Biology subjects

Young, N. L.

Publications and source records attributed to Young, N. L..

7 recordsLinked to original sources

bioRxiv2026

H2AX C-Terminal Dipeptide Truncation: A Master Switch of the DNA Damage Response.

Phosphorylation of histone H2AX at serine 139 ({gamma}H2AX) by ATM/ATR kinases is a central marker of the DNA damage response (DDR), widely used to detect DNA double-strand breaks. However, the molecular basis for tissue- and context-specific variation in {gamma}H2AX signaling remains poorly defined. Here we discover a post-translational truncation of H2AX, catalyzed by lysine demethylase 4A (KDM4A), which removes two C-terminal amino acids critical for ATM/ATR-dependent phosphorylation. This truncation renders H2AX refractory to {gamma}H2AX formation, effectively bypassing canonical DDR signaling. Truncated H2AX accumulates in select cell lines, primary cells, solid tumors, and normal tissues. Genetic knockdown or pharmacologic inhibition of KDM4A reduces H2AX truncation, restores {gamma}H2AX induction, and enhances DNA repair capacity. Conversely, KDM4A overexpression promotes H2AX truncation, impairs {gamma}H2AX signaling, and exacerbates DNA damage accumulation. This previously unrecognized regulatory axis implicates KDM4A catalyzed H2AX truncation as a superseding mechanism that represses the canonical DDR and disrupts the correlation between {gamma}H2AX and DNA damage. This dioxygenase-based protease mechanism represents a new class of proteases and is the first example of c-terminal dipeptide protein truncation. This discovery has broad implications in the basic science of genome maintenance, wound healing, cancer, combinatorial therapy, precision medicine, and technologies such as gene editing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/723272v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1b4050forg.highwire.dtl.DTLVardef@1b8333aorg.highwire.dtl.DTLVardef@2b27c5org.highwire.dtl.DTLVardef@13ff699_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Normalized and Directional Interplay Scoring for the Interrogation of Proteoform Data

Histone proteoforms, often presenting multiple co-occurring post-translational modifications (PTMs), are central to chromatin regulation and gene expression. A proteoform is a specific form of a protein that includes variations arising from genetic changes, alternative RNA splicing, proteolytic processing, and PTMs. Genomic context-dependent histone proteoforms define the histone code, influencing cellular phenotype by dictating interactions with DNA and chromatin-associated proteins. Understanding the dynamics of histone proteoforms is essential for elucidating chromatin-based regulatory mechanisms. Advances in middle-down and top-down proteomics methods enable accurate identification and quantitation of hundreds to thousands of proteoforms in a single run. However, the resulting data complexity presents significant challenges for analysis and visualization. Here, we introduce new computational methods to analyze the dynamics of histone PTMs and demonstrate their use in mouse organs during aging. We have developed and benchmarked two novel PTM crosstalk scores. The score that we term Normalized Interplay addresses limitations of the original crosstalk score Interplay providing a more complete and accurate measure of PTM crosstalk. The second score, delta I or Directional Interplay is an asymmetric measure quantifying the magnitude and directionality of crosstalk between PTMs. Applying our two-stage scoring approach to data from CrosstalkDB, a community resource that curates proteoform-level data, reveals the dynamics of histone H3 modifications during aging. The source code is available under an Apache license at https://github.com/k-p4/ptm_interplay_scoring.

biochemistry↗

bioRxiv2024

H3K18 & H3K23 acetylation directs establishment of MLL-mediated H3K4 methylation

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA, but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin "switch" governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects, and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression.

biochemistry↗

bioRxiv2023

TFEB degradation is regulated by an IKK/β-TrCP2 phosphorylation-ubiquitination cascade

Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy that plays a key role in the regulation of cellular clearance pathways. TFEB is regulated via a complex array of post-translational modifications (PTMs), but the exact molecular mechanism that regulates TFEB stability has remained elusive. Here, we show that TFEB levels are critically regulated by a defined phosphorylation-ubiquitination cascade. A human kinome screen identifies IKK (inhibitor of {kappa}B kinase) as a TFEB modifier, and a combination of phosphorylation assays, mass spectrometry analyses, and site-specific mutagenesis unveils a previously unrecognized TFEB phospho-degron (423SPFPSLS429) as the target of IKK. We show that IKK-mediated phosphorylation of TFEB triggers ubiquitination of adjacent lysine residues (K430 and K431) by the E3 ligase {beta}-TrCP2 ({beta}-Transducin repeat-containing protein 2), thereby tagging TFEB for degradation. Modified TFEB constructs that abolish these PTMs show much increased stability and expression levels but remain equally sensitive to autophagy- or stress- related stimuli while maintaining the capability to promote the expression of TFEB target genes and the clearance of Alzheimers associated tau in a cellular model of disease. Our results therefore uncover an IKK/{beta}-TrCP2 phosphorylation-ubiquitination cascade as a major mechanism that governs TFEB stability independently of other TFEB regulators.

cell biology↗

bioRxiv2023

The epigenetic response of mouse brown adipose tissue to cold stress: histone proteoform, DNA methylation, and RNA expression

Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top- down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28.8{degrees}C), mild cold/room temperature (RT, 22{degrees}C), or severe cold (SC, 8{degrees}C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression data suggest a role for epigenetic modification of DNA in gene regulation in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. Taken together, our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research.

systems biology↗

bioRxiv2022

Regulation of ATAD2B bromodomain binding activity by the histone code

The ATPase family AAA+ domain containing 2 (ATAD2) protein, and its paralog ATAD2B, have a C-terminal bromodomain that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2 and ATAD2B bromodomains to select acetylated lysine among multiple histone post-translational modifications. Isothermal titration calorimetry experiments revealed that the ATAD2 and ATAD2B bromodomains selectively recognize distinct patterns of acetylated lysine residues on the N-terminal tails of histone proteins. Adjacent methylation or phosphorylation marks were found to either enhance or weaken the recognition of acetylated lysine by the ATAD2/B bromodomains. Complementary structural studies provide mechanistic insights into how residues within the bromodomain binding pocket coordinate the acetyllysine group in the context of adjacent post- translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands, either as onco mutations or as histone variants, impact the recognition of an adjacent acetylated lysine residue. In summary, our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2 and ATAD2B bromodomains, resulting in distinct binding modes of the two bromodomains. KEY POINTSO_LIMultiple independent ATAD2 gene duplication events are evident during metazoan evolution, indicating expansion of functionality in the ATAD2 gene family and suggesting distinct functions for ATAD2 and ATAD2B. C_LIO_LIHigh-resolution structures of the ATAD2 and ATAD2B bromodomains in complex with their histone ligands demonstrate how multiple post-translational modifications are coordinated. C_LIO_LIRecognition of different subsets acetylated histone ligands by the ATAD2 and ATAD2B bromodomains is driven by unique features within the binding pockets of these paralogous proteins. C_LIO_LIOnco-histone mutations and histone variants that change the amino acid sequence of the histone tails modulate the ATAD2 and ATAD2B bromodomain activity. C_LIO_LIThis study demonstrates how the combinatorial activity of multiple post- translational modifications forms a histone code and influences the recognition of acetylated lysine by bromodomain-containing proteins. C_LI

biochemistry↗

bioRxiv2022

An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g., K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has broader extension. Here we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation chromatin switch on the H3 tail that modulates read-write accessibility in nucleosomes and resolve the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.

biochemistry↗