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2-Hydroxyisobutyrylation on Histone H4K8 Is Regulated by Glucose Homeostasis in Saccharomyces Cerevisiae

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2-Hydroxyisobutyrylation on Histone H4K8 Is Regulated by Glucose Homeostasis in Saccharomyces Cerevisiae 2-Hydroxyisobutyrylation on histone H4K8 is regulated by glucose homeostasis in Saccharomyces cerevisiae Jing Huanga,b,1, Zhouqing Luoa,b,1, Wantao Yingc,1, Qichen Caoc, He Huangd, Junkai Donga, Qingyu Wua, Yingming Zhaod, Xiaohong Qianc,2, and Junbiao Daia,b,2 aMinistry of Education Key Laboratory of Bioinformatics, Centre for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China; bCenter for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China; cState Key Laboratory of Proteomics, National Protein Science Center, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China; and dBen May Department of Cancer Research, University of Chicago, Chicago, IL 60637 Edited by Jef D. Boeke, New York University Langone Medical Center, New York, NY, and approved July 10, 2017 (received for review February 3, 2017) New types of modifications of histones keep emerging. Recently, and histone deacetylases (HDACs) have been reported to be histone H4K8 2-hydroxyisobutyrylation (H4K8hib) was identified as able to catalyze additional acylation or deacylation reactions in an evolutionarily conserved modification. However, how this mod- addition to acetylation or deacetylation (6, 24–26), hinting that ification is regulated within a cell is still elusive, and the enzymes the regulation and function of these new histone acylations may adding and removing 2-hydroxyisobutyrylation have not been also be similar to or perhaps redundant with histone acetylation. found. Here, we report that the amount of H4K8hib fluctuates in However, until now, little about the regulation and function of response to the availability of carbon source in Saccharomyces cer- these new acylations on histones is known. evisiae and that low-glucose conditions lead to diminished modifi- Lysine 2-hydroxyisobutyrylation (Khib) is a newly identified cation. The removal of the 2-hydroxyisobutyryl group from H4K8 is histone mark conserved from yeast to humans; specifically, mediated by the histone lysine deacetylase Rpd3p and Hos3p H4K8 2-hydroxyisobutyrylation (H4K8hib) has been detected in in vivo. In addition, eliminating modifications at this site by alanine actively transcribed genes in mouse meiotic and postmeiotic cells substitution alters transcription in carbon transport/metabolism (10). Here we report the discovery of a carbon stress-related genes and results in a reduced chronological life span (CLS). Further- function and regulation of H4K8hib in Saccharomyces cerevisiae. more, consistent with the glucose-responsive H4K8hib regulation, We found that H4K8hib was a stress-responsive modification and BIOCHEMISTRY proteomic analysis revealed that a large set of proteins involved identified its modifying enzymes. In addition, we showed that the in glycolysis/gluconeogenesis are modified by lysine 2-hydroxyiso- nonmodifiable substrate mutant, H4K8A, leads to a reduced butyrylation. Cumulatively, these results established a functional and chronological life span (CLS). Finally, we performed Khib pro- regulatory network among Khib, glucose metabolism, and CLS. teomics analysis in Saccharomyces cerevisiae and identified 1,458 modified sites on 369 proteins, revealing an enrichment of protein posttranslational modifications | lysine acetylation | lysine 2- this modification in the glycolysis/gluconeogenesis pathway. hydroxyisobutyrylation | chronological life span | histone deacetylase Results osttranslational modifications (PTMs) of proteins influence H4K8hib Is Dynamically Regulated by the Availability of a Carbon Ptheir properties, such as cellular localization, stability, in- Source. Histone Khib has been reported as a dynamic mark teraction, and enzymatic activity (1–5). Over the past decade, enriched in active chromatin, but its regulatory mechanism re- several new types of PTMs have been identified on lysine residues, mains elusive (10). To identify potential modulators and dissect including propionylation, butyrylation, crotonylation, succinylation, its functions, we monitored changes of this modification under malonylation, glutarylation, and 2-hydroxyisobutyrylation, which, different stress conditions using the H4K8hib-specific antibody collectively, are termed as lysine acylation (6–11). The possible donors of these acylations are, presumably, their corresponding Significance acyl-CoAs, the intermediates of many cellular metabolic processes (12). Several recent studies indicated that these new acylations oc- DNA–histone complexes are packed into the eukaryotic genome cur on thousands of proteins in various cellular metabolic processes and fit into the nucleus of the cell. One mechanism to access the and play important roles in metabolic regulation (11, 13–20). genetic information is to disrupt the complexes through post- Acylations were also found on histones (6, 8, 10, 21), the major translational modification of histones. Recently, histone H4K8 protein components of chromatin, which, together with a fragment 2-hydroxyisobutyrylation (H4K8hib) was identified as an evolution- of DNA, form the basic building block of a eukaryotic genome arily conserved active mark. However, how this modification is (22). Modifications on histones have important functions in regulated and what are the enzymes to modulate this modification transcriptional regulation, DNA repair, replication, and chromatin within a cell remain a mystery. In this study, we discover that this condensation (23). Two major mechanisms have been proposed. modification is regulated by the availability of a carbon source in One mechanism is to alter the charge state of the modified resi- Saccharomyces cerevisiae and identify the enzymes catalyzing the dues, which might interfere with the histone–histone and/or his- removal of this active mark in vivo. This discovery provides insight tone–DNA electrostatic interactions, leading to a chromatin state into the function and regulation of the histone mark H4K8hib. transition. The other mechanism is to act as “bait” to recruit ef- Author contributions: Y.Z., X.Q., and J. Dai designed research; J.H., Z.L., W.Y., Q.C., H.H., fector proteins to chromatin (23). A previous study of histone and J. Dong performed research; J.H., Z.L., W.Y., H.H., Q.W., Y.Z., X.Q., and J. Dai analyzed crotonylation suggested that it is functionally distinct from lysine data; and J.H., Z.L., and J. Dai wrote the paper. acetylation (Kac) and marks active testis-specific genes in post- Conflict of interest statement: Y.Z. is on the science advisory board of PTM Biolabs. meiotic cells (8). Furthermore, histone crotonylation was proved This article is a PNAS Direct Submission. to stimulate transcription to a greater extent than histone acety- 1J.H., Z.L., and W.Y. contributed equally to this work. lation in a cell-free transcription system (24). 2To whom correspondence may be addressed. Email: [email protected] or Although quite a few new modifications have been identified, [email protected]. identifying the enzymes that write or erase the modification has This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. largely lagged behind. Some histone acetyltransferases (HATs) 1073/pnas.1700796114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1700796114 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 that was developed, characterized, and reported in a previous H4K8hib, we supplied different sugars back into the SC-D me- study (10). We found that H4K8hib exhibited no or little response dium and monitored the recovery of H4K8hib, respectively. As upon most treatments such as DNA damage, temperature, and shown in Fig. 1D, not only glucose, but also fructose, is able to redox stresses (Fig. 1A). In contrast, we detected significant re- restore the H4K8hib level rapidly after glucose starvation. In duction of H4K8 after cells were incubated in water (Fig. 1 A contrast, glycerol, ethanol, and even galactose that is ferment- hib “ ” and B). During water treatment, cells were challenged by mul- able but a secondary carbon source failed to rescue H4K8hib, tiple stresses including osmotic pressure and severe nutrient suggesting that a preferred fermentable sugar is required for this starvation. Since 1 M sodium chloride (NaCl, osmotic stress) and modification. Taken together, these data indicate that H4K8hib is synthetic defined medium lacking nitrogen (SD-N, nitrogen a dynamic modification directly regulated by glucose/fructose availability and establishes a link between histone modification starvation) treatment had little effect on H4K8hib (Fig. 1A), we asked whether glucose deprivation might be the cause. As shown and carbon metabolism. in Fig. 1B, treating the cells in synthetic complete medium lacking glucose (SC-D) resulted in a decreased H4K8 level Glycolysis Is Required to Restore H4K8hib but Dispensable for Its hib Establishment and Maintenance. Given that H4K8 is a type of comparable to that after water treatment. In addition, supple- hib glucose-regulated modification, we next asked how glucose is menting glucose in both water and SC-D could restore the able to modulate H4K8 Glucose and fructose are preferen- amount of H4K8 (Fig. 1B). Together, these results strongly hib. hib tially used by many unicellular organisms since they can directly argue that the presence of glucose in the medium is the major enter the glycolytic pathway. Therefore, we hypothesized that
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