alleviates ventricular arrhythmias in pathologically remodeled hearts. Conclusions: Our findings unveil this new gene regulatory avenue as a new antiarrhythmic target where repurposing of the antiepileptic drug valproic acid as an antiarrhythmic is supported.

Fas-associated death domain protein interacts with methyl-CpG binding domain protein 4

Fas-associated death domain protein (FADD) is an adaptor protein bridging death receptors with initiator caspases. Thus, its function and localization are assumed to be cytoplasmic, although the localization of endogenous FADD has not been reported. Surprisingly, the data presented here demonstrate that FADD is mainly nuclear in several adherent cell lines. Its accumulation in the nucleus and export to the cytoplasm required the phosphorylation site Ser-194, which was also required for its interaction with the nucleocytoplasmic shuttling protein exportin-5. Within the nucleus, FADD interacted with the methyl-CpG binding domain protein 4 (MBD4), which excises thymine from GT mismatches in methylated regions of chromatin. The MBD4-interacting mismatch repair factor MLH1 was also found in a complex with FADD. The FADD–MBD4 interaction involved the death effector domain of FADD and a region of MBD4 adjacent to the glycosylase domain. The FADD-binding region of MBD4 was downstream of a frameshift mutation that occurs in a significant fraction of human colorectal carcinomas. Consistent with the idea that MBD4 can signal to an apoptotic effector, MBD4 regulated DNA damage-, Fas ligand-, and cell detachment-induced apoptosis. The nuclear localization of FADD and its interaction with a genome surveillance/DNA repair protein that can regulate apoptosis suggests a novel function of FADD distinct from direct participation in death receptor signaling complexes.