Biology subjects

Kraemer, C.

Publications and source records attributed to Kraemer, C..

4 recordsLinked to original sources

bioRxiv2026

Epigenetic splicing regulation of hTERT mediated by hTAPAS

Telomerase activity is primarily controlled by expression of the catalytic subunit hTERT, yet the mechanisms coordinating its transcriptional and post-transcriptional regulation remain incompletely understood. Paradoxically, hypermethylation of a CpG-rich region upstream of the hTERT transcription start site, termed the TERT Hypermethylated Oncological Region (THOR), is strongly associated with hTERT activation. Recent studies suggest that THOR methylation influences the expression of hTAPAS, a long non-coding RNA transcribed antisense to the hTERT promoter that represses hTERT expression. Here, we investigated the relationship between DNA methylation and alternative splicing of hTERT. Using CRISPR-Cas9-mediated targeted enrichment combined with Nanopore sequencing, we generated high-resolution DNA methylation maps across several kilobases surrounding the hTERT promoter and intronic regions encompassing exons 6-8 in multiple human cell lines. These analyses revealed distinct methylation signatures that correlate with specific hTERT splice isoform profiles. Functional perturbation experiments further demonstrated that altering the epigenetic state of the locus influences hTERT splicing. Overexpression of the antisense lncRNA hTAPAS, as well as treatment with the DNA demethylating agent 5'-Azacytidine, reduced CpG methylation at the hTERT promoter and induced a shift in hTERT splice isoform distribution. Together, our findings identify DNA methylation as an upstream regulator of hTERT alternative splicing and implicate the antisense lncRNA hTAPAS in shaping this regulatory landscape. The results point to an epigenetic mechanism linking hTAPAS to hTERT promoter methylation and splice isoform selection at the hTERT locus and provide new insight into how telomerase regulation may be remodeled during development and in cancer.

molecular biology↗

bioRxiv2024

Metabolic insights into microbially induced calcite formation by Bacillaceae for application in bio-based construction materials

Microbially induced calcite precipitation (MICP) offers promising solutions for sustainable, low-cement infrastructure materials. While it is known how urea catabolism leads to biomineralization, the non-ureolytic pathways of MICP are less clear. This limits the use of the latter in biotechnology, despite its clear benefit of avoiding toxic ammonia release. To address this knowledge gap, the present study explored the interdependence between carbon source utilization and non-ureolytic MICP. We show that acetate can serve as the carbon source driving calcite formation in several environmental Bacillaceae isolates. This effect was particularly clear in a Solibacillus silvestris strain, which could precipitate almost all provided calcium when provided with a 2:1 acetate-to-calcium molar ratio, and we show that this process was independent of active cell growth. Genome sequencing and gene expression analyses revealed an apparent link between acetate catabolism and calcite precipitation in this species, suggesting MICP may be a calcium stress response. Development of a simple genetic system for S. silvestris led to deletion of a proposed calcium binding protein, although this showed minimal effects on MICP. Taken together, this study provides insights into the physiological processes leading to non-ureolytic MICP, paving the way for targeted optimization of biomineralization for sustainable materials development.

microbiology↗

bioRxiv2022

Striated muscle-specific base editing enables correction of mutations causing dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of DCM patients harbor heritable mutations which are amenable to CRISPR-based gene therapy1. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart2. We employed a combination of the viral gene transfer vector AAVMYO with superior targeting specificity of heart muscle tissue3 and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive and arrhythmogenic DCM4. Using optimized conditions, we could improve splice defects in human iPSC-derived cardiomyocytes (iPSC-CMs) and repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restored the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reached wildtype levels. Single-nuclei RNA sequencing (snRNA-seq) uncovered restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing (WGS) revealed no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.

genetics↗

bioRxiv2022

MHC class II-restricted antigen presentation is required to prevent dysfunction of cytotoxic T cells by blood-borne myeloids in brain tumors

Cancer immunotherapy critically depends on fitness of cytotoxic and helper T cell responses. Dysfunctional cytotoxic T cell states in the tumor microenvironment (TME) are a major cause of resistance to immunotherapy. Intratumoral myeloid cells, particularly blood-borne myeloids (bbm), are key drivers of T cell dysfunction in the TME. We show here that major histocompatibility complex (MHC) class II (MHCII)-restricted antigen presentation on bbm is essential to control the growth of brain tumors. Loss of MHCII on bbm drives dysfunctional intratumoral tumor-reactive CD8+ T cell states through increased chromatin accessibility and expression of Tox, a critical regulator of T cell exhaustion. Mechanistically, MHCII-dependent activation of CD4+ T cells restricts myeloid-derived osteopontin that triggers a chronic activation of nuclear factor of activated T cells (Nfat)2 in tumor-reactive CD8+ T cells. In summary, we provide evidence that MHCII-restricted antigen presentation on bbm is a key mechanism to directly maintain functional cytotoxic T cell states in brain tumors. Highlights- MHCII on intratumoral blood-borne myeloid cells is required for sustained anti-glioma T cell response - Loss of myeloid MHCII drives dysfunction of CD8+ T cells by activating TOX - Tox expression is induced by osteopontin-triggered chronic NFAT2 signaling in tumor-reactive CD8+ T cells - steopontin production is restricted upon intratumoral T helper cell activation via MHCII - MHCII expression on tumor-infiltrating bbm correlates with osteopontin expression and cytotoxic T cell dysfunction in human glioblastoma tissue O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/495502v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1fbedc7org.highwire.dtl.DTLVardef@b7dc3aorg.highwire.dtl.DTLVardef@db7fe6org.highwire.dtl.DTLVardef@aa06b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗