Ant-icipating Change: An Epigenetic Switch in Reprogramming the Social Lives of Ants.

Glastad et al. (2019) describe a role for the neuronal CoREST corepressor and changes in juvenile hormone (JH) and ecdysone signaling during the reprogramming of social behavioral phenotypes in ants that are reflective of a natural mechanism differentiating "Major" and "Minor" worker ants.

Histone H3 lysine 4 methylation signature associated with human undernutrition.

Chronically undernourished children become stunted during their first 2 years and thereafter bear burdens of ill health for the rest of their lives. Contributors to stunting include poor nutrition and exposure to pathogens, and parental history may also play a role. However, the epigenetic impact of a poor environment on young children is largely unknown. Here we show the unfolding pattern of histone H3 lysine 4 trimethylation (H3K4me3) in children and mothers living in an urban slum in Dhaka, Bangladesh. A pattern of chromatin modification in blood cells of stunted children emerges over time and involves a global decrease in methylation at canonical locations near gene start sites and increased methylation at ectopic sites throughout the genome. This redistribution occurs at metabolic and immune genes and was specific for H3K4me3, as it was not observed for histone H3 lysine 27 acetylation in the same samples. Methylation changes in stunting globally resemble changes that occur in vitro in response to altered methylation capacity, suggesting that reduced levels of one-carbon nutrients in the diet play a key role in stunting in this population. A network of differentially expressed genes in stunted children reveals effects on chromatin modification machinery, including turnover of H3K4me3, as well as posttranscriptional gene regulation affecting immune response pathways and lipid metabolism. Consistent with these changes, reduced expression of the endocytic receptor gene LDL receptor 1 (LRP1) is a driver of stunting in a mouse model, suggesting a target for intervention.

Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

Reelin is a target of polyglutamine expanded ataxin-7 in human spinocerebellar ataxia type 7 (SCA7) astrocytes.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant neurodegenerative disorder that results from polyglutamine expansion of the ataxin-7 (ATXN7) protein. Remarkably, although mutant ATXN7 is expressed throughout the body, pathology is restricted primarily to the cerebellum and retina. One major goal has been to identify factors that contribute to the tissue specificity of SCA7. Here we describe the development and use of a human astrocyte cell culture model to identify reelin, a factor intimately involved in the development and maintenance of Purkinje cells and the cerebellum as a whole, as an ATXN7 target gene. We found that polyglutamine expansion decreased ATXN7 occupancy, which correlated with increased levels of histone H2B monoubiquitination, at the reelin promoter. Treatment with trichostatin A, but not other histone deacetylase inhibitors, partially restored reelin transcription and promoted the accumulation of mutant ATXN7 into nuclear inclusions. Our findings suggest that reelin could be a previously unknown factor involved in the tissue specificity of SCA7 and that trichostatin A may ameliorate deleterious effects of the mutant ATXN7 protein by promoting its sequestration away from promoters into nuclear inclusions.

Direct inhibition of Gcn5 protein catalytic activity by polyglutamine-expanded ataxin-7.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion within the N-terminal region of the ataxin-7 protein, a known subunit of the SAGA complex. Although the mechanisms of SCA7 pathogenesis remain poorly understood, previous studies have shown perturbations in SAGA histone acetyltransferase function and transcriptional alterations. We sought to determine whether and how polyQ-expanded ataxin-7 affects SAGA catalytic activity. Here, we determined that polyQ-expanded ataxin-7 directly bound the Gcn5 catalytic core of SAGA while in association with its regulatory proteins, Ada2 and Ada3. This caused a significant decrease in Gcn5 histone acetyltransferase activity in vitro and in vivo at two SAGA-regulated galactose genes, GAL1 and GAL7. However, Gcn5 occupancy at the GAL1 and GAL7 promoters was increased in these cells, revealing a dominant-negative phenotype of the polyQ-expanded ataxin-7-incorporated, catalytically inactive SAGA. These findings suggest a dominant mechanism of polyQ-mediated SAGA inhibition that potentially contributes to SCA7 disease pathogenesis.

Gcn5 loss-of-function accelerates cerebellar and retinal degeneration in a SCA7 mouse model.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by expansion of a CAG repeat encoding a polyglutamine tract in ATXN7, a component of the SAGA histone acetyltransferase (HAT) complex. Previous studies provided conflicting evidence regarding the effects of polyQ-ATXN7 on the activity of Gcn5, the HAT catalytic subunit of SAGA. Here, we report that reducing Gcn5 expression accelerates both cerebellar and retinal degeneration in a mouse model of SCA7. Deletion of Gcn5 in Purkinje cells in mice expressing wild-type (wt) Atxn7, however, causes only mild ataxia and does not lead to the early lethality observed in SCA7 mice. Reduced Gcn5 expression strongly enhances retinopathy in SCA7 mice, but does not affect the known transcriptional targets of Atxn7, as expression of these genes is not further altered by Gcn5 depletion. These findings demonstrate that loss of Gcn5 functions can contribute to the time of onset and severity of SCA7 phenotypes, and suggest that non-transcriptional functions of SAGA may play a role in neurodegeneration in this disease.

The role of DNA methylation and histone modifications in transcriptional regulation in humans.

Although the field of genetics has grown by leaps and bounds within the last decade due to the completion and availability of the human genome sequence, transcriptional regulation still cannot be explained solely by an individual's DNA sequence. Complex coordination and communication between a plethora of well-conserved chromatin modifying factors are essential for all organisms. Regulation of gene expression depends on histone post translational modifications (HPTMs), DNA methylation, histone variants, remodeling enzymes, and effector proteins that influence the structure and function of chromatin, which affects a broad spectrum of cellular processes such as DNA repair, DNA replication, growth, and proliferation. If mutated or deleted, many of these factors can result in human disease at the level of transcriptional regulation. The common goal of recent studies is to understand disease states at the stage of altered gene expression. Utilizing information gained from new high-throughput techniques and analyses will aid biomedical research in the development of treatments that work at one of the most basic levels of gene expression, chromatin. This chapter will discuss the effects of and mechanism by which histone modifications and DNA methylation affect transcriptional regulation.

Globally elevated levels of histone H3 lysine 9 trimethylation in early infancy are associated with poor growth trajectory in Bangladeshi children.

BACKGROUND: Stunting is a global health problem affecting hundreds of millions of children worldwide and contributing to 45% of deaths in children under the age of five. Current therapeutic interventions have limited efficacy. Understanding the epigenetic changes underlying stunting will elucidate molecular mechanisms and likely lead to new therapies. RESULTS: We profiled the repressive mark histone H3 lysine 9 trimethylation (H3K9me3) genome-wide in peripheral blood mononuclear cells (PBMCs) from 18-week-old infants (n = 15) and mothers (n = 14) enrolled in the PROVIDE study established in an urban slum in Bangladesh. We associated H3K9me3 levels within individual loci as well as genome-wide with anthropometric measurements and other biomarkers of stunting and performed functional annotation of differentially affected regions. Despite the relatively small number of samples from this vulnerable population, we observed globally elevated H3K9me3 levels were associated with poor linear growth between birth and one year of age. A large proportion of the differentially methylated genes code for proteins targeting viral mRNA and highly significant regions were enriched in transposon elements with potential regulatory roles in immune system activation and cytokine production. Maternal data show a similar trend with child's anthropometry; however, these trends lack statistical significance to infer an intergenerational relationship. CONCLUSIONS: We speculate that high H3K9me3 levels may result in poor linear growth by repressing genes involved in immune system activation. Importantly, changes to H3K9me3 were detectable before the overt manifestation of stunting and therefore may be valuable as new biomarkers of stunting.

Transcriptional Profile of Exercise-Induced Protection Against Relapse to Cocaine Seeking in a Rat Model.

Background: Exercise has shown promise as a treatment for cocaine use disorder; however, the mechanism underlying its efficacy has remained elusive. Methods: We used a rat model of relapse (cue-induced reinstatement) and exercise (wheel running, 2 hours/day) coupled with RNA sequencing to establish transcriptional profiles associated with the protective effects of exercise (during early withdrawal [days 1-7] or throughout withdrawal [days 1-14]) versus noneffective exercise (during late withdrawal [days 8-14]) against cocaine-seeking and sedentary conditions. Results: As expected, cue-induced cocaine seeking was highest in the sedentary and late-withdrawal exercise groups; both groups also showed upregulation of a Grin1-associated transcript and enrichment of Drd1-Nmdar1 complex and glutamate receptor complex terms. Surprisingly, these glutamate markers were also enriched in the early- and throughout-withdrawal exercise groups, despite lower levels of cocaine seeking. However, a closer examination of the Grin1-associated transcript revealed a robust loss of transcripts spanning exons 9 and 10 in the sedentary condition relative to saline controls that was normalized by early- and throughout-withdrawal exercise, but not late-withdrawal exercise, indicating that these exercise conditions may normalize RNA mis-splicing induced by cocaine seeking. Our findings also revealed novel mechanisms by which exercise initiated during early withdrawal may modulate glutamatergic signaling in dorsomedial prefrontal cortex (e.g., via transcripts associated with non-NMDA glutamate receptors or those affecting signaling downstream of NMDA receptors), along with mechanisms outside of glutamatergic signaling such as circadian rhythm regulation and neuronal survival. Conclusions: These findings provide a rich resource for future studies aimed at manipulating these molecular networks to better understand how exercise decreases cocaine seeking.

Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes.

Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt-Ada-Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.

Epigenetic methodologies for behavioral scientists.

Hormones are essential regulators of many behaviors. Steroids bind either to nuclear or membrane receptors while peptides primarily act via membrane receptors. After a ligand binds, the conformational change in the receptor initiates changes in cell signaling cascades (membrane receptors) or direct alternations in DNA transcription (steroid receptors). Changes in gene transcription that result are responsible for protein production and ultimately behavioral modifications. A significant part of how hormones affect DNA transcription is via epigenetic modifications of DNA and/or the chromatin in which it is entwined. These alterations lead to transcriptional changes that ultimately define the phenotype and function of a given cell. Importantly we now know that environmental stimuli influence epigenetic marks, which in the context of neuroendocrinology can lead to behavioral changes. Importantly tracking epigenetic states and profiling the epigenome within cells require the use of epigenetic methodologies and subsequent data analysis. Here we describe the techniques of particular importance in the mapping of DNA methylation, histone modifications and occupancy of chromatin bound effector proteins that regulate gene expression. For researchers wanting to move into these levels of analysis we discuss the application of modern sequencing technologies applied in assays such as chromatin immunoprecipitation and the bioinformatics analysis involved in the rich datasets generated.

Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation.

The specific post-translational modifications to histones influence many nuclear processes including gene regulation, DNA repair and replication. Recent studies have identified effector proteins that recognize patterns of histone modification and transduce their function in downstream processes. For example, histone acetyltransferases (HATs) have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. Yeast SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous and conserved multi-subunit HAT complexes, which preferentially acetylate histones H3 and H2B and deubiquitinate histone H2B. Here we identify the chromatin remodelling protein Chd1 (chromo-ATPase/helicase-DNA binding domain 1) as a component of SAGA and SLIK. Our findings indicate that one of the two chromodomains of Chd1 specifically interacts with the methylated lysine 4 mark on histone H3 that is associated with transcriptional activity. Furthermore, the SLIK complex shows enhanced acetylation of a methylated substrate and this activity is dependent upon a functional methyl-binding chromodomain, both in vitro and in vivo. Our study identifies the first chromodomain that recognizes methylated histone H3 (Lys 4) and possibly identifies a larger subfamily of chromodomain proteins with similar recognition properties.

The biochemical and genetic discovery of the SAGA complex.

One of the major advances in our understanding of gene regulation in eukaryotes was the discovery of factors that regulate transcription by controlling chromatin structure. Prominent among these discoveries was the demonstration that Gcn5 is a histone acetyltransferase, establishing a direct connection between transcriptional activation and histone acetylation. This breakthrough was soon followed by the purification of a protein complex that contains Gcn5, the SAGA complex. In this article, we review the early genetic and biochemical experiments that led to the discovery of SAGA and the elucidation of its multiple activities.

MYBL2-Driven Transcriptional Programs Link Replication Stress and Error-prone DNA Repair With Genomic Instability in Lung Adenocarcinoma.

It has long been recognized that defects in cell cycle checkpoint and DNA repair pathways give rise to genomic instability, tumor heterogeneity, and metastasis. Despite this knowledge, the transcription factor-mediated gene expression programs that enable survival and proliferation in the face of enormous replication stress and DNA damage have remained elusive. Using robust omics data from two independent studies, we provide evidence that a large cohort of lung adenocarcinomas exhibit significant genome instability and overexpress the DNA damage responsive transcription factor MYB proto-oncogene like 2 (MYBL2). Across two studies, elevated MYBL2 expression was a robust marker of poor overall survival and disease-free survival outcomes, regardless of disease stage. Clinically, elevated MYBL2 expression identified patients with aggressive early onset disease, increased lymph node involvement, and increased incidence of distant metastases. Analysis of genomic sequencing data demonstrated that MYBL2 High lung adenocarcinomas had elevated somatic mutation burden, widespread chromosomal alterations, and alterations in single-strand DNA break repair pathways. In this study, we provide evidence that impaired single-strand break repair, combined with a loss of cell cycle regulators TP53 and RB1, give rise to MYBL2-mediated transcriptional programs. Omics data supports a model wherein tumors with significant genomic instability upregulate MYBL2 to drive genes that control replication stress responses, promote error-prone DNA repair, and antagonize faithful homologous recombination repair. Our study supports the use of checkpoint kinase 1 (CHK1) pharmacological inhibitors, in targeted MYBL2 High patient cohorts, as a future therapy to improve lung adenocarcinoma patient outcomes.

Mechanisms underlying the efficacy of exercise as an intervention for cocaine relapse: a focus on mGlu5 in the dorsal medial prefrontal cortex.

RATIONALE: Exercise shows promise as a treatment option for addiction; but in order to prevent relapse, it may need to be introduced early in the course of treatment. OBJECTIVE: We propose that exercise, by upregulating dorsal medial prefrontal cortex (dmPFC)-nucleus accumbens (NAc) transmission, offsets deficits in pathways targeting glutamate, BDNF, and dopamine during early abstinence, and in doing so, normalizes neuroadaptations that underlie relapse. METHODS: We compared the effects of exercise (wheel running, 2-h/day) during early (days 1-7), late (days 8-14), and throughout abstinence (days 1-14) to sedentary conditions on cocaine-seeking and gene expression in the dmPFC and NAc core of male rats tested following 24-h/day extended-access cocaine (up to 96 infusions/day) or saline self-administration and protracted abstinence (15 days). Based on these data, we then used site-specific manipulation to determine whether dmPFC metabotropic glutamate receptor5 (mGlu5) underlies the efficacy of exercise. RESULTS: Exercise initiated during early, but not late abstinence, reduced cocaine-seeking; this effect was strongly associated with dmPFC Grm5 expression (gene encoding mGlu5), and modestly associated with dmPFC Grin1 and Bdnf-IV expression. Activation of mGlu5 in the dmPFC during early abstinence mimicked the efficacy of early-initiated exercise; however, inhibition of these receptors prior to the exercise sessions did not block its efficacy indicating that there may be redundancy in the mechanisms through which exercise reduces cocaine-seeking. CONCLUSION: These findings indicate that addiction treatments, including exercise, should be tailored for early versus late phases of abstinence since their effectiveness will vary over abstinence due to the dynamic nature of the underlying neuroadaptations.

The Novel ReNu Region of TAF12 Regulates Gcn5 Nucleosomal Acetylation.

The post-translational acetylation of the histone components of chromatin mediates numerous DNA-templated events, including transcriptional activation, DNA repair, and genomic replication. The conserved SAGA (Spt-Ada-Gcn5 Acetyltranferase) and SLIK (SAGA-Like) Histone Acetyltransferase (HAT) complexes are required for transcriptional activation of a subset of yeast genes and contain multiple subunits including the histone fold-containing TBP- Associated Factors (TAFs): 6, 9, 10, and 12. These TAFs are also components of the TFIID complex and are consequently involved in most RNA polymerase II-transcription in yeast. Here we identify a novel conserved region of TAF12, termed ReNu, outside of its histone fold, which is required for SAGA and SLIK-directed nucleosomal acetylation. We demonstrate that this region is not required for chromatin association, but show that this region plays an important role for histone H3 acetylation at specific SAGA and SLIK-regulated promoters. Our data suggests that the ReNu region of TAF12 regulates Gcn5 acetylation of specific substrates within the SAGA super-family of HAT complexes.

The novel SLIK histone acetyltransferase complex functions in the yeast retrograde response pathway.

The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAF(II)s), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II.

Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression.

Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae. Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail. Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo. Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain. These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription.

Multi-tasking on chromatin with the SAGA coactivator complexes.

Over the past 10 years, much progress has been made to understand the roles of the similar, yet distinct yeast SAGA and SLIK coactivator complexes involved in histone post-translational modification and gene regulation. Many different groups have elucidated functions of the SAGA complexes including identification of novel components, which have conferred additional distinct functions. Together, recent studies demonstrate unique attributes of the SAGA coactivator complexes in histone acetylation, methylation, phosphorylation, and deubiquitination. In addition to roles in transcriptional activation with the 19S proteasome regulatory particle, recent evidence also suggests functions for SAGA in elongation and mRNA export. The modular nature of SAGA allows this approximately 1.8 MDa complex to organize its functions and carry out multiple roles during transcription, particularly under conditions of cellular stress.

Poly(Q) Expansions in ATXN7 Affect Solubility but Not Activity of the SAGA Deubiquitinating Module.

Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.