Histone Gene Regulation Research Articles

KMT2D-mediated H3K4me1 recruits YBX1 to facilitate triple-negative breast cancer progression through epigenetic activation of c-Myc.

Lysine methyltransferase 2D (KMT2D) mediates mono-methylation of histone H3 lysine 4 (H3K4me1) in mammals. H3K4me1 mark is involved in establishing an active chromatin structure to promote gene transcription. However, the precise molecular mechanism underlying the KMT2D-mediated H3K4me1 mark modulates gene expression in triple-negative breast cancer (TNBC) progression is unresolved. We recognized Y-box-binding protein 1 (YBX1) as a "reader" of the H3K4me1 mark, and a point mutation of YBX1 (E121A) disrupted this interaction. We found that KMT2D and YBX1 cooperatively promoted cell growth and metastasis of TNBC cells in vitro and in vivo. The expression levels of KMT2D and YBX1 were both upregulated in tumour tissues and correlated with poor prognosis for breast cancer patients. Combined analyses of ChIP-seq and RNA-seq data indicated that YBX1 was co-localized with KMT2D-mediated H3K4me1 in the promoter regions of c-Myc and SENP1, thereby activating their expressions in TNBC cells. Moreover, we demonstrated that YBX1 activated the expressions of c-Myc and SENP1 in a KMT2D-dependent manner. Our results suggest that KMT2D-mediated H3K4me1 recruits YBX1 to facilitate TNBC progression through epigenetic activation of c-Myc and SENP1. These results together unveil a crucial interplay between histone mark and gene regulation in TNBC progression, thus providing novel insights into targeting the KMT2D-H3K4me1-YBX1 axis for TNBC treatment. YBX1 is a KMT2D-mediated H3K4me1-binding effector protein and mutation of YBX1 (E121A) disrupts its binding to H3K4me1. KMT2D and YBX1 cooperatively promote TNBC proliferation and metastasis by activating c-Myc and SENP1 expression in vitro and in vivo. YBX1 is colocalized with H3K4me1 in the c-Myc and SENP1 promoter regions in TNBC cells and increased YBX1 expression predicts a poor prognosis in breast cancer patients.

Calcineurin/NFATc1 pathway represses cellular cytotoxicity by modulating histone H3 expression

Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones are degraded by the proteasome mediated via the DNA damage response factor Rad53. Histone expression, therefore, is tightly regulated at the protein level. Our understanding of the transcriptional regulation of histone genes is far from complete. In this study, we found that calcineurin inhibitor treatment increased histone protein levels, and that the transcription factor NFATc1 (nuclear factor of activated T cells 1) repressed histone transcription and acts downstream of the calcineurin. We further revealed that NFATc1 binds to the promoter regions of many histone genes and that histone transcription is downregulated in a manner dependent on intracellular calcium levels. Indeed, overexpression of histone H3 markedly inhibited cell proliferation. Taken together, these findings suggest that NFATc1 prevents the detrimental effects of histone H3 accumulation by inhibiting expression of histone at the transcriptional level.

Gene loss and cis-regulatory novelty shaped core histone gene evolution in the apiculate yeast Hanseniaspora uvarum.

Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here, we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation-mediated by the trans-regulator Spt10-is ancient, likely emerging between 320 and 380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage, which coincided with the loss of 1 copy of its paralogous core histone genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora fast-evolving lineage.

Organ-specific characteristics govern the relationship between histone code dynamics and transcriptional reprogramming during nitrogen response in tomato

Environmental stimuli trigger rapid transcriptional reprogramming of gene networks. These responses occur in the context of the local chromatin landscape, but the contribution of organ-specific dynamic chromatin modifications in responses to external signals remains largely unexplored. We treated tomato seedlings with a supply of nitrate and measured the genome-wide changes of four histone marks, the permissive marks H3K27ac, H3K4me3, and H3K36me3 and repressive mark H3K27me3, in shoots and roots separately, as well as H3K9me2 in shoots. Dynamic and organ-specific histone acetylation and methylation were observed at functionally relevant gene loci. Integration of transcriptomic and epigenomic datasets generated from the same organ revealed largely syngenetic relations between changes in transcript levels and histone modifications, with the exception of H3K27me3 in shoots, where an increased level of this repressive mark is observed at genes activated by nitrate. Application of a machine learning approach revealed organ-specific rules regarding the importance of individual histone marks, as H3K36me3 is the most successful mark in predicting gene regulation events in shoots, while H3K4me3 is the strongest individual predictor in roots. Our integrated study substantiates a view that during plant environmental responses, the relationships between histone code dynamics and gene regulation are highly dependent on organ-specific contexts.

THU558 Combination Therapy Of Acetyl-CoA Synthetase Inhibitor And Fulvestrant Reverse Epigenetic Alterations Of Metastatic Breast Cancer Cells And Reduce Cancer Cell Proliferation

Abstract Disclosure: A.N. Mogol: None. Z. Madak Erdogan: None. Endocrine therapies exploit the hormone dependencies of the cancer cells in various ways, including by targeting estrogen receptor alpha (ERα) that is expressed by approximately 70% of human breast cancers. After treatment, recurrence and metastasis are still observed in 30-40% of ER+ patients and the 5-year relative overall survival rate for these patients is just 24%. Acetyl-CoA production significantly regulates histone acetylation and gene regulation, which support tumor progression. In our previous study, we showed the synergistic effect of the ERα agonist Fulvestrant (Fulv) and Acetyl-CoA Synthase Inhibitor (ACSI) in reducing cell viability in ER+ metastatic breast cancer (MBC) cells in vitro. To study the synergistic contribution of ACSS2 to endocrine resistance, we used ChIP analysis, which showed that ACSS2 was recruited to ERα binding sites with Fulv treatment in cell line xenograft tumors and PDX samples. We used the CUT&RUN method to investigate specific protein (H3k27ac, ERα and ACSS2) and DNA interactions in ER+ MBC cells globally. Enriched chromatin samples were then used for qPCR and sequencing. Combination therapy of the ACSI and Fulv significantly altered the epigenetic landscape of the breast cancer cells in a manner to decrease cell viability. High histone acetylation at lysine 27 residue was observed in Veh treatment group and combination therapy of ACSI+Fulv further decreased the enrichment of this histone modification in classical ER binding sites. Interestingly, ACSS2 binding was increased with Fulv treatment. Targeting epigenetic alterations caused by cancer cell adaptations is a novel approach that could potentially lead to new therapies to reduce metastasis mortality. Presentation: Thursday, June 15, 2023

AMPK Associates with Chromatin and Phosphorylates the TAF-1 Subunit of the Transcription Initiation Complex to Regulate Histone Gene Expression in ALL Cells.

Fully delineating the protein interactome by which AMPK regulates adaptive survival responses to energy/metabolic stress, either via epigenetic gene regulation or other mechanisms, will allow the rational development of strategies to overcome de novo or acquired resistance in ALL and other cancers.

Cardiovascular Mechano-Epigenetics: Force-Dependent Regulation of Histone Modifications and Gene Regulation.

The local mechanical microenvironment impacts on the cell behavior. In the cardiovascular system, cells in both the heart and the vessels are exposed to continuous blood flow, blood pressure, stretching forces, and changing extracellular matrix stiffness. The force-induced signals travel all the way to the nucleus regulating epigenetic changes such as chromatin dynamics and gene expression. Mechanical cues are needed at the very early stage for a faultless embryological development, while later in life, aberrant mechanical signaling can lead to a range of pathologies, including diverse cardiovascular diseases. Hence, an investigation of force-generated epigenetic alteration at different time scales is needed to understand fully the phenotypic changes in disease onset and progression. That being so, cardiovascular mechano-epigenetics emerges as an attractive field of study. Given the rapid advances in this emergent field of research, this short review aims to provide an analysis of the state of knowledge of force-induced epigenetic changes in the cardiovascular field.

AMPK Downregulates Histone Gene Expression Via Dissociation of a Chromatin-Associated Transcription Complex in Response to Metabolic Stress in Acute Lymphoblastic Leukemia

We and others have reported that activation of the AMP-activated protein kinase (AMPK) mediates cell death in ALL. AMPK has been reported to interact with chromatin-associated proteins to epigenetically regulate gene expression in response to environmental/energy/metabolic stress, however, little is known about AMPK's epigenetic targets. To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we performed ChIP-seq with AMPKα2 and RNA pol II antibodies in Bp-ALL (KASUMI-2) and T-ALL (KE-37) cells treated with or without glucose or AICAR, a well known AMPK activator. ChIP-seq identified 132 and 167 significant AMPKα2 peaks located at gene promoters in AICAR-treated KASUMI-2 and KE-37 cells, respectively. GO terms analysis in KASUMI-2 control cells showed that AMPKα2 target set was enriched for genes involved in regulation of hematopoietic cell differentiation, DNA metabolism, DNA topology, and histone H3-K4 methylation, whereas in AICAR-treated cells it included the histone H3-K4 methylation, protein localization, Notch signaling, and mRNA destabilization. In AICAR-treated cells, we found that AMPKα2 was recruited to regions that were enriched for TAF1 and MYC targets, and to the promoter of the epigenetic regulators KMT2A and SETD1A. Among AMPKα2 targets, we identified a cluster of histone genes in which recruitment of AMPKα2 was significantly decreased at gene promoters. ChIP-seq data were validated by ChIP-qPCR on histone genes H1-2, H1-3, and H4C4, and the occupancy of AMPKα2 on histone gene loci in other Bp- and T-ALL cells confirmed. To correlate the level of gene mRNA expression and recruitment of AMPKα2 to chromatin we performed RNA-seq in KASUMI-2 and KE-37 cells treated ± AICAR for 24h. RNA-seq identified 1342 differential expressed genes in at least one of the cell lines, of which 57 were histone genes and 1285 non-histone genes. RT-qPCR confirmed histone gene mRNA downregulation in AICAR-treated cells. In addition, consistent with an epigenetic role for AMPK in ALL cells in response to energy/metabolic stress, we found that expression of the H3-K4 histone methyltransferase KMT2A and SETD1A genes were upregulated in KASUMI-2 cells treated with AICAR. To further investigate the effect of AMPK on histone gene expression, we generated HEK293 cells expressing single knockout (KO) or double knockout (DKO) of AMPKα1 and AMPKα2 genes using CRISPR/Cas9, and determined the level of selected histone gene mRNA expression using RT-qPCR. Our data show that both AMPKα1 and AMPKα2 participate in regulating histone gene expression, and these findings were confirmed using MEF WT, AMPKα1/KO, AMPKα2/KO, and AMPKα1/α2/DKO cells. These data support our previous findings indicating that AMPK kinase activity was required for histone genes’ regulation in response to metabolic stress. Next, we examined the cluster of non-histone genes altered by AICAR, and found that downregulated genes were enriched in MYC, Myc-associated factor X, and transcription initiation factor TFIID subunit 1 (TAF1) targets, and involved in pathways such as gene expression and protein metabolism. In contrast, the upregulated genes were enriched in RUNX1, SPI1, and GATA1 targets, and involved in pathways related to immune system signaling, regulation by TP53, and apoptosis. Using HEK293 CRISPR AMPKα1/KO, AMPKα2/KO and AMPKα1/α2/DKO cells, we demonstrated that AMPK is also required for MYC expression. In conclusion, our data show that (i) in response to metabolic stress, AMPKα2 is recruited to or released from a chromatin-associated complex to regulate gene transcription in ALL, (ii) AMPKα2 targets include the histone, MYC-target, and the epigenetic regulator KMT2A and SETD1A genes (H3-K4 histone methyltransferases), and (iii) AICAR treatment represses histone gene transcription via an AMPK-dependent mechanism. Further elucidation of AMPK's interactions with members of a putative AMPK/chromatin-associated transcription complex may lead to unique opportunities for epigenetic-based therapeutic interventions to exploit synthetic lethality in relapse/refractory ALL and other hematological malignancies.

SMN controls neuromuscular junction integrity through U7 snRNP.

SUMMARYThe neuromuscular junction (NMJ) is an essential synapse whose loss is a key hallmark of the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that activity of the SMA-determining SMN protein in the assembly of U7 small nuclear ribonucleoprotein (snRNP)—which functions in the 3′-end processing of replication-dependent histone mRNAs—is required for NMJ integrity. Co-expression of U7-specific Lsm10 and Lsm11 proteins selectively enhances U7 snRNP assembly, corrects histone mRNA processing defects, and rescues key structural and functional abnormalities of neuromuscular pathology in SMA mice—including NMJ denervation, decreased synaptic transmission, and skeletal muscle atrophy. Furthermore, U7 snRNP dysfunction drives selective loss of the synaptic organizing protein Agrin at NMJs innervating vulnerable muscles of SMA mice. These findings reveal a direct contribution of U7 snRNP dysfunction to neuromuscular pathology in SMA and suggest a role for histone gene regulation in maintaining functional synaptic connections between motor neurons and muscles.

PB1969: FUNCTIONAL CHARACTERIZATION OF SUSCEPTIBILITY LOCI TO MULTIPLE MYELOMA

Background: Multiple myeloma (MM) is the second most common blood malignancy, caused by an uncontrolled growth of plasma cells in the bone marrow, accounting for 20% of all newly diagnosed hematological cancers. Although the current 5-year survival rate is ranging between 40-60%, MM is still considered an incurable disease since most of the patients eventually relapse. While the causes of MM are incompletely understood, several genome-wide association studies (GWAS) have been conducted to identify germline variants that predispose to MM. Up to date, a total of 24 loci were found to be associated with MM risk, but very little information is available about their functional role. Aims: The principal goal is to explore in silico the function of the germline variants associated with MM risk. As we do not know if the GWAS-identified SNPs are the causal variants or just markers of risk, the functional characterization of the causal risk variants would lead to a better understanding of disease development. Methods: GWAS design takes advantage of the linkage disequilibrium (LD) structure of the human genome, thus the main GWAS findings are single-nucleotide polymorphisms (SNPs) that show the strongest association with MM risk (measured as the lowest p-values), but they are not necessarily the functionally causal variants. In this project, we used bioinformatics tools (GTEx, HaploReg v4.1, Roadmap, LDlinke, SNPnexus, RegulomeDB 2.0.3, SNP2TFBS, miRNASNP v3, GeneMANIA) to perform fine mapping of all GWAS-identified loci and to prioritize in each locus the polymorphism with the highest chance of being functionally relevant. In particular, we focused on the loci with the smallest number of SNPs in high LD (r2>0.8) in order to maximize the probability to capture the casual variant. Results: Four of the 24 MM risk loci had a relatively small number of SNPs in high LD and within them we found that the locus located at chromosome 16 contained the greatest number of functionally annotated SNPs. Of particular functional interest was rs3747481 (chr16:30666367 C/T) due to the following reasons: it is a missense variant (protein change: P359L), it is located in the PRR14 gene, that contributes to chromatin hierarchical organization and has a role in gene regulation, has a high CADD PHRED score (22.1). Additionally, according to GTEx portal, rs3747481 is associated with the expression level of the RNF40 gene (which plays a central role in histone code and gene regulation) in whole blood cells (p=3.02-12). It has a score of “1d” in RegulomeDB (eQTL+ TF binding + any motif + DNase peak), meaning that this variant has a high likelihood to affect binding of transcription factors. Some other SNPs (like rs35629860 and rs67128646) in the same LD block show the co-occurrence of H3K4me3 and H3K27me3 histone marks (associated with gene activation and repression, respectively) in promoters and enhancers in B lymphocytes. rs6565197 is predicted to affect the binding of the KLF4 and KLF5 transcription factors which play key roles in cell cycle regulation. Summary/Conclusion: Through a fine mapping of MM risk loci by bioinformatics tools, we found a variant in the locus 16p11.2 that shows in silico a very high probability to have biological role in the risk disease.

MSL2 Localization to Histone Loci in Drosophila Species

Nuclear bodies are membraneless structures containing concentrated regulatory factors that coordinate nuclear processes such as gene expression. The histone locus body (HLB) is a discrete nuclear body that is the main site of histone mRNA production. While many factors of the HLB are known, there are many unknown factors. In addition, while HLB function is highly conserved, it is also unknown how the HLB has changed and evolved in different species. The histone locus (HL) of the model organism Drosophila melanogaster contains ~100 tandem arrays of the five histone genes. While D. melanogaster has one HL, the related species Drosophila virilis, has two HL. We observe localization of the male‐specific dosage compensation protein MSL2 to one of the two D. virilishistone loci using polytene chromosome immunofluorescence, which we do not observe in other Drosophila species. In order to confirm our immunofluorescence observations, we mapped existing MSL2 ChIP‐seq data and discovered that when MSL2 from either species is overexpressed, it targets the H2a‐H2b promoter. We immunostained transgenic flies carrying an H2a‐H2b promoter transgene to determine if MSL2 targets this region when outside the context of the histone locus. We do not see the same HL localization when MSL2 is expressed at wild‐type levels. Finally, we performed RNA isolation and qPCR analysis on four Drosophila species to compare expression levels of msl2. Our results indicate that MSL2 may be playing a different role at the HL in different Drosophila species. Overall, this gives us better insight on histone gene regulation.

Heterochromatin extension: a possible cytogenetic fate of primary amenorrhea along with normal karyotype

Primary amenorrhoea (PA) is considered to be one of the challenging and taxing problems for the gynaecologist. Previous studies suggested that different numerical and structural chromosome abnormalities are associated with this. Heterochromatin polymorphisms are considered to be normal variant but considering the recent research on crucial cellular effects of heterochromatin, we have aimed to find out the prevalence of heteromorphism along with other standard chromosomal abnormalities. This was an observational study which was conducted in Diamond Harbour Govt. Medical College and Hospital, West Bengal during March 2019–February 2021. Clinical features of 178 patients were noted and peripheral venous blood was taken following informed consent. This comprehensive study reveals that there are 10.11% of the females among 178 females having a heterochromatin extension which is significantly high. We hence suggest that heteromorphism may be associated with ovarian dysfunction leading to amenorrhoea as the region of heterochromatin acts as a key part in chromosome structure, histone modification and gene regulation. Analysis at the molecular level may be needed to unveil any relationship between heteromorphism and PA. Impact Statement What is already known on this subject? Primary amenorrhoea (PA) is a menstrual abnormality found in females with the prevalence of 1–3%. It may be associated with different types of numerical and structural chromosomal anomalies. Among them Turner’s syndrome (pure and in variant form) is the commonest chromosomal aberration associated with PA. Some patients with PA are found to have a normal karyotype with heterochromatin extension on the large arm (q) of either chromosome 9 or chromosome 16. Chromosomal polymorphism with increase in heterochromatin region consists of highly repetitive sequences of satellite DNA, which normally does not encode any protein and thus considered to be a normal variant. What do the results of this study add? This comprehensive study reveals that there are 10.11% of the females among 178 females having a heterochromatin extension which is significantly high. PA and certain association of phenotypical stigmata like short stature in these patients with heterochromatin extension can be explained on the basis of histone modification and gene regulation by heterochromatin. What are the implications of these findings for clinical practice and/or further research? We will be able to know about involved transcription factors those are responsible for the histone modification directly linked to the heterochromatin extension by further molecular study. That will definitely help to find out the reason for PA as well as implementation of gene therapy in these cases.

Mitochondrial superoxide targets energy metabolism to modulate epigenetic regulation of NRF2-mediated transcription

Mitochondria are central to the metabolic circuitry that generates superoxide radicals/anions (O2•-) as a by-product of oxygen metabolism. By regulating superoxide levels, manganese superoxide dismutase plays important roles in numerous biochemical and molecular events essential for the survival of aerobic life. In this study, we used MitoParaquat (mPQ) to generate mitochondria-specific O2•- and stable isotope-resolved metabolomics tracing in primary human epidermal keratinocytes to investigate how O2•- generated in mitochondria regulates gene expression. The results reveal that isocitrate is blocked from conversion to α-ketoglutarate and that acetyl-coenzyme A (CoA) accumulates, which is consistent with a reduction in oxygen consumption rate and inactivation of isocitrate dehydrogenase (IDH) activity. Since acetyl-CoA is linked to histone acetylation and gene regulation, we determined the effect of mPQ on histone acetylation. The results demonstrate an increase in histone H3 acetylation at lysines 9 and 14. Suppression of IDH increased histone acetylation, providing a direct link between metabolism and epigenetic alterations. The activity of histone acetyltransferase p300 increased after mPQ treatment, which is consistent with histone acetylation. Importantly, mPQ selectively increased the nuclear levels and activity of the oxidative stress-sensitive nuclear factor erythroid 2-related factor 2. Together, the results establish a new paradigm that recognizes O2•- as an initiator of metabolic reprogramming that activates epigenetic regulation of gene transcription in response to mitochondrial dysfunction.

RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells.

In eukaryotic nuclei, a number of phase‐separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post‐transcriptional regulation of small nuclear RNA and histone genes. By live‐cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC–CB–HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC–HLBs with CBs and nucleation of PCs near preformed CB–HLBs. Coilin knockout increased the co‐localization rate between PCs and HLBs, whereas the number, nucleation timing and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6‐hexanediol revealed that PCs were more liquid‐like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs.

Methylxanthines and Neurodegenerative Diseases: An Update.

Methylxanthines (MTX) are purine derived xanthine derivatives. Whereas naturally occurring methylxanthines like caffeine, theophylline or theobromine are widely consumed in food, several synthetic but also non-synthetic methylxanthines are used as pharmaceuticals, in particular in treating airway constrictions. Besides the well-established bronchoprotective effects, methylxanthines are also known to have anti-inflammatory and anti-oxidative properties, mediate changes in lipid homeostasis and have neuroprotective effects. Known molecular mechanisms include adenosine receptor antagonism, phosphodiesterase inhibition, effects on the cholinergic system, wnt signaling, histone deacetylase activation and gene regulation. By affecting several pathways associated with neurodegenerative diseases via different pleiotropic mechanisms and due to its moderate side effects, intake of methylxanthines have been suggested to be an interesting approach in dealing with neurodegeneration. Especially in the past years, the impact of methylxanthines in neurodegenerative diseases has been extensively studied and several new aspects have been elucidated. In this review we summarize the findings of methylxanthines linked to Alzheimer´s disease, Parkinson’s disease and Multiple Sclerosis since 2017, focusing on epidemiological and clinical studies and addressing the underlying molecular mechanisms in cell culture experiments and animal studies in order to assess the neuroprotective potential of methylxanthines in these diseases.

Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH–NPAT and YARP–NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple -helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single -helix that makes multiple contacts with -helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.

Exogenous pyruvate represses histone gene expression and inhibits cancer cell proliferation via the NAMPT-NAD+-SIRT1 pathway.

Pyruvate is a glycolytic metabolite used for energy production and macromolecule biosynthesis. However, little is known about its functions in tumorigenesis. Here, we report that exogenous pyruvate inhibits the proliferation of different types of cancer cells. This inhibitory effect of pyruvate on cell growth is primarily attributed to its function as a signal molecule to repress histone gene expression, which leads to less compact chromatin and misregulation of genome-wide gene expression. Pyruvate represses histone gene expression by inducing the expression of NAD+ biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT) via myocyte enhancer factor 2C (MEF2C), which then increases NAD+ levels and activates the histone deacetylase activity of SIRT1. Chromatin immunoprecipitation analysis indicates that pyruvate enhances SIRT1 binding at histone gene promoters where it reduces histone acetylation. Although pyruvate delays cell entry into S phase, pyruvate represses histone gene expression independent of cell cycle progression. Moreover, we find that administration of pyruvate reduces histone expression and retards tumor growth in xenograft mice without significant side effects. Using tissues from cervical and lung cancer patients, we find intracellular pyruvate concentrations inversely correlate with histone protein levels. Together, we uncover a previously unknown function of pyruvate in regulating histone gene expression and cancer cell proliferation.

Histone H2B Ubiquitylation Regulates Histone Gene Expression by Suppressing Antisense Transcription in Fission Yeast.

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymeraseII transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+ -a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.

Abstract 375: Acetyl-CoA Production by Specific Metabolites Promotes Cardiac Repair After Myocardial Infarction via Mediating Histone Acetylation

In this study, we set out to identify an interlinked metabolic and epigenetic network that produces acetyl-CoA for histone acetylation and gene regulation, and determine whether this network promotes heart repair and protection after I/R injury. Myocardial ischemic reperfusion (I/R) injury induces dramatic metabolic changes and is accompanied with extensive epigenetic changes. Recent studies reveal that energy metabolism and chromatin epigenetics are intimately linked in cellular functions. In particular, acetyl-CoA is a building block for energy metabolism and histone acetylation. However, the acetyl-CoA mediated regulatory machinery integrating metabolic pathways and chromatin modifications has been under-explored in heart repair and protection. We conducted a screen of energy metabolites in promoting histone acetylation and heart repair both in vivo and in vitro. Our screen identified that acetate, pyruvate, and octanoic acid (8C) but not citrate and nonanoic acid (9C) improved heart function after MI in rats. In particular, 8C administration resulted in the most significant heart functional recovery after MI. More importantly, in a more clinically relevant setting, 8C injection at the time of reperfusion 45 minutes after left anterior descending coronary (LAD) ligation showed comparable repair effect to that of 8C administration before LAD ligation, suggesting that 8C could be a very effective metabolic natural product to treat MI. Mechanistically, 8C promoted histone acetylation in CM chromatin after IR injury and inhibited CM death by activating expression of anti-oxidant genes Nrf2, HO1, and NQO1. We further established that the 8C promoted histone acetylation and heart repair was transduced by metabolic enzyme medium-chain acyl-CoA dehydrogenase (MCAD) and histone acetylase GCN5/KAT2A. Therefore, this study has elucidated an interlinked metabolic/epigenetic network comprising 8C, acetyl-CoA, MCAD, and KAT2A in stimulating histone acetylation and anti-oxidative stress gene expression to combat heart injury. This study provides a novel strategy for treating myocardial I/R disease at the interface of metabolism and epigenetics.

(De)Toxifying the Epigenetic Code.

Cells are continuously subjected to an array of reactive/toxic chemical species which are produced both endogenously through metabolic pathways and taken up exogenously by diet and exposure to drugs or toxins. As a result, proteins often undergo non-enzymatic covalent modifications (NECMs) by these species, which can alter protein structure, function, stability, and binding partner affinity. NECMs accumulate over time and are linked to various diseases such as Alzheimer's disease, cancer, and diabetes. In the cellular proteome, histones have some of the longest half-lives, making them prime targets for NECMs. In addition, histones have emerged as key regulators of transcription, a function that is primarily controlled by modificationoftheir tails. These modifications are usually installed or removed enzymatically, but recent evidence suggests that some may also occur non-enzymatically. Despite the vast knowledge detailing the relationship between histone modifications and gene regulation, NECMs on histones remain poorly explored. A major reason for this difference stems from the fact that, unlike their enzymatically installed counterparts, NECMs are difficult to both control and test in vivo. Here, we review advances in our understanding of the effect non-enzymatic covalent modifications (NECMs) have on the epigenetic landscape, cellular fate, and their implications in disease. Cumulatively, this illustrates how the epigenetic code is directly toxified by chemicals and detoxified by corresponding eraser enzymes.