Biology subjects

Melters, D. P.

Publications and source records attributed to Melters, D. P..

7 recordsLinked to original sources

bioRxiv2025

Linker Histone H1.5 Contributes to Centromere Integrity in Human Cells

Mammalian H1 linker histones comprise a group of 11 non-allelic variants which have key roles in modulating chromatin. H1 variant specific genomic distribution contributes to fine tuning regulation of gene expression and chromatin architecture. Contradictory reports on the presence and role of H1 histones at centromeres led us to further investigate whether H1s impact centromeric chromatin. In this study, we focused on H1.5 and by in vitro assays we showed that H1.5 directly interacts with centromeric-protein A (CENP-A) mononucleosomes. Notably, our in vitro findings revealed that H1 variants H1.0 and H1.2 can also bind CENP-A nucleosomes, although with differing affinities and signatures, asserting centromeric localization may not be unique to H1.5. In human cells, H1.5 localized to the centromere and chromatin immuno-precipitation revealed an interaction between H1.5 with CENP-A nucleosomes. Knocking down of H1.5 resulted in the loss of centromeric -satellite transcription, reduction in loading of new CENP-A, and the accumulation of mitotic defects. These data point to an unreported role for histone H1 in the regulation of mitotic integrity in human cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/657682v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@26a35borg.highwire.dtl.DTLVardef@aecc44org.highwire.dtl.DTLVardef@1e49939org.highwire.dtl.DTLVardef@107a485_HPS_FORMAT_FIGEXP M_FIG Graphical AbstractProposed model of H1.5 function at the centromere. When H1.5 is bound to the CENP-A nucleosomes at the centromere, the centromeric chromatin allows RNAPII based transcription to generate -satellite transcripts, which in turn allows for de novo CENP-A loading. The absence of H1.5 blocks this transcription, leading to an accumulation of mitotic defects. C_FIG

biochemistry↗

bioRxiv2024

Incorporating histone H2B variants into chromatin modifies chromatin accessibility to induce epithelial to mesenchymal transition in breast cancer

Histones scaffold genomic DNA and regulate access to the transcriptional machinery. However, naturally occurring histone variants can alter histone-DNA interactions, DNA and histone modifications, and the chromatin interactome. Hence, alterations in histone variant deposition can disrupt chromatin, and are increasingly recognized as a way to trigger various disease, including cancer. While significant attention has been placed on the biochemical and functional roles of H2A, H3, and H4 histone variants, the variants of H2B remain largely understudied. Here, we show that H2B variants are dysregulated in breast cancer and that certain variants are associated with specific breast cancer subtypes. HIST1H2BO overexpression (in particular) is more common in Asian, African American/Black, and young female populations and is associated with a worse prognosis. In vitro studies show that H2B1O compacts nucleosome structure. Incorporating H2B1O into chromatin activates pro-inflammatory and oncogenic pathways, induces the epithelial-to-mesenchymal transition (EMT), and generates resistance to first-line chemotherapeutic agents. Thus, H2B1O acts much like an onco-histone, with H2B variant expression being a prognostic biomarker for breast cancer and a potential new target for drug therapies to enhance treatment efficacy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/627414v1_ufig1.gif" ALT="Figure 1"> View larger version (106K): org.highwire.dtl.DTLVardef@12c21dcorg.highwire.dtl.DTLVardef@631f3aorg.highwire.dtl.DTLVardef@1972244org.highwire.dtl.DTLVardef@14ab3b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Aberrant expression of histone H2B variants reshape chromatin and alter oncogenic gene expression programs

Chromatin architecture governs DNA accessibility and gene expression. Thus, any perturbations to chromatin can significantly alter gene expression programs and promote disease. Prior studies demonstrate that every amino acid in a histone is functionally significant, and that even a single amino acid substitution can drive specific cancers. We previously observed that naturally occurring H2B variants are dysregulated during the epithelial to mesenchymal transition (EMT) in bronchial epithelial cells. Naturally occurring H2B variants differ from canonical H2B by only a few amino acids, yet single amino acid changes in other histone variants (e.g., H3.3) can drive cancer. We therefore hypothesized that H2B variants might function like oncohistones, and investigated how they modify chromatin architecture, dynamics, and function. We find that H2B variants are frequently dysregulated in many cancers, and correlate with patient prognosis. Despite high sequence similarity, mutations in each H2B variant tend to occur at specific "hotspots" in cancer. Some H2B variants cause tighter DNA wrapping around nucleosomes, leading to more compact chromatin structures and reduced transcription factor accessibility to nucleosomal DNA. They also altered genome-wide accessibility to oncogenic regulatory elements and genes, with concomitant changes in oncogenic gene expression programs. Although we did not observe changes in cell proliferation or migration in vitro, our Gene Ontology (GO) analyses of ATAC-seq peaks and RNA-seq data indicated significant changes in oncogenic pathways. These findings suggest that H2B variants may influence early-stage, cancer-associated regulatory mechanisms, potentially setting the stage for oncogenesis later on. Thus, H2B variant expression could serve as an early cancer biomarker, and H2B variants might be novel therapeutic targets.

cancer biology↗

bioRxiv2023

A hyper-quiescent chromatin state formed during aging is reversed by regeneration

Epigenetic alterations are a key hallmark of aging but have been limitedly explored in tissues. Here, using naturally aged murine liver as a model and extending to other quiescent tissues, we find that aging is driven by temporal chromatin alterations that promote a refractory cellular state and compromise cellular identity. Using an integrated multi-omics approach, and the first direct visualization of aged chromatin we find that globally, old cells show H3K27me3-driven broad heterochromatinization and transcription suppression. At the local level, site-specific loss of H3K27me3 over promoters of genes encoding developmental transcription factors leads to expression of otherwise non-hepatocyte markers. Interestingly, liver regeneration reverses H3K27me3 patterns and rejuvenates multiple molecular and physiological aspects of the aged liver.

genomics↗

bioRxiv2022

Mobility of nucleosomes is regulated by their binding partners

Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated in the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about chromatin accessibility in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the MSD and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. Thus, changes which alter chromatin accessibility in vitro, also correspondingly alter transcription in vivo. These data suggest a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance mobility.

biophysics↗

bioRxiv2019

CENP-C stabilizes the conformation of CENP-A nucleosomes within the inner kinetochore at human centromere

The centromere is the chromosomal locus that seeds the kinetochore, allowing for a physical connection between the chromosome and the mitotic spindle. At the heart of the centromere is the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle the constitutive centromere associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome dynamics in vivo is unknown. Here, we purify kinetochore associated native centromeric chromatin and analyze its biochemical features using a combinatorial approach. We report that kinetochore bound chromatin has strongly reduced DNA accessibility and a distinct stabilized nucleosomal configuration. Disrupting the balance between CENP-A and CENP-C result in reduced centromeric occupancy of RNA polymerase 2 and impaired de novo CENP-A loading on the centromeric chromatin fiber, correlating with significant mitotic defects. CENP-A mutants that restore the ratio rescue the mitotic defects. These data support a model in which CENP-C bound centromeric nucleosomes behave as a barrier to the transcriptional machinery and suggest that maintaining the correct ratio between CENP-A and CENP-C levels is critical for centromere homeostasis.

biochemistry↗

bioRxiv2018

Elastic and Rigidified CENP-A Nucleosomes govern Centromeric Chromatin Plasticity

Histone variants fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether and how nucleosome variants encode unique mechanical properties to their cognate chromatin structures remains elusive. Here, using novel in silico and in vitro nanoindentation methods, extending to in vivo dissections, we report that histone variant nucleosomes are intrinsically more elastic than their canonical counterparts. Furthermore, binding proteins which discriminate between histone variant nucleosomes suppress this innate elasticity and also compact chromatin. Interestingly, when we overexpress the binding proteins in vivo, we also observe increased compaction of chromatin enriched for histone variant nucleosomes, correlating with diminished access. Together, these data suggest a plausible link between innate mechanical properties possessed by histone variant nucleosomes, the adaptability of chromatin states in vivo, and the epigenetic plasticity of the underlying locus.\n\nSignificanceNucleosomes are the base unit which organize eukaryotic genomes. Besides the canonical histone, histone variants create unique local chromatin domains that fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. We developed computational and single-molecule nanoindentation tools to determine mechanical properties of histone variant nucleosomes. We found that the CENP-A nucleosome variant is more elastic than the canonical H3 nucleosome but becomes stiffer when bound to its partner CENP-C. In addition, CENP-C induces cross-array clustering, creating a chromatin state that less accessible. These data suggest that innate material properties of nucleosomes can influence the ultimate chromatin state, thereby influence biological outcomes.

biochemistry↗