Biology subjects

Whedon, S. D.

Publications and source records attributed to Whedon, S. D..

3 recordsLinked to original sources

bioRxiv2026

Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

Repressive histone methyltransferases carry a catalytic ("write") domain and a separate domain specialized for recognizing ("reading") the reaction product. This read-write configuration acts as a positive feedback mechanism for epigenetic maintenance and the growth of repressive chromatin domains. Feedback exhibits as catalytic stimulation and is understood to act towards a proximal (trans) nucleosome. Whether this stimulation affects a specific methylation transition and whether it is restricted to trans-stimulation remains opaque. Here, we dissect the positive feedback in the heterodimeric histone 3 lysine 9 (H3K9) mono- and dimethlyase G9a-GLP, which carries two catalytic SET and two product-reading Ankyrin repeat (ANK) domains. We find that reading by both ANK domains is required for H3K9 di-, but not monomethylation on nucleosomes and for tight binding to them. As this read-writing occurs on dilute mononucleosomes, we propose that intranucleosomal feedback occurs for G9a-GLP. Swapping the ANK domains results in loss of dimethylation while maintaining nucleosome binding, indicating catalytic coupling of nucleosome methylation intermediates to reading. Crosslinking mass spectrometry reveals specific G9a surfaces that contact nucleosomal methylation intermediates. Structural approaches reveal how these surfaces position the G9a ANK domain on the methylation-intermediate nucleosome and stabilize G9a-GLP on chromatin during the reaction.

biochemistry↗

bioRxiv2025

Balancing Activation and Repression: CoREST-p300 Antagonism Controls Retinoic Acid-Driven Differentiation in AML

The histone demethylase KDM1A (LSD1), a component of the CoREST corepressor complex, is highly expressed in hematologic malignancies and regulates hematopoietic differentiation. Despite its essential developmental role, LSD1 inhibition has emerged as a promising strategy to enhance retinoic acid (RA)-responsive gene expression in subsets of acute myeloid leukemia (AML). Here, we show that LSD1 physically interacts with RAR/RXR heterodimers at specific genomic loci, restricting chromatin accessibility and transcriptional activation of differentiation programs. Single-agent inhibition of LSD1 or HDACs promotes only partial differentiation. In contrast, Corin, a dual LSD1/CoREST inhibitor, synergizes with all-trans retinoic acid (ATRA) to induce robust myeloid differentiation and apoptosis. Corin treatment alone does not significantly increase H3K4me3 levels; however, in combination with ATRA, it disrupts CoREST-RAR/RXR complexes and facilitates the recruitment of the coactivator p300. Together, they shift chromatin to an active state, enhancing H3K4me3 via increased transcriptional engagement and coactivator recruitment. Our findings identify the functional antagonism between CoREST and p300 as a regulatory axis of RA signaling in AML. Targeting this mechanism with Corin and ATRA re-sensitizes non-APL AML cells to RA-induced differentiation, suggesting a broader therapeutic approach for overcoming resistance in ATRA-refractory leukemias. SignificanceCo-inhibition of LSD1 and HDACs with Corin, combined with ATRA, reprograms chromatin by replacing CoREST with p300, uniquely activating differentiation-associated genes and offering a targeted strategy for treating AML. Graphical abstract created with BioRender (https://biorender.com). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/682235v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1216c81org.highwire.dtl.DTLVardef@15ac53aorg.highwire.dtl.DTLVardef@4a96d2org.highwire.dtl.DTLVardef@f3caaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

A circular engineered sortase for interrogating histone H3 in chromatin

Reversible modification of the histone H3 N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex "cut-and-paste" middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetics discovery and therapeutic development.

biochemistry↗