Early detection of pancreatic cancer: Study design and analytical considerations in biomarker discovery and early phase validation studies.

Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease that is challenging to detect at an early stage. Biomarkers are needed that can detect PDAC early in the course of disease when interventions lead to the best outcomes. We highlight study design and statistical considerations that inform pancreatic cancer early detection biomarker evaluation.

Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programmes and repeats in pluripotent cells.

H3K9me3 heterochromatin, established by lysine methyltransferases (KMTs) and compacted by heterochromatin protein 1 (HP1) isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3 heterochromatin stability is presently limited to individual domains and DNA repeats. Here we engineered Suv39h2-knockout mouse embryonic stem cells to degrade remaining two H3K9me3 KMTs within 1 hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours.

Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programs and repeats in pluripotent cells.

H3K9me3-heterochromatin, established by lysine methyltransferases (KMTs) and compacted by HP1 isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3-heterochromatin stability is presently limited to individual domains and DNA repeats. We engineered KO mouse embryonic stem cells to degrade remaining two H3K9me3-KMTs within one hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours.

The yin and yang of pioneer transcription factors: Dual roles in repression and activation.

Pioneer transcription factors, by virtue of their ability to target nucleosomal DNA in silent chromatin, play crucial roles in eliciting cell fate decisions during development and cellular reprogramming. In addition to their well-established role in chromatin opening to activate gene expression programs, recent studies have demonstrated that pioneer factors have the complementary function of being able to silence the starting cell identity through targeted chromatin repression. Given recent discoveries regarding the repressive aspect of pioneer function, we discuss the basis by which pioneer factors can suppress alternative lineage programs in the context of cell fate control.

Heterochromatin protein ERH represses alternative cell fates during early mammalian differentiation.

During development, H3K9me3 heterochromatin is dynamically rearranged, silencing repeat elements and protein coding genes to restrict cell identity. Enhancer of Rudimentary Homolog (ERH) is an evolutionarily conserved protein originally characterized in fission yeast and recently shown to be required for H3K9me3 maintenance in human fibroblasts, but its function during development remains unknown. Here, we show that ERH is required for proper segregation of the inner cell mass and trophectoderm cell lineages during mouse development by repressing totipotent and alternative lineage programs.

Cellular reprogramming in vivo initiated by SOX4 pioneer factor activity.

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that SOX4 is sufficient to initiate hepatobiliary metaplasia in the adult mouse liver, closely mimicking metaplasia initiated by toxic damage to the liver.

TXNRD1 drives the innate immune response in senescent cells with implications for age-associated inflammation.

Sterile inflammation, also known as 'inflammaging', is a hallmark of tissue aging. Cellular senescence contributes to tissue aging, in part, through the secretion of proinflammatory factors collectively known as the senescence-associated secretory phenotype (SASP). The genetic variability of thioredoxin reductase 1 (TXNRD1) is associated with aging and age-associated phenotypes such as late-life survival, activity of daily living and physical performance in old age.

Pioneer factors: roles and their regulation in development.

Pioneer factors are a subclass of transcription factors that can bind and initiate opening of silent chromatin regions. Pioneer factors subsequently regulate lineage-specific genes and enhancers and, thus, activate the zygotic genome after fertilization, guide cell fate transitions during development, and promote various forms of human cancers. As such, pioneer factors are useful in directed cell reprogramming.

Chromatin expansion microscopy reveals nanoscale organization of transcription and chromatin.

Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo.

Different chromatin-scanning modes lead to targeting of compacted chromatin by pioneer factors FOXA1 and SOX2.

Pioneer transcription factors interact with nucleosomes to scan silent, compact chromatin, enabling cooperative events that modulate gene activity. While at a subset of sites pioneer factors access chromatin by assisted loading with other transcription factors, the nucleosome-binding properties of pioneer factors enable them to initiate zygotic genome activation, embryonic development, and cellular reprogramming. To better understand nucleosome targeting in vivo, we assess whether pioneer factors FoxA1 and Sox2 target stable or unstable nucleosomes and find that they target DNase-resistant, stable nucleosomes, whereas HNF4A, a non-nucleosome binding factor, targets open, DNase-sensitive chromatin.

Diverse heterochromatin states restricting cell identity and reprogramming.

Heterochromatin is defined as a chromosomal domain harboring repressive H3K9me2/3 or H3K27me3 histone modifications and relevant factors that physically compact the chromatin. Heterochromatin can restrict where transcription factors bind, providing a barrier to gene activation and changes in cell identity. While heterochromatin thus helps maintain cell differentiation, it presents a barrier to overcome during efforts to reprogram cells for biomedical purposes.

Subcellular omics: a new frontier pushing the limits of resolution, complexity and throughput.

We argue that the study of single-cell subcellular organelle omics is needed to understand and regulate cell function. This requires and is being enabled by new technology development.

Physiological reprogramming mediated by Sox4 pioneer factor activity.

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver.

Two ways to skin a new cell fate.

To induce cell fate changes, do transcription factors engage open domains of chromatin or elicit chromatin opening in a pioneering fashion? In this issue of Developmental Cell, Delás et al. show that the same sonic hedgehog (Shh) inducing signal can yield different neural tube fates by either modality.

A pioneer factor locally opens compacted chromatin to enable targeted ATP-dependent nucleosome remodeling.

To determine how different pioneer transcription factors form a targeted, accessible nucleosome within compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated nucleosome arrays in vitro with a central nucleosome containing binding sites for the hematopoietic E-Twenty Six (ETS) factor PU.1 and Basic Leucine Zipper (bZIP) factors C/EBPα and C/EBPβ. Our long-read sequencing reveals that each factor can expose a targeted nucleosome on linker histone-compacted arrays, but with different nuclease sensitivity patterns.

Structures and consequences of pioneer factor binding to nucleosomes.

Pioneer transcription factors are able to bind a partially exposed motif on the surface of a nucleosome, enabling the proteins to target sites in silent regions of chromatin that have been compacted by linker histone. The targeting of nucleosomal DNA by pioneer factors has been observed in vitro and in vivo, where binding can promote local nucleosome exposure that allows other transcription factors, nucleosome remodelers, and histone modifiers to engage the chromatin and elicit gene activation or further repression. Pioneer factors thereby establish new gene expression programs during cell fate changes that occur during embryonic development, regeneration, and cancer.

ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage.

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and is thus relevant to the treatment of ischaemic diseases, injury-induced regeneration and solid tumour growth. Previously, we demonstrated that ETV2 is an essential transcription factor for the development of cardiac, endothelial and haematopoietic lineages. Here we report that ETV2 functions as a pioneer factor that relaxes closed chromatin and regulates endothelial development.

Maintaining Transcriptional Specificity Through Mitosis.

Virtually all cell types have the same DNA, yet each type exhibits its own cell-specific pattern of gene expression. During the brief period of mitosis, the chromosomes exhibit changes in protein composition and modifications, a marked condensation, and a consequent reduction in transcription. Yet as cells exit mitosis, they reactivate their cell-specific programs with high fidelity.

THSB2 as a prognostic biomarker for patients diagnosed with metastatic pancreatic ductal adenocarcinoma.

Patients newly diagnosed with metastatic pancreatic ductal adenocarcinoma generally have poor survival, with heterogeneous rates of progression. Biomarkers that could predict progression and/or survival would help inform patients and providers as they make care decisions. In a previous retrospective study, we discovered that circulating thrombospondin-2 (THBS2) could, in combination with CA19-9, better distinguish patients with PDAC versus healthy controls.

EU-RNA-seq for in vivo labeling and high throughput sequencing of nascent transcripts.

The protocol allows for labeling nascent RNA without isolating nuclei. The cell-permeable uridine analog, 5-ethynyluridine (EU), is added to media to allow labeling of nascent transcripts. Cells are lysed, total RNA is collected, and biotin is conjugated to EU-labeled RNAs.