Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) share key features, including accumulation of the RNA-binding protein TDP-43. TDP-43 regulates RNA homeostasis, but it remains unclear whether RNA stability is affected in these disorders. We use Bru-seq and BruChase-seq to assess genome-wide RNA stability in ALS patient-derived cells, demonstrating profound destabilization of ribosomal and mitochondrial transcripts. This pattern is recapitulated by TDP-43 overexpression, suggesting a primary role for TDP-43 in RNA destabilization, and in postmortem samples from ALS and FTD patients. Proteomics and functional studies illustrate corresponding reductions in mitochondrial components and compensatory increases in protein synthesis. Collectively, these observations suggest that TDP-43 deposition leads to targeted RNA instability in ALS and FTD, and may ultimately cause cell death by disrupting energy production and protein synthesis pathways.

Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3’ end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3’ processing are deployed across human tissues, with nearly half of multitranscript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that promotes stemness and cell survival in cancers such as prostate cancer. Most prostate malignancies are adenocarcinomas with luminal differentiation. However, some tumors undergo cellular reprogramming to a more lethal subset termed neuroendocrine prostate cancer (NEPC) with neuronal differentiation. The frequency of NEPC is increasing since the widespread use of potent androgen receptor signaling inhibitors. Currently, there are no effective treatments for NEPC. We previously determined that LSD1 promotes survival of prostate adenocarcinoma tumors. However, the role of LSD1 in NEPC is unknown. Here, we determined that LSD1 is highly upregulated in NEPC versus adenocarcinoma patient tumors. LSD1 suppression with RNAi or allosteric LSD1 inhibitors — but not catalytic inhibitors — reduced NEPC cell survival. RNA-Seq analysis revealed that LSD1 represses pathways linked to luminal differentiation, and TP53 was the top reactivated pathway. We confirmed that LSD1 suppressed the TP53 pathway by reducing TP53 occupancy at target genes while LSD1’s catalytic function was dispensable for this effect. Mechanistically, LSD1 inhibition disrupted LSD1-HDAC interactions, increasing histone acetylation at TP53 targets. Finally, LSD1 inhibition suppressed NEPC tumor growth in vivo. These findings suggest that blocking LSD1’s noncatalytic function may be a promising treatment strategy for NEPC.

Lysine (K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in pancreatic ductal adenocarcinoma (PDAC). However, the impact of KDM6A loss on the PDAC tumor immune microenvironment is not known. This study used a genetically engineered, pancreas-specific Kdm6a knockout (KO) PDAC mouse model and human PDAC tissue samples to demonstrate that KDM6A loss correlates with increased tumor-associated neutrophils and neutrophil extracellular traps (NET) formation, which are known to contribute to PDAC progression. Genome-wide bromouridine sequencing analysis to evaluate nascent RNA synthesis showed that the expression of many chemotactic cytokines, especially CXC motif chemokine ligand 1 (CXCL1), was upregulated in KDM6A KO PDAC cells. KDM6A-deficient PDAC cells secreted higher levels of CXCL1 protein, which in turn recruited neutrophils. Furthermore, in a syngeneic orthotopic mouse model, treatment with a CXCL1 neutralizing antibody blocked the chemotactic and NET-promoting properties of KDM6A-deficient PDAC cells and suppressed tumor growth, confirming CXCL1 as a key mediator of chemotaxis and PDAC growth driven by KDM6A loss. These findings shed light on how KDM6A regulates the tumor immune microenvironment and PDAC progression and suggests that the CXCL1–CXCR2 axis may be a candidate target in PDAC with KDM6A loss. Significance: KDM6A loss in pancreatic cancer cells alters the immune microenvironment by increasing CXCL1 secretion and neutrophil recruitment, providing a rationale for targeting the CXCL1–CXCR2 signaling axis in tumors with low KDM6A.