Biology subjects

Fleck, K.

Publications and source records attributed to Fleck, K..

5 recordsLinked to original sources

bioRxiv2026

Developmental expression of the skeletal muscle determination gene, MyoD, is regulated by novel enhancer elements that interact with the core enhancer and distal regulatory region

MyoD plays a central role in determining the skeletal muscle lineage in vertebrate embryos. The core enhancer (CE) and distal regulatory region (DRR) are the only known MyoD enhancers, and together, they recapitulate all major aspects of MyoD expression in the embryo. However, knocking out each enhancer individually has only modest effects on MyoD expression. Here, we show that embryos lacking both enhancers maintain muscle-specific MyoD expression, indicating the existence of unknown MyoD regulatory elements. Precision run-on sequencing together with available ChIP-seq and DNase I hypersensitivity datasets identified three new candidate enhancer regions within 96 kb of MyoD 5 flanking sequences. Transgenic analysis revealed that DNA elements at -36 and -60 kb are active in all muscle-forming regions, each recapitulating aspects of endogenous MyoD expression. Enhancer activities in muscle regulatory factor-deficient mice suggest that they are components of the auto- and cross-regulatory circuitry that maintains MyoD expression. Analysis of Hi-C data showed that the entire -96 kb region constitutes a loop domain, within which multiple interactions between elements and with the MyoD gene were detected. A larger loop domain delimited by CTCF sites was also identified from -96 kb to +220 kb relative to the MyoD transcriptional start site. These data indicate that the newly identified enhancers are key components of a cis regulatory network that controls the activation and maintenance of MyoD expression in the embryo. One sentence summaryCis regulation of MyoD transcription during development

developmental biology↗

bioRxiv2025

Disentangling spatial organization and splicing of rare intron classes in the human genome

Three-dimensional (3D) genome organization influences transcription and RNA processing, yet how the spatial positioning of genes contributes to pre-mRNA splicing has only recently come into focus. Despite these advances, it remains unclear how introns, particularly rare intron classes, are organized within the 3D genome and whether this organization influences their splicing. Here, we mapped the spatial organization of six intron classes including major, minor, minor-like, hybrid, major-like and non-canonical across four human cell lines (K562, H1, HCT116, and HFFc6) using Hi-C, TSA-seq, and DamID-seq data. This revealed minor intron enrichment in active compartments A and speckle-associated domains (SPADs) and depletion from lamina-associated domains (LADs), whereas hybrid and non-canonical introns showed the opposite trend. Integrating TSA-seq with RNA-seq data suggested that splicing efficiency depends on intron identity rather than nuclear positioning. For example, major-like, minor-like, and non-canonical introns in SPADs were less efficiently spliced than major and minor introns despite their proximity to nuclear speckles. These patterns were consistent across cancer (K562, HCT116) and stem cells (H1) but not fibroblasts (HFFc6). Comparison of minor intron splicing in and out of SPADs across cell lines revealed that, relative to fibroblasts, minor introns outside of SPADs in cancer cells were more efficiently spliced. This suggests that increased efficiency of minor intron splicing in cancer cell lines is not necessarily due to 3D positioning. In all, these findings reveal that intron subclasses show distinct nuclear organization, yet for minor introns, identity rather than position governs splicing efficiency.

bioinformatics↗

bioRxiv2024

Functional associations of evolutionarily recent human genes exhibit sensitivity to the 3D genome landscape and disease

Genome organization is intricately tied to regulating genes and associated cell fate decisions. Here, we examine the positioning and functional significance of human genes, grouped by their lineage restriction level, within the 3D organization of the genome. We reveal that genes of different lineage restriction levels have distinct positioning relationships with both domains and loop anchors, and remarkably consistent relationships with boundaries across cell types. While the functional associations of each group of genes are primarily cell type-specific, associations of conserved genes maintain greater stability across 3D genomic features and disease than recently evolved genes. Furthermore, the expression of these genes across various tissues follows an evolutionary progression, such that RNA levels increase from young lineage restricted genes to ancient genes present in most species. Thus, the distinct relationships of gene evolutionary age, function, and positioning within 3D genomic features contribute to tissue-specific gene regulation in development and disease.

evolutionary biology↗

bioRxiv2022

An apicomplexan bromodomain, TgBDP1 associates with diverse epigenetic factors to regulate essential transcriptional processes in Toxoplasma gondii.

The protozoan pathogen Toxoplasma gondii relies on tight regulation of gene expression to invade and establish infection in its host. The divergent gene regulatory mechanisms of Toxoplasma and related apicomplexan pathogens rely heavily on regulators of chromatin structure and histone modifications. The important contribution of histone acetylation for Toxoplasma in both acute and chronic infection has been demonstrated, where histone acetylation increases at active gene loci. However, the direct consequences of specific histone acetylation marks and the chromatin pathway that influences transcriptional regulation in response to the modification is unclear. As a reader of lysine acetylation, the bromodomain serves as a mediator between the acetylated histone and transcriptional regulators. Here we show that the bromodomain protein TgBDP1 which is conserved amongst Apicomplexa and within the Alveolata superphylum, is essential for Toxoplasma asexual proliferation. Using CUT&TAG we demonstrate that TgBDP1 is recruited to transcriptional start sites of a large proportion of parasite genes. Transcriptional profiling during TgBDP1 knockdown revealed that loss of TgBDP1 leads to major dysregulation of gene expression, implying multiple roles for TgBDP1 in both gene activation and repression. This is supported by interactome analysis of TgBDP1 demonstrating that TgBDP1 forms a core complex with two other bromodomain proteins and an ApiAP2 factor. This core complex appears to interact with other epigenetic factors such as nucleosome remodelling complexes. We conclude that TgBDP1 interacts with diverse epigenetic regulators to exert opposing influences on gene expression in the Toxoplasma tachyzoite. SummaryHistone acetylation is critical for proper regulation of gene expression in the single celled eukaryotic pathogen Toxoplasma gondii. Bromodomain proteins are "readers" of histone acetylation and may link the modified chromatin to transcription factors. Here, we show that the bromodomain protein TgBDP1 is essential for parasite survival and that loss of TgBDP1 results in global dysregulation of gene expression. TgBDP1 is recruited to the promoter region of a large proportion of parasite genes, forms a core complex with two other bromodomain proteins and interacts with different transcriptional regulatory complexes. We conclude that TgBDP1 is a key factor for sensing specific histone modifications to influence multiple facets of transcriptional regulation in Toxoplasma gondii.

microbiology↗

bioRxiv2020

Uncovering temporally sensitive targeting motifs for traumatic brain injury via phage display

The heterogeneous injury pathophysiology of traumatic brain injury (TBI) is a barrier to developing highly sensitive and specific diagnostic tools. Embracing neural injury complexity is critical for the development and advancement of diagnostics and therapeutics. The current study employs a unique discovery pipeline to identify targeting motifs that recognize specific phases of TBI pathology. This pipeline entails in vivo biopanning with a domain antibody (dAb) phage display library, next generation sequencing (NGS) analysis, and peptide synthesis. Here, we identify targeting motifs based on the HCDR3 structure of dAbs for acute (1 day) and subacute (7 days) post-injury timepoints using a mouse controlled cortical impact model. Their bioreactivity was validated using immunohistochemistry and candidate target epitopes were identified via immunoprecipitation-mass spectrometry. The acute targeting motif recognizes targets associated with metabolic and mitochondrial dysfunction whereas the subacute motif was largely associated with neurodegenerative processes. This phage display biomarker discovery pipeline for TBI successfully achieved discovery of temporally specific TBI targeting motif/epitope pairs that will advance the TBI diagnostics and therapeutics.

bioengineering↗