Biology subjects

Banaszynski, L. A.

Publications and source records attributed to Banaszynski, L. A..

5 recordsLinked to original sources

bioRxiv2025

A transcriptional biosensor reveals mechanisms of α-ketoglutarate signaling to chromatin

Alpha-ketoglutarate (KG) is required for chromatin demethylation but mechanisms controlling KG abundance in the nucleus are poorly defined. Therefore, we designed a biosensor system to monitor this metabolite pool in human cells using an KG-responsive cyanobacterial transcription factor, NtcA. We then coupled this system with a genetic screen to identify genes that regulate KG in the nucleus, defining an inter-organelle pathway in which sequential mitochondrial activities of the GPT2 transaminase and SLC25A11 transporter supply nuclear KG. Using a mouse model of GPT2 deficiency, a human inborn error of metabolism, we found that this pathway controls chromatin methylation in the developing brain. Our work provides a tool to assess KG signaling to chromatin and a framework for leveraging forward genetics to study nuclear metabolite pools.

synthetic biology↗

bioRxiv2023

G1 length dictates heterochromatin landscape

Stem cells have lower facultative heterochromatin as defined by trimethylation of histone H3 lysine 27 (H3K27me3) compared to differentiated cells. However, the mechanisms underlying these differential H3K27me3 levels remain elusive. Because H3K27me3 levels are diluted two-fold in every round of replication and then restored through the rest of the cell cycle, we reasoned that the cell cycle length could be a key regulator of total H3K27me3 levels. Here, we propose that a fast cell cycle restricts H3K27me3 levels in stem cells. To test this model, we determined changes to H3K27me3 levels in mESCs globally and at specific loci upon G1 phase lengthening - accomplished by thymidine block or growth in the absence of serum (with the "2i medium"). H3K27me3 levels in mESC increase with G1 arrest when grown in serum and in 2i medium. Additionally, we observed via CUT&RUN and ChIP-seq that regions that gain H3K27me3 in G1 arrest and 2i media overlap, supporting our model of cell cycle length as a critical regulator of the stem cell epigenome and cellular identity. Furthermore, we demonstrate the inverse effect - that G1 shortening in differentiated cells results in a loss of H3K27me3 levels. Finally, in tumor cells with extreme H3K27me3 loss, lengthening of the G1 phase leads to H3K27me3 recovery despite the presence of the dominant negative, sub-stoichiometric H3.1K27M mutation. Our results indicate that G1 length is an essential determinant of H3K27me3 landscapes across diverse cell types.

molecular biology↗

bioRxiv2022

H3.3 contributes to chromatin accessibility and transcription factor binding at promoter-proximal regulatory elements

BackgroundThe histone variant H3.3 is enriched at active regulatory elements such as promoters and enhancers in mammalian genomes. These regions are highly accessible, creating an environment that is permissive to transcription factor binding and the recruitment of transcriptional coactivators that establish a unique chromatin post-translational landscape. How H3.3 contributes to the establishment and function of chromatin states at these regions is poorly understood. ResultsWe performed genomic analyses of features associated with active promoter chromatin in mouse embryonic stem cells (ESCs) and found evidence of subtle yet widespread promoter dysregulation in the absence of H3.3. Loss of H3.3 deposition at promoters reduces chromatin accessibility and transcription factor (TF) footprinting for nearly all TFs expressed in ESCs. H3.3 deletion leads to reduced promoter enrichment of the transcriptional coactivator and histone acetyltransferase, p300. Subsequently, histone H3 acetylation at lysine 27 (H3K27ac) is reduced at promoters in the absence of H3.3, along with reduced enrichment of the bromodomain-containing protein BRD4, an acetyl lysine reader. Despite the observed chromatin dysregulation, H3.3 KO ESCs maintain transcription from ESC-specific genes. However, upon undirected differentiation, H3.3 KO cells retain footprinting of ESC-specific TFs motifs and fail to generate footprints of lineage-specific TF motifs, in line with their diminished capacity to differentiate. ConclusionsH3.3 facilitates DNA accessibility, TF binding, and histone post-translational modification at active promoters. While H3.3 is not required for maintaining transcription in ESCs, it is required for TF binding at new promoters during differentiation.

genomics↗

bioRxiv2022

Loss of heterochromatin at endogenous retroviruses creates competition for transcription factor binding

The mammalian genome is partitioned into active and inactive regions, broadly termed euchromatin and heterochromatin, respectively. The majority of heterochromatin consists of repetitive elements, including endogenous retroviruses (ERVs). ERVs are enriched in regulatory elements containing transcription factor (TF) binding sites with individual families containing hundreds to thousands of distinct copies scattered throughout the genome. We hypothesized that epigenetic derepression of ERVs (such as that observed during early development) may alter the stoichiometry between TFs and their euchromatic target sites, with ERVs effectively competing for these factors. To test this, we modeled acute heterochromatin loss using inducible deletion of the co-repressor KAP1 in mouse embryonic stem cells (ESCs). Upon KAP1 deletion, we observe clear reductions in chromatin accessibility, histone acetylation, and TF binding at euchromatic regions. To directly test the concept of global binding site competition, we designed exogenous binding site arrays (EBSAs) to introduce upwards of 1500 copies of the OCT4 TF binding motif into ESCs. OCT4 EBSAs specifically reduce chromatin accessibility at POU family motifs and result in reduced transcription of the pluripotency machinery with subsequent differentiation. Overall, these data support a model in which heterochromatin at ERVs promotes euchromatic TF binding and transcriptional homoeostasis. We propose that regulated ERV derepression during pre-implantation may serve as a developmental siphon to weaken the robustness of ongoing transcription programs in favor of the plasticity required for cell fate specification.

genomics↗

bioRxiv2022

A cell cycle-linked mechanism for the glutamine driven establishment of stem cell fate

The cell cycle offers a unique opportunity for stem cells to sample metabolic and signaling cues to establish cell fate. Molecular pathways that integrate and convey these signals to cell cycle machinery to license cell fate transitions and drive terminal differentiation remain unknown. Here, we describe a signaling role of mitochondrial glutamine metabolism in driving exit from cell cycle-linked self-renewal to generate differentiation competent progenitors. In proliferating stem cells, mitochondrial glutamine metabolism opposes the WDR5-linked self-renewal network via acetylation and nuclear translocation of its upstream regulator, PASK. Nuclear PASK disrupts the mitotic WDR5-anaphase-promoting complex (APC/C) interaction to drive exit from self-renewal. Consistent with these roles, loss of PASK or inhibition of glutamine metabolism preserves stemness in vitro and in vivo during muscle regeneration. Our results suggest a mechanism whereby the proliferative functions of glutamine metabolism are co- opted by stem cells to establish cell fate.

cell biology↗