Biology subjects

Padhan, J.

Publications and source records attributed to Padhan, J..

4 recordsLinked to original sources

bioRxiv2025

Aromatic cage-directed azide-methyllysine photochemistry for profiling non-histone interacting partners of the MeCP2 methyl-CpG binding domain

Methyl-CpG binding protein 2 (MeCP2) is a well-characterized DNA methylation reader that has recently been shown to interact with methylated histones. MeCP2 recognizes tri-methylated lysine 27 on histone H3 (H3K27me3) through an aromatic cage within its methyl-CpG binding domain (MBD), and this interaction contributes to the regulation of MeCP2 target genes. However, whether MeCP2 can bind methylated non-histone proteins remains unknown. In this study, we sought to identify novel, aromatic cage-dependent non-histone interacting partners of MeCP2 using an unnatural amino acid-based photocrosslinking chemoproteomic strategy. We engineered the aromatic cage of MeCP2-MBD by incorporating the photocrosslinkable amino acid 4-azido-L-phenylalanine (AzF). The AzF-incorporated MeCP2-MBD variants efficiently crosslinked with histone H3 and with methylated histone marks such as H3K4me3 and H3K27me3. MeCP2-MBD-F142AzF was subsequently used to crosslink proteins from HEK293T cell lysates, and the enriched complexes were analyzed by mass spectrometry. This approach identified numerous previously unrecognized interacting partners of MeCP2, many of which are involved in key cellular processes including chromatin regulation, RNA processing, translation, and metabolism. These findings reveal that MeCP2 functions extend beyond DNA methylation reading and transcriptional repression, highlighting a broader role for MeCP2 in coordinating cellular homeostasis.

biochemistry↗

bioRxiv2025

Molecular Insights into Methyllysine Recognition by the Methyl-CpG-Binding Domain of MeCP2

Methyl-CpG binding protein 2 (MeCP2) is a DNA methylation reader, which is highly expressed in the central nervous system. Loss of function mutation of MeCP2, which impairs DNA binding, causes Rett syndrome, while gain of MeCP2 function causes MeCP2 duplication syndrome. MeCP2 functions as both transcription activator and repressor but its full range of activity remains largely unknown. Recent study suggests that beyond DNA binding, MBD domain of MeCP2 also interacts with methylated lysine residues on histone H3 tail. In this study we aimed to characterize the methyllysine binding pocket of MeCP2-MBD. Structural analysis reveals the presence of an aromatic cage formed by five residues, namely, W104, F132, Y141, F142, and F155, where the trimethyllysine of H3K27me3 binds, as confirmed in docking studies. Alanine substitution of these residues abolished binding of MeCP2-MBD with its reported ligand H3K27me3 in ITC and pull-down experiments. Genomic analysis of publicly available ChIP-seq data reveals that MeCP2 localizes with canonical histone methylation marks like H3K4me3, H3K9me3, H3K27me3 and H4K20me3 and regulate critical biological and cancer related pathways. Promoters of MeCP2 regulated genes, show signature of H3K4me3 and H3K27me3. In the genomic level, mutation of aromatic cage residue W104A, significantly reduced chromatin occupancy of MeCP2 as demonstrated by ChIP-qPCR experiments. Furthermore, our qRT-PCR experiment, shows that this aromatic cage dependent binding is necessary for regulating expression of MeCP2 target genes. Overall, our study establishes MeCP2 as a histone methylation reader and highlights the functional significance of its histone binding capacity in chromatin association and gene regulation.

biochemistry↗

bioRxiv2022

Identification of novel natural product inhibitors of BRD4 using high throughput virtual screening and MD simulation

Bromodomains are evolutionarily conserved structural motifs that recognize acetylated lysine residues on histone tails. They play a crucial role in shaping chromatin architecture and regulating gene expression in various biological processes. Mutations in bromodomains containing proteins leads to multiple human diseases, which makes them attractive target for therapeutic intervention. Extensive studies have been done on BRD4 as a target for several cancers, such as Acute Myeloid Leukemia (AML) and Burkitt Lymphoma. Several potential inhibitors have been identified against the BRD4 bromodomain. However, most of these inhibitors have drawbacks such as nonspecificity and toxicity, decreasing their appeal and necessitating the search for novel non-toxic inhibitors. This study aims to address this need by virtually screening natural compounds from the NPASS database against the Kac binding site of BRD4-BD1 using high throughput molecular docking followed by similarity clustering, pharmacokinetic screening, MD simulation, and MM-PBSA binding free energy calculations. Using this approach, we identified five natural product inhibitors having a similar or better binding affinity to the BRD4 bromodomain compared to JQ1 (previously reported inhibitor of BRD4). Further systematic analysis of these inhibitors resulted in the top three hits: NPC268484 (Palodesangren-B), NPC295021 (Candidine), and NPC313112 (Buxifoliadine-D). Collectively, our in silico results identified some promising natural products that have the potential to act as potent BRD4-BD1 inhibitors and can be considered for further validation through future in vitro and in vivo studies.

bioinformatics↗

bioRxiv2022

Molecular insights into the recognition of acetylated histone modifications by the BRPF2 bromodomain

HBO1 (HAT bound to ORC), a member of the MYST family of histone acetyltransferases (HATs), was initially identified as a binding partner of the origin recognition complex (ORC) that acetylates free histone H3, H4, and nucleosomal H3. It functions as a quaternary complex with the BRPF (BRPF1/2/3) scaffolding protein and two accessory proteins, ING4/5 and Eaf6. BRPF2 interaction with HBO1 has been shown to be important for regulating H3K14 acetylation during embryonic development. However, how the BRPF2 directs the HBO1 HAT complex to chromatin to regulate its HAT activity towards nucleosomal substrates remains unclear. Our findings reveal novel interacting partners of the BRPF2 bromodomain that recognizes different acetyllysine residues on the N-terminus of histone H4, H3, and H2A and preferentially binds to H4K5ac, H4K8ac, and H4K5acK12ac modifications. Further, mutational analysis of BRPF2 bromodomain coupled with ITC binding and pull-down assays on the histone substrates identified critical residues responsible for acetyllysine binding. Moreover, the BRPF2 bromodomain could enrich H4K5ac mark-bearing mononucleosomes compared to other acetylated H4 marks. Consistent with this, ChIP-seq analysis revealed that BRPF2 strongly co-localizes with HBO1 at histone H4K5ac and H4K8ac marks near the TSS in the genome. Together, our study provides novel insights into how the histone binding function of the BRPF2 bromodomain directs the recruitment of the HBO1 HAT complex to chromatin to regulate gene expression.

biochemistry↗