Biology subjects

Sturgill, D.

Publications and source records attributed to Sturgill, D..

8 recordsLinked to original sources

bioRxiv2024

U2AF regulates the translation and localization of nuclear-encoded mitochondrial mRNAs

The mechanisms underlying molecular targeting to mitochondria remain enigmatic, yet this process is crucial for normal cellular function. The RNA binding proteins U2AF1/2 form a heterodimer (U2AF) that shuttles between the nucleus and cytoplasm, regulating splicing in the nucleus and translation in the cytoplasm. Our study identifies an unexpected role for U2AF in mitochondrial function. We demonstrate that U2AF interacts with nuclear-transcribed mitochondrial mRNAs and proteins, inhibits translation, localizes to the outer mitochondrial membrane, and regulates mRNA localization to mitochondria. Moreover, an oncogenic point-mutation in U2AF1(S34F) disrupts this regulation, leading to altered mitochondrial structure, increased translation, and OXPHOS-dependent metabolic rewiring, recapitulating changes observed in bone marrow progenitors from patients with myelodysplastic syndromes. These findings reveal a non-canonical role for U2AF, where it modulates multiple aspects of mitochondrial function by regulating the translation and mitochondrial targeting of nuclear-encoded mRNAs.

molecular biology↗

bioRxiv2024

Predictive Prioritization of Enhancers Associated with Pancreas Disease Risk

Genetic and epigenetic variations in regulatory enhancer elements increase susceptibility to a range of pathologies. Despite recent advances, linking enhancer elements to target genes and predicting transcriptional outcomes of enhancer dysfunction remain significant challenges. Using 3D chromatin conformation assays, we generated an extensive enhancer interaction dataset for the human pancreas, encompassing more than 20 donors and five major cell types, including both exocrine and endocrine compartments. We employed a network approach to parse chromatin interactions into enhancer-promoter tree models, facilitating a quantitative, genome-wide analysis of enhancer connectivity. With these tree models, we developed a machine learning algorithm to estimate the impact of enhancer perturbations on cell type-specific gene expression in the human pancreas. Orthogonal to our computational approach, we perturbed enhancer function in primary human pancreas cells using CRISPR interference and quantified the effects at the single-cell level through RNA FISH coupled with high-throughput imaging. Our enhancer tree models enabled the annotation of common germline risk variants associated with pancreas diseases, linking them to putative target genes in specific cell types. For pancreatic ductal adenocarcinoma, we found a stronger enrichment of disease susceptibility variants within acinar cell regulatory elements, despite ductal cells historically being assumed as the primary cell-of-origin. Our integrative approach--combining cell type-specific enhancer-promoter interaction mapping, computational models, and single-cell enhancer perturbation assays--produced a robust resource for studying the genetic basis of pancreas disorders.

genomics↗

bioRxiv2020

Activation of the Crtc2/Creb1 transcriptional network in skeletal muscle enhances weight loss during intermittent fasting

The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc 2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, supported by maintenance of higher energy expenditure during the fast. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

physiology↗

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

TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide

The mechanisms supporting dynamic regulation of CTCF binding sites remain poorly understood. Here we describe the TET-catalyzed 5-methylcytosine derivative, 5-carboxylcytosine (5caC) as a factor driving new CTCF binding within genomic DNA. Through a combination of in vivo and in vitro approaches, we reveal that 5caC generally strengthens CTCF association with DNA and facilitates binding to suboptimal sequences. Dramatically, profiling of CTCF binding in a cellular model that accumulates genomic 5caC identified [~]13,000 new CTCF sites. The new sites were enriched for overlapping 5caC and were marked by an overall reduction in CTCF motif strength. As CTCF has multiple roles in gene expression, these findings have wide-reaching implications and point to induced 5caC as a potential mechanism to achieve differential CTCF binding in cells.

molecular biology↗

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↗

bioRxiv2017

Independence Between Pre-mRNA Splicing And De Novo DNA Methylation In An Isogenic Minigene Resource

Actively transcribed genes adopt a unique chromatin environment with characteristic patterns of enrichment. Within gene bodies, H3K36me3 and cytosine DNA methylation are elevated at exons of spliced genes and have been implicated in the regulation of pre-mRNA splicing. H3K36me3 is further responsive to splicing, wherein splicing inhibition led to a redistribution and general reduction over gene bodies. In contrast, little is known of the mechanisms supporting elevated DNA methylation at actively spliced genic locations. Recent evidence associating the de novo DNA methyltransferase Dnmt3b with H3K36me3-rich chromatin raises the possibility that genic DNA methylation is influenced by splicing-associated H3K36me3. Here, we report the generation of an isogenic resource to test the direct impact of splicing on chromatin. A panel of minigenes of varying splicing potential were integrated into a single FRT site for inducible expression. Profiling of H3K36me3 confirmed the established relationship to splicing, wherein levels were directly correlated with splicing efficiency. In contrast, DNA methylation was equivalently detected across the minigene panel, irrespective of splicing and H3K36me3 status. In addition to revealing a degree of independence between genic H3K36me3 and DNA methylation, these findings highlight the generated minigene panel as a flexible platform for the query of splicing-dependent chromatin modifications.

molecular biology↗

bioRxiv2017

CENP-A associated lncRNAs influence chromosome segregation in human cells

Transcription occurs ubiquitously throughout non-coding parts of the genome, including at repetitive -satellite DNA elements which comprise the majority of human centromeres. The function of temporally regulated centromeric transcription, and transcripts, is consequently a topic of intense investigation. In this study, we use high throughput approaches to identify and describe lncRNAs associated with the centromere specific histone variant CENP-A that arise from the transcription of specific centromeres at early G1, which we then show are physically associated with centromeres, and which are functionally necessary for accurate chromosome segregation. Targeted depletion of one such centromeric RNA, which originates from a single centromere, is sufficient to increase the frequency of chromosome segregation defects. These data support the emerging paradigm of the necessity of centromere-specific lncRNAs in the integrity of faithful chromosome segregation.

cell biology↗