A moonlighting metabolic protein influences repair at DNA double-stranded breaks.

Catalytically active proteins with divergent dual functions are often described as 'moonlighting'. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair

Obtención de un sistema de análisis para genes transcritos por RNA polimerasa III en Trichomonádidos : estudio del gen del RNAr 5S / tesis que para obtener el grado de Doctor en Ciencias Biomédicas, presenta Ana Lilia Torres Machorro ; asesor María Imelda López Villaseñor

. 103, [46] páginas : ilustraciones. Doctorado en Ciencias Biomédicas UNAM, Instituto de Investigaciones Biomédicas, 201

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix–Loop–Helix Transcription Factors

The basic helix–loop–helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell’s fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed

Bypassing the Requirement for an Essential MYST Acetyltransferase

Histone acetylation is a key regulatory feature for chromatin that is established by opposing enzymatic activities of lysine acetyltransferases (KATs/HATs) and deacetylases (KDACs/HDACs). Esa1, like its human homolog Tip60, is an essential MYST family enzyme that acetylates histones H4 and H2A and other nonhistone substrates. Here we report that the essential requirement for ESA1 in Saccharomyces cerevisiae can be bypassed upon loss of Sds3, a noncatalytic subunit of the Rpd3L deacetylase complex. By studying the esa1∆ sds3∆ strain, we conclude that the essential function of Esa1 is in promoting the cellular balance of acetylation. We demonstrate this by fine-tuning acetylation through modulation of HDACs and the histone tails themselves. Functional interactions between Esa1 and HDACs of class I, class II, and the Sirtuin family define specific roles of these opposing activities in cellular viability, fitness, and response to stress. The fact that both increased and decreased expression of the ESA1 homolog TIP60 has cancer associations in humans underscores just how important the balance of its activity is likely to be for human well-being

A moonlighting metabolic protein influences repair at DNA double-stranded breaks.

Catalytically active proteins with divergent dual functions are often described as 'moonlighting'. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair

Chromatin Regulation by the NuA4 Acetyltransferase Complex Is Mediated by Essential Interactions Between Enhancer of Polycomb (Epl1) and Esa1

Enzymes that modify and remodel chromatin act in broadly conserved macromolecular complexes. One key modification is the dynamic acetylation of histones and other chromatin proteins by opposing activities of acetyltransferase and deacetylase complexes. Among acetyltransferases, the NuA4 complex containing Tip60 or its Saccharomyces cerevisiae ortholog Esa1 is of particular significance because of its roles in crucial genomic processes including DNA damage repair and transcription. The catalytic subunit Esa1 is essential, as are five noncatalytic NuA4 subunits. We found that of the noncatalytic subunits, deletion of Enhancer of polycomb (Epl1), but not the others, can be bypassed by loss of a major deacetylase complex, a property shared by Esa1 Noncatalytic complex subunits can be critical for complex assembly, stability, genomic targeting, substrate specificity, and regulation. Understanding the essential role of Epl1 has been previously limited, a limitation now overcome by the discovery of its bypass suppression. Here, we present a comprehensive in vivo study of Epl1 using the powerful tool of suppression combined with transcriptional and mutational analyses. Our results highlight functional parallels between Epl1 and Esa1 and further illustrate that the structural role of Epl1 is important for promotion of Esa1 activity. This conclusion is strengthened by our dissection of Epl1 domains required in vivo for interaction with specific NuA4 subunits, histone acetylation, and chromatin targeting. These results provide new insights for the conserved, essential nature of Epl1 and its homologs, such as EPC1/2 in humans, which is frequently altered in cancers

The Set3 Complex Antagonizes the MYST Acetyltransferase Esa1 in the DNA Damage Response

Acetylation is a dynamic posttranslational modification that contributes to chromatin-regulated processes, including DNA replication, repair, recombination, and gene expression. Acetylation is controlled by complexes containing opposing lysine and histone acetyltransferase (KAT and HAT) and deacetylase (KDAC and HDAC) activities. The essential MYST family Esa1 KAT acetylates core histones and many nonhistone substrates. Phenotypes of esa1 mutants include transcriptional silencing and activation defects, impaired growth at high temperatures, and sensitivity to DNA damage. The KDAC Rpd3 was previously identified as an activity opposing Esa1, as its deletion suppresses growth and silencing defects of esa1 mutants. However, loss of Rpd3 does not suppress esa1 DNA damage sensitivity. In this work, we identified Hos2 as a KDAC counteracting ESA1 in the damage response. Deletion of HOS2 resulted in changes of esa1's transcriptional response upon damage. Further, loss of HOS2 or components of the Set3 complex (Set3C) in which it acts specifically suppressed damage sensitivity and restored esa1 histone H4 acetylation. This rescue was mediated via loss of either Set3C integrity or of its binding to dimethylated histone H3K4. Our results thus add new insight into the interactions of an essential MYST acetyltransferase with diverse deacetylases to respond specifically to environmental and physiological challenges

The Set3 Complex Antagonizes the MYST Acetyltransferase Esa1 in the DNA Damage Response

Acetylation is a dynamic posttranslational modification that contributes to chromatin-regulated processes, including DNA replication, repair, recombination, and gene expression. Acetylation is controlled by complexes containing opposing lysine and histone acetyltransferase (KAT and HAT) and deacetylase (KDAC and HDAC) activities. The essential MYST family Esa1 KAT acetylates core histones and many nonhistone substrates. Phenotypes of esa1 mutants include transcriptional silencing and activation defects, impaired growth at high temperatures, and sensitivity to DNA damage. The KDAC Rpd3 was previously identified as an activity opposing Esa1, as its deletion suppresses growth and silencing defects of esa1 mutants. However, loss of Rpd3 does not suppress esa1 DNA damage sensitivity. In this work, we identified Hos2 as a KDAC counteracting ESA1 in the damage response. Deletion of HOS2 resulted in changes of esa1's transcriptional response upon damage. Further, loss of HOS2 or components of the Set3 complex (Set3C) in which it acts specifically suppressed damage sensitivity and restored esa1 histone H4 acetylation. This rescue was mediated via loss of either Set3C integrity or of its binding to dimethylated histone H3K4. Our results thus add new insight into the interactions of an essential MYST acetyltransferase with diverse deacetylases to respond specifically to environmental and physiological challenges

Short-Term Exposure to Wood Smoke Increases the Expression of Pro-Inflammatory Cytokines, Gelatinases, and TIMPs in Guinea Pigs

Exposure to air pollutants in wildfire smoke and indoor pollution causes lung diseases. Short-term exposure to wood smoke (WS) is partially known to alter the expression of human matrix metalloproteinases (MMPs), inflammatory cytokines, and tissue inhibitors of metalloproteinases (TIMPs). Accordingly, we investigated the effect of exposing guinea pigs to WS for two and four three-hour periods on different days. The daily content of particles reported by indoor pollution was produced by 60 g of pinewood. We analyzed the cell profile and collagen content in bronchoalveolar lavages (BAL). The mRNA expression of pro-inflammatory cytokines, MMPs, and TIMPs was studied in lung tissue. Cytokines and gelatinolytic activity were analyzed in BAL and serum. The results showed that total cells, macrophages, neutrophils, and collagen increased in BAL, whereas neutrophils and lymphocytes decreased. TGF-β1, TNF-α, IFN-γ, IL-1β, IL-6, IL-8, MMP-2, MMP-9, TIMP-1, and TIMP-2 were upregulated in lungs, downregulating IL-12. TNF-α, IFN-γ, TGF-β1, IL-1β, IL-6, and IL-8 were increased in BAL and serum, decreasing IL-12. Gelatinase activity was increased in serum. Thus, guinea pigs exposed to short-term domestic doses of WS overexpressed pro-inflammatory cytokines, MMPs, and TIMPs. These results are similar to ECM remodeling and pulmonary and systemic inflammation reported in humans

Phosphorylation of the 19S regulatory particle ATPase subunit, Rpt6, modifies susceptibility to proteotoxic stress and protein aggregation

<div><p>The ubiquitin proteasome system (UPS) is a highly conserved and tightly regulated biochemical pathway that degrades the majority of proteins in eukaryotic cells. Importantly, the UPS is responsible for counteracting altered protein homeostasis induced by a variety of proteotoxic stresses. We previously reported that Rpt6, the ATPase subunit of the 19S regulatory particle (RP) of the 26S proteasome, is phosphorylated in mammalian neurons at serine 120 in response to neuronal activity. Furthermore, we found that Rpt6 S120 phosphorylation, which regulates the activity and distribution of proteasomes in neurons, is relevant for proteasome-dependent synaptic remodeling and function. To better understand the role of proteasome phosphorylation, we have constructed models of altered Rpt6 phosphorylation in <i>S</i>. <i>cerevisiae</i> by introducing chromosomal point mutations that prevent or mimic phosphorylation at the conserved serine (S119). We find that mutants which prevent Rpt6 phosphorylation at this site (<i>rpt6-S119A</i>), had increased susceptibility to proteotoxic stress, displayed abnormal morphology and had reduced proteasome activity. Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington’s disease (HD) related huntingtin (Htt) protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead (<i>rpt6-S119A)</i> and phospho-mimetic (<i>rpt6-S119D</i>) mutants. We showed Htt103Q aggregate size to be significantly larger in <i>rpt6-S119A</i> mutants compared to wild-type or <i>rpt6-S119D</i> strains. Furthermore, we observed that phosphorylation of endogenous Rpt6 at S119 is increased in response to various stress conditions. Together, these data suggest that Rpt6 phosphorylation at S119 may play an important function in proteasome-dependent relief of proteotoxic stress that can be critical in protein aggregation pathologies.</p></div