scholarly journals Class I Histone Deacetylases (HDAC1–3) are Histone Lysine Delactylases

2021 ◽  
Author(s):  
Carlos Moreno-Yruela ◽  
Di Zhang ◽  
Wei Wei ◽  
Michael Bæk ◽  
Jinjun Gao ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Chemical Reactivity ◽  
Regulatory Elements ◽  
Systematic Evaluation ◽  
Full Characterization ◽  
Regulatory Enzymes ◽  
Chain Acyl ◽  
The Warburg Effect

AbstractLysine l-lactylation [K(l-la)] is a newly discovered histone mark that can be stimulated under conditions of high glycolysis, such as the Warburg effect. K(l-la) is associated with functions that are different from the widely studied histone acetylation. While K(l-la) can be introduced by the acetyltransferase p300, histone delactylase enzymes remain unknown. Here, we report the systematic evaluation of zinc- and NAD+-dependent HDACs for their ability to cleave ε-N-l-lactyllysine marks. Our screens identified HDACs 1–3 and SIRT1–3 as delactylases in vitro. HDACs 1–3 show robust activity toward not only K(l-la) but also K(d-la) and diverse short-chain acyl modifications. We further confirmed the de-l-lactylase activity of HDACs 1 and 3 in cells. Identification of p300 and HDAC3 as regulatory enzymes suggests that histone lactylation is installed and removed by enzymes as opposed to spontaneous chemical reactivity. Our results therefore represent an important step toward full characterization of this pathway’s regulatory elements.

scholarly journals Kinetic characterization of inhibitors of histone deacetylases (HDACs) and sirtuins

2019 ◽  
Author(s):  
Carlos Moreno-Yruela ◽  
Andreas Stahl Madsen ◽  
Christian A. Olsen
Keyword(s):  
Histone Deacetylases ◽  
Hdac Inhibitors ◽  
Kinetic Properties ◽  
Kinetic Characterization ◽  
Class Iii ◽  
Vitro Assay ◽  
Substrate Conversion ◽  
Fluorogenic Peptide Substrate

Abstract Histone deacetylase (HDAC) inhibitors are employed for the treatment of lymphoma and are under development against multiple other types of cancer and neurodegenerative diseases. Here, we describe a robust and uncomplicated in vitro assay for HDAC inhibitor kinetic profiling. Enzyme and fluorogenic peptide substrate are incubated together with a small amount of protease “assay developer”, which enables continuous recording of substrate conversion under steady-state conditions. Assay progression curves upon addition of an inhibitors at varying concentrations permit determination of kinetic constants and overall inhibitor potency. This assay helped provide new insight into the kinetic properties of known HDAC inhibitors as well as the kinetic characterization of both inhibitors and substrates of sirtuin enzymes, which are class III HDACs involved in metabolic control and oncogene regulation.

scholarly journals The HosA Histone Deacetylase Regulates Aflatoxin Biosynthesis Through Direct Regulation of Aflatoxin Cluster Genes

2019 ◽  
Vol 32 (9) ◽  
pp. 1210-1228 ◽  
Author(s):  
Huahui Lan ◽  
Lianghuan Wu ◽  
Ruilin Sun ◽  
Nancy P. Keller ◽  
Kunlong Yang ◽  
...  
Keyword(s):  
Secondary Metabolite ◽  
Histone Deacetylases ◽  
Trichostatin A ◽  
Secondary Metabolite Production ◽  
Fungal Development ◽  
Metabolite Production ◽  
Vitro Analysis ◽  
Aflatoxin B

Histone deacetylases (HDACs) always function as corepressors and sometimes as coactivators in the regulation of fungal development and secondary metabolite production. However, the mechanism through which HDACs play positive roles in secondary metabolite production is still unknown. Here, classical HDAC enzymes were identified and analyzed in Aspergillus flavus, a fungus that produces one of the most carcinogenic secondary metabolites, aflatoxin B1 (AFB1). Characterization of the HDACs revealed that a class I family HDAC, HosA, played crucial roles in growth, reproduction, the oxidative stress response, AFB1 biosynthesis, and pathogenicity. To a lesser extent, a class II family HDAC, HdaA, was also involved in sclerotia formation and AFB1 biosynthesis. An in vitro analysis of HosA revealed that its HDAC activity was considerably diminished at nanomolar concentrations of trichostatin A. Notably, chromatin immunoprecipitation experiments indicated that HosA bound directly to AFB1 biosynthesis cluster genes to regulate their expression. Finally, we found that a transcriptional regulator, SinA, interacts with HosA to regulate fungal development and AFB1 biosynthesis. Overall, our results reveal a novel mechanism by which classical HDACs mediate the induction of secondary metabolite genes in fungi.

scholarly journals The regulatory enzymes and protein substrates for the lysine β-hydroxybutyrylation pathway

2021 ◽  
Vol 7 (9) ◽  
pp. eabe2771 ◽  
Author(s):  
He Huang ◽  
Di Zhang ◽  
Yejing Weng ◽  
Kyle Delaney ◽  
Zhanyun Tang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Functional Study ◽  
Histone Deacetylase 1 ◽  
Major Mechanism ◽  
Cellular Processes ◽  
Regulatory Enzymes ◽  
Substrate Proteins

Metabolism-mediated epigenetic changes represent an adapted mechanism for cellular signaling, in which lysine acetylation and methylation have been the historical focus of interest. We recently discovered a β-hydroxybutyrate–mediated epigenetic pathway that couples metabolism to gene expression. However, its regulatory enzymes and substrate proteins remain unknown, hindering its functional study. Here, we report that the acyltransferase p300 can catalyze the enzymatic addition of β-hydroxybutyrate to lysine (Kbhb), while histone deacetylase 1 (HDAC1) and HDAC2 enzymatically remove Kbhb. We demonstrate that p300-dependent histone Kbhb can directly mediate in vitro transcription. Moreover, a comprehensive analysis of Kbhb substrates in mammalian cells has identified 3248 Kbhb sites on 1397 substrate proteins. The dependence of histone Kbhb on p300 argues that enzyme-catalyzed acylation is the major mechanism for nuclear Kbhb. Our study thus reveals key regulatory elements for the Kbhb pathway, laying a foundation for studying its roles in diverse cellular processes.

scholarly journals The human SOX18 gene: Expression analysis and characterization of its 5’ flanking region

2007 ◽  
Vol 59 (4) ◽  
pp. 267-272 ◽  
Author(s):  
Isidora Petrovic ◽  
Milena Stevanovic
Keyword(s):  
Transcriptional Regulation ◽  
Regulatory Elements ◽  
Model System ◽  
Suitable Model ◽  
Flanking Region ◽  
Vitro Model ◽  
The Given ◽  
First Time

The aim of this study was to establish an adequate in vitro model system for studying transcriptional regulation of the human SOX18 gene. The paper presents an analysis of expression of this gene in cultured cell lines and characterization of its 5' flanking region. Using RT-PCR, Northern and Western blot analysis, we demonstrated SOX18 expression in HeLa cells, indicating that this cell line provides a suitable model system for studying transcriptional regulation of the given gene. We also cloned, sequenced and for the first time characterized the human SOX18 5? flanking region. It is shown that the region 892 bp in size immediately upstream from the start codone harbors regulatory elements sufficient for transcription and represents an SOX18 promoter region.

scholarly journals Evolution of ASC Immunophenotypical Subsets During Expansion In Vitro

2020 ◽  
Vol 21 (4) ◽  
pp. 1408 ◽  
Author(s):  
Qiuyue Peng ◽  
Hiva Alipour ◽  
Simone Porsborg ◽  
Trine Fink ◽  
Vladimir Zachar
Keyword(s):  
Stem Cells ◽  
Early Stage ◽  
Clinical Use ◽  
Cross Sectional ◽  
Full Characterization ◽  
Surface Molecules ◽  
Surface Marker Expression ◽  
Stromal Stem Cells

Adipose-derived stromal/stem cells (ASCs) are currently being considered for clinical use for a number of indications. In order to develop standardized clinical protocols, it is paramount to have a full characterization of the stem cell preparations. The surface marker expression of ASCs has previously been characterized in multiple studies. However, most of these studies have provided a cross-sectional description of ASCs in either earlier or later passages. In this study, we evaluate the dynamic changes of 15 different surface molecules during culture. Using multichromatic flow cytometry, ASCs from three different donors each in passages 1, 2, 4, 6, and 8 were analyzed for their co-expression of markers associated with mesenchymal stem cells, wound healing, immune regulation, ASC markers, and differentiation capacity, respectively. We confirmed that at an early stage, ASC displayed a high heterogeneity with a plethora of subpopulations, which by culturing became more homogeneous. After a few passages, virtually all ASCs expressed CD29, CD166 and CD201, in addition to canonical markers CD73, CD90, and CD105. However, even at passage 8, there were several predominant lineages that differed with respect to the expression of CD34, CD200 and CD271. Although the significance of remaining subpopulations still needs to be elucidated, our results underscore the necessity to fully characterize ASCs prior to clinical use.

scholarly journals In Vitro Osteogenic Properties of Two Dental Implant Surfaces

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Marta Monjo ◽  
Christiane Petzold ◽  
Joana Maria Ramis ◽  
Staale Petter Lyngstadaas ◽  
Jan Eirik Ellingsen
Keyword(s):  
Gene Expression ◽  
Cell Viability ◽  
Dental Implant ◽  
Temporal Gene Expression ◽  
Full Characterization ◽  
Igf I ◽  
Implant Therapy ◽  
Osteoblast Markers

Current dental implant research aims at understanding the biological basis for successful implant therapy. The aim of the study was to perform a full characterization of the effect of two commercial titanium (Ti) surfaces, OsseoSpeed and TiOblast, on the behaviour of mouse preosteoblast MC3T3-E1 cells. The effect of these Ti surfaces was compared with tissue culture plastic (TCP). In vitro experiments were performed to evaluate cytotoxicity, cell morphology and proliferation, alkaline phosphatase activity, gene expression, and release of a wide array of osteoblast markers. No differences were observed on cell viability and cell proliferation. However, changes were observed in cell shape after 2 days, with a more branched morphology on OsseoSpeed compared to TiOblast. Moreover, OsseoSpeed surface increased BMP-2 secretion after 2 days, and this was followed by increased IGF-I, BSP, and osterix gene expression and mineralization compared to TiOblast after 14 days. As compared to the gold standard TCP, both Ti surfaces induced higher osteocalcin and OPG release than TCP and differential temporal gene expression of osteogenic markers. The results demonstrate that the gain of using OsseoSpeed surface is an improved osteoblast differentiation and mineralization, without additional effects on cell viability or proliferation.

scholarly journals Genome-wide Functional Characterization of Escherichia coli Promoters and Regulatory Elements Responsible for their Function

2020 ◽  
Author(s):  
Guillaume Urtecho ◽  
Kimberly D. Insigne ◽  
Arielle D. Tripp ◽  
Marcia Brinck ◽  
Nathan B. Lubock ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Functional Characterization ◽  
Regulatory Elements ◽  
Machine Learning Algorithms ◽  
Regulatory Sequences ◽  
Primary Sequence ◽  
Entire Genome ◽  
E Coli ◽  
Full Characterization

SummaryDespite decades of intense genetic, biochemical, and evolutionary characterizations of bacterial promoters, we still lack the basic ability to identify or predict transcriptional activities of promoters using primary sequence. Even in simple, well-characterized organisms such as E. coli there is little agreement on the number, location, and strength of promoters. Here, we use a genomically-encoded massively parallel reporter assay to perform the first full characterization of autonomous promoter activity across the E. coli genome. We measure promoter activity of >300,000 sequences spanning the entire genome and precisely map 2,228 promoters active in rich media. We show that antisense promoters have a profound effect on global transcription and how codon usage has adapted to encode intragenic promoters. Furthermore, we perform a scanning mutagenesis of 2,057 promoters to uncover regulatory sequences responsible for regulating promoter activity. Finally, we show that despite these large datasets and modern machine learning algorithms, the task of predicting promoter activity from primary sequence sequence is still challenging.

Characterization of the Epstein–Barr virus BRRF1 gene, located between early genes BZLF1 and BRLF1

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1791-1799 ◽  
Author(s):  
Carine Segouffin-Cariou ◽  
Géraldine Farjot ◽  
Alain Sergeant ◽  
Henri Gruffat
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Regulatory Elements ◽  
Barr Virus ◽  
Viral Genes ◽  
Epstein Barr

The switch from latency to a productive cycle in Epstein–Barr virus (EBV)-infected B cells proliferating in vitro is thought to be due to the transcriptional activation of two viral genes, BZLF1 and BRLF1, encoding two transcription factors called EB1 and R respectively. However, a third gene, BRRF1 is contained in the BZLF1/BRLF1 locus, overlapping with BRLF1 but in inverse orientation. We have characterized the 5′ end of the BRRF1 mRNA and the promoter, PNa, at which BRRF1 pre-mRNA is initiated. We show that although a single BRRF1 mRNA species is induced by 12-O-tetradecanoylphorbol 13-acetate/sodium butyrate in several EBV-infected B cell lines, in Akata cells treated with anti-IgG two BRRF1 mRNAs can be detected. Transcription initiated at the BRRF1 promoter was activated by EB1 but not by R, and EB1-binding sites which contribute to the EB1-activated transcription have been mapped to between positions −469 and +1. A 34 kDa protein could be translated from the BRRF1 mRNA both in vitro and in vivo, and was found predominantly in the nucleus of HeLa cells transfected with a BRRF1 expression vector. Thus there are three promoters in the region of the EBV chromatin containing the BZLF1/BRLF1 genes, two of which, PZ and PNa, potentially share regulatory elements.

Molecular characterization of the lam locus and sequences involved in regulation by the AmdR protein of Aspergillus nidulans

1992 ◽  
Vol 12 (1) ◽  
pp. 337-346
Author(s):  
I B Richardson ◽  
M E Katz ◽  
M J Hynes
Keyword(s):  
Aspergillus Nidulans ◽  
Regulatory Gene ◽  
Regulatory Elements ◽  
Specific Factor ◽  
Carbon And Nitrogen ◽  
In Vitro Binding ◽  
Vitro Binding

The lam locus of Aspergillus nidulans consists of two divergently transcribed genes, lamA and lamB, involved in the utilization of lactams such as 2-pyrrolidinone. Both genes are under the control of the positive regulatory gene amdR and are subject to carbon and nitrogen metabolite repression. The lamB gene and the region between the two genes have been sequenced, and the start points of transcription have been determined. Within the lam locus are two sequences with homology to elements, required for AmdR regulation, found in the 5' regions of the coregulated genes amdS and gatA. In vitro and in vivo assays were used to investigate the lam and gatA regulatory elements. One of the three gatA elements and one of the two lam elements were shown to bind AmdR protein in vivo and activate transcription. With a gel shift mobility assay, in vitro binding of AmdR protein to the functional gatA element was detected. Both the functional gatA and lam boxes contain within them a CAAT sequence. In vitro binding analysis indicates that a CCAAT-specific factor(s) binds at these sequences, adjacent to or overlapping the AmdR protein-binding site.

Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

2004 ◽  
Vol 287 (4) ◽  
pp. G822-G829 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Hamid M. Said
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Critical Role ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Minimal Promoter Region

Transcriptional regulation of expression of the human thiamin transporter-2 (the product of the SLC19A3 gene) is unknown. In this study, we cloned the 5′-regulatory region of the human SLC19A3 gene (2,016 bp), identified the minimal promoter region required for basal activity, demonstrated a critical role for specific cis-regulatory elements in determining the promoter activity, and confirmed activity and physiological relevance of the cloned SLC19A3 promoter in vivo. With the use of transiently transfected human intestinal epithelial Caco-2 cells and 5′-deletion analysis, the minimal promoter region required for basal activity of the SLC19A3 promoter was found to be encoded in a sequence between −77 and +59 by using the start of transcription initiation as position 1. This minimal region was found to contain a number of putative cis-regulatory elements, with a critical role for a stimulating protein-1 (SP1)/GC-box binding site (at position −48/−45 bp) established by means of mutational analysis. With the use of EMSA and supershift assays, the binding of SP1 and SP3 to the minimal promoter region was also demonstrated. In transiently transfected Drosophila SL2 cells, both SP1 and SP3 transactivated the SLC19A3 minimal promoter in a dose-dependent manner and in combination demonstrated an additive stimulatory effect. Functionality of the full-length SLC19A3 promoter was confirmed in vivo in transgenic mice expressing the promoter-luciferase reporter gene. These studies report the first characterization of the SLC19A3 promoter in vitro and in vivo and demonstrate the importance of an SP1 cis-regulatory element in regulating promoter activity of this important human gene.

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