Browsing by Subject "Histones"
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Item Carbon Starvation Metabolically Regulates Chromatin for Transcriptome Rewiring(2022-05) Hsieh, Wen-Chuan; Conrad, Nicholas; Kraus, W. Lee; Orth, Kim; Tu, BenjaminCells robustly rewire their transcriptomes to survive under stress conditions. Yet, how does such reprogramming of gene expression occur? Under favorable nutrient conditions, acetyl-CoA normally promotes histone acetylation to activate genes required for cell growth. However, glucose starvation significantly reduces the availability of acetyl-CoA. And it is unclear how such a change impacts genome-wide histone acetylation and gene expression. In this study, I set up a robust glucose starvation model in budding yeast to discover a mechanism by which cells preserve acetyl-CoA, a key intermediate in energy metabolism, in order to sustain histone acetylation for gene activation even under stress conditions. I demonstrate a dramatic redistribution of histone acetylation upon glucose starvation. Mechanistically, I determined that a major histone deacetylase (HDAC) releases acetyl groups from histones at growth-promoting genes, which can subsequently be used to acetylate histones at a distinctive set of stress-responsive genes. Strikingly, bioinformatic analysis revealed these genes to be required for gluconeogenic and fat metabolism, which are metabolic pathways that generate acetyl-CoA for oxidation and ATP synthesis. Genetic deletion of histone modifiers mediating this reallocation, including the key HDAC or histone acetyltransferase (HAT), disrupts proper transcriptome rewiring for survival. Given the importance of acetate for recycling the acetyl- group, I next characterize acetyl-CoA synthetases (Acs), metabolic enzymes that convert acetate to acetyl-CoA. I demonstrate that Acs2 is required for maintaining global histone acetylation, yet its nuclear localization appears to be dispensable for such regulation. I observe that the catalytic activity of Acs2 governs the intracellular acetyl-CoA level and global histone acetylation amounts. Compromising its activity leads to up-regulation of ergosterol biosynthetic pathways in addition to gluconeogenic and fat metabolism genes upon glucose starvation. In summary, I reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting. I have elucidated how transcriptome rewiring is driven by reallocation of histone acetylation. My findings present a mechanism by which cells recycle acetyl groups to differentially acetylate histones for activation of key genes required for metabolism and survival.Item Control of Regulatory Element Function by Histone H3.3(2022-05) Tafessu, Amanuel Melesse; Yu, Hongtao; Banaszynski, Laura; Chiang, Cheng-Ming; Xu, JianIn eukaryotic cells, DNA is wrapped around histone proteins to form nucleosomes, the fundamental repeating unit of chromatin. While chromatin functions in part to organize a large amount of genomic material within the confines of the nucleus, regulatory DNA sequences consequently become masked to transcription machinery. Such regulatory sequences are enriched in specific histone variants and post-translational modifications (PTMs). The histone variant H3.3 is enriched at transcriptionally active regulatory elements such as promoters and enhancers. While recent studies have revealed a role for H3.3 in silencing repetitive elements and repressing developmentally regulated promoters, it is unclear how H3.3 contributes to chromatin states at active promoters and enhancers. In this study, we performed genomic analyses of chromatin features associated with active regulatory elements in mouse embryonic stem cells (ESCs) and found evidence of subtle yet widespread dysregulation in the absence of H3.3. Loss of H3.3 or HIRA- the chaperone responsible for H3.3 deposition to transcriptionally active regions- reduces chromatin accessibility and transcription factor (TF) footprinting at promoters. Further, H3.3 KO ESCs show reduced promoter enrichment of p300- a transcriptional coactivator responsible for H3 acetylation at lysine 27 (H3K27ac). Consequently, H3.3 KO ESCs show reduced H3K27ac at promoters, along with reduced enrichment of the acetyllysine reader BRD4. Despite the enrichment of H3.3 at both promoters and enhancers, it appears to play distinct roles at these regions. ESCs lacking H3.3 or HIRA are able to maintain both accessibility and TF footprinting at enhancers, but still show reduced H3K27ac. Unlike promoters, enhancers show no deficit of p300 enrichment in the absence of H3.3. The loss of H3K27ac observed at enhancers of H3.3 KO ESCs can be attributed to reduced catalytic activity of p300. In particular, phosphorylation of Ser31, the only residue unique to the N-terminal tail of H3.3, facilitates p300 activity and H3K27ac enrichment. In spite of extensive chromatin dysregulation and reduced active RNA polymerase II (RNAPII) engagement, ESCs maintain transcription from ESC-specific genes in the absence of H3.3. However, H3.3 KO ESCs are unable to initiate lineage-specific transcription upon undirected differentiation. In line with their differentiation defect, H3.3 KO ESCs retain footprinting of ESC-specific TFs and fail to generate footprints of lineage-specific TFs. Further, H3.3 KO ESCs fail to "open" and acetylate developmentally regulated enhancers. Overall, our study shows that H3.3 facilitates the establishment of transcriptionally permissive chromatin at regulatory elements, with context-dependent outcomes for transcriptional output. While H3.3 is not required for maintaining transcription in ESCs, it plays a key role in activating promoters and enhancers during differentiation.Item Identification and Characterization of the Multifunctional Epigenetic Regulator CFP1 as an ERK1/2 Substrate(2014-11-21) Klein, Aileen Melanie; Sternweis, Paul C.; Cobb, Melanie H.; Goodman, Joel M.; Conrad, NicholasEpigenetic regulation of gene transcription occurs as an integration of multiple layers of signals at a genetic locus. These signals can include local chromatin structure, covalent modifications to both histone proteins and DNA, the presence of transcription factors, and modification directly to the transcriptional machinery. Our lab is interested in the control of cellular processes by the mitogen activated protein kinases ERK1/2. In a yeast two-hybrid screen with activated ERK2 (extracellular signal-regulated kinase 2) to find novel interacting partners, our lab identified CFP1 (CxxC finger protein 1), a DNA-binding protein that is a vital component of the H3K4 trimethylating Set1A/B complexes to promote gene transcription. CFP1 has also been shown to interact physically and functionally with the major maintenance DNA methyltransferase DNMT1. We are interested in defining how substrate targeting of CFP1 by ERK1/2 regulates downstream transcriptional outcomes. Interaction between ERK2 and CFP1 in cells was validated by co-immunoprecipitation from isolated mononucleosomes. Active ERK2 can phosphorylate CFP1 on multiple sites in vitro, an observation supported by studies in cells. Some of the most likely in vivo ERK1/2 phosphorylation sites include serine 224 and threonine 227. CFP1 is essential for focusing trimethylation of H3K4 at promoters, a histone modification that supports transcription from these loci. We hypothesized that phosphorylation of CFP1 by ERK1/2 during mitogenic signaling may support trimethylation of H3K4 and transcription of ERK1/2-regulated target genes. Introduction of CFP1 containing the mutation T227V into HeLa cells blocked global H3K4 trimethylation to a similar extent as CFP1 depletion. On the other hand, CFP1 S224A shows diminished transactivation capacity against a model transcriptional substrate. Neither of these mutants fail to interact with Set1B in a pulldown, suggesting that these sites may be important for Set1 complex targeting or activity towards chromatin. Consistently, CFP1 knockdown hinders induction of several ERK1/2-regulated immediate early gene targets in response to serum treatment. It will be of interest to test whether this is dependent on stable or inducible H3K4 trimethylation and what impact overexpression of point mutants will play in their transcription. Regulation of H3K4 trimethylation through CFP1 phosphorylation might represent a novel regulatory input to support transcription of ERK1/2-regulated genes.Item Regulation of Reparative Macrophage Transition by the B-cell Adapter for PI3K (BCAP)(2021-05-01T05:00:00.000Z) Irizarry-Caro, Ricardo A.; Satterthwaite, Anne B.; Pasare, Chandrashekhar; Street, Nancy E.; Tagliabracci, Vincent S.; Zaki, HasanMacrophages respond to microbial ligands and various noxious cues by initiating an inflammatory response aimed at eliminating the original pathogenic insult. Transition of macrophages from a pro-inflammatory state to a reparative state, however, is vital for resolution of inflammation and return to homeostasis. The molecular players governing this transition remain poorly defined. Here, we find that the reparative macrophage transition is dictated by B-cell adapter for PI3K (BCAP). Mice harboring a macrophage specific deletion of BCAP fail to recover from and succumb to DSS-induced colitis due to prolonged intestinal inflammation and impaired tissue repair. Following microbial stimulation, gene expression in WT macrophages switches from an early inflammatory signature to a late reparative signature, a process that is hampered in BCAP deficient macrophages. We find that absence of BCAP hinders inactivation of FOXO1 and GSK3b that contributes to their enhanced inflammatory state. BCAP deficiency also results in defective aerobic glycolysis and reduced lactate production. This translates into reduced histone lactylation and decreased expression of reparative macrophage genes. Thus, our results reveal BCAP to be critical cell intrinsic switch that regulates transition of inflammatory macrophages to reparative macrophages by imprinting epigenetic changes.Item The Role of Class I Histone Deacetylases in Cardiovascular Development and Disease(2008-05-13) Montgomery, Rusty Lee; Olson, Eric N.Histone acetylation/deacetylation is a dynamic process that coordinates proper gene expression through the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibitors continue to show promise in a multitude of pathological settings such as cancer and heart disease, however the role of individual HDACs remains largely unexplored. Genetic studies have shown class II HDACs regulate developmental and physiological processes through the interaction with and repression of myocyte enhancer factor 2, MEF2, however the biological functions of class I HDACs have not been determined. To potentially understand the role of individual class I HDACs in development and disease, we generated conditional knockout alleles for HDAC1, HDAC2, and HDAC3. Through global and tissue specific analyses, we hope to identify specific roles of these enzymes in developmental, physiological, and pathological settings. Here I show that HDAC1 and HDAC2 act redundantly in controlling cardiac growth, morphogenesis, and contractility. Mice with cardiac-specific deletion of either HDAC1 or HDAC2 are viable and lack obvious phenotypes, however cardiac-specific deletion of both HDAC1 and HDAC2 results in lethality by two weeks of age. These mice show cardiac arrhythmias, dilated cardiomyopathy, and increased expression of calcium channels and skeletal muscle-specific contractile proteins. HDAC3 is an independent regulator of cardiac development. Global deletion of HDAC3 results in embryonic lethality, whereas cardiac-specific deletion of HDAC3 results in massive cardiac hypertrophy by 3 months of age and lethality by 16 weeks. These mice show metabolic abnormalities including up-regulation of genes involved in fatty acid uptake and oxidation, down-regulation of the glucose utilization pathway, and ligand induced myocardial lipid accumulation. Additionally, these hearts show mitochondrial dysfunction and decreased cardiac efficiency. These studies have identified HDAC3 as a central regulator of myocardial energy metabolism. In addition to cardiac studies, tissue-specific deletions in multiple cell-types have led to the discovery that functional redundancy of HDAC1 and HDAC2 is not restricted to postnatal cardiomyocytes, but extends to early cardiomyocytes, endothelial cells, smooth muscle cells, chondrocytes, and neurons. Deletion of HDAC1 or HDAC2 individually in these cell types does not evoke a phenotype, however deletion of both HDAC1 and HDAC2 results in embryonic lethality or neonatal lethality. Taken together, these studies identify HDAC1 and HDAC2 as redundant regulators of multiple cell types during development. Collectively, these studies have identified distinct and specific roles for HDAC1, HDAC2, and HDAC3 during development and disease. Furthermore, these genetic studies have provided mechanistic insight into the pathways regulated by each enzyme. Additional analyses on these mice will prove instrumental to the development of more specific inhibitors for the treatment of a wide array of pathological conditions.Item Structural and Biochemical Studies of Multiple Importin-Histone Interactions(2016-04-13) Soniat, Michael Maurice, II; Rizo-Rey, José; Chook, Yuh Min; Jiang, Youxing; Goodman, Joel M.; Li, BingMultiple Importins can bind the N-terminal tails of histones H3 and H4, and import them into the nucleus to be assembled into the nucleosomes. However, it is not known what sequence elements in the histone tails are recognized by each of the Importins. Through structural and quantitative biochemical analysis, I identified binding determinants in the N-terminal tails of histones H3 and H4 for each of seven different human Importins (Impα, Impβ, Kapβ2, Imp4, Imp5, Imp7, Imp9). Crystal structure of the H3 tail bound to Kapβ2 identified H3 tail residues 11-27 as the important binding element, which resembles a PY-NLS that is missing the canonical proline-tyrosine motif. This same N-terminal basic segment of H3 is also important for binding Impβ, Imp4, Imp5, Imp7, Imp9, and Impα. In addition, a C-terminal IK-NLS-like motif at residues 35-40 of H3 is also used to bind Imp5, Imp7, Imp9 and Impα. Interactions of the H4 tail with the same Importins show a similar trend of relative affinities as the H3 tail, though at least 10-fold weaker. Similar to the H3 tail, the H4 tail also uses one or two basic regions to bind the Importins. I also studied the effects of histone tail acetylation on Importin-histone interactions and showed that acetylation of Lys14 of the H3 tail impairs binding to all six Importins and Impα while acetylation of Lys18 of H3 tail and acetylation of Lys5 and Lys12 of the H4 tail had only mild effects on binding to the Importins. Lastly, I studied Importin binding to the H3/H4 dimer and showed that only one Importin molecule binds each H3/H4 dimer. Furthermore, the Importin-binding trend with the H3/H4 dimer is very different than with the N-terminal tails alone suggesting additional interactions with the histone fold domains of H3 and H4. Overall, I have mapped Importin-binding determinants for the H3 and H4 and revealed acetylation effects on Importin-histone binding.