Azmani, Z. | bioRxiv Science

bioRxiv2024

The Integrator complex prevents premature neuronal differentiation through global control of transcription elongation.

Transcription pause-release constitutes a key step in the regulation of gene expression. Pathogenic variants in various pausing regulators have been identified in neurodevelopmental disorders, but how transcription pause-release regulates neural development remains largely unknown. Here we show that a patient-specific in-frame deletion in Integrator subunit 8 (INTS8) that prevents association of this subunit with the Integrator complex results in a global increase in nascent transcription and precocious expression of neuronal genes in progenitor cells. Consequently, differentiating neural progenitors fail to sustain a proliferating progenitor cell population during cortical neuronal network formation, resulting in network collapse. We show that attenuation of RNApol2 pause-release by targeted degradation of BRD4 can revert neuronal gene activation and prevent premature progenitor cell loss. Our data demonstrate that a fine-tuned balance between RNApol2 pausing and elongation is crucial for normal neuronal development and suggest transcription pause-release as a druggable target for neurological defects in developmental disorders.

developmental biology↗

bioRxiv2021

High throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

BackgroundDNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum-concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery. ResultsThe MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell(R) RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QIAsymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (P<0.001). Vacuum-concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4, were validated by quantitative methylation specific PCR. ConclusionsThe MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.

genomics↗