Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila

Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, our understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, we have monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). We show that the scrib mutation impacts the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch dedicated transcription factor. The Notch-dependent neoplasms require however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1, and bZIP factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally our work highlights some Notch/scrib specificities, in particular the role of the PAR domain containing bZIP transcription factor and Notch direct target Pdp1 for neoplastic growth.

Context-dependent transcriptional regulations of YAP/TAZ in stem cell and differentiation

Hippo pathway is initially identified as a master regulator for cell proliferation and organ size control, and the subsequent researches show this pathway is also involved in development, tissue regeneration and homeostasis, inflammation, immunity and cancer. YAP/TAZ, the downstream effectors of Hippo pathway, usually act as coactivators and are dependent on other transcription factors to mediate their transcriptional outputs. In this review, we will first provide an overview on the core components and regulations of Hippo pathway in mammals, and then systematically summarize the identified transcriptional factors or partners that are responsible for the transcriptional output of YAP/TAZ in stem cell and differentiation. More than that, we will discuss the potential applications and future directions based on these findings.

The MLL3/4 complexes and MiDAC act antagonistically as genome-wide regulators of H4K20ac to control a specific gene expression program

The mitotic deacetylase complex MiDAC has recently been shown to play a vital physiological role in embryonic development and neurite outgrowth. However, how MiDAC functionally intersects with other chromatin-modifying regulators is poorly understood. Here, we describe a physical interaction between the histone H3K27 demethylase UTX, a complex-specific subunit of the enhancer-associated MLL3/4 complexes, and MiDAC. We demonstrate that UTX bridges the association of the MLL3/4 complexes and MiDAC by interacting with ELMSAN1, a scaffolding subunit of MiDAC. Our data shows that MiDAC constitutes a negative genome-wide regulator of H4K20ac, an activity which is counteracted by the MLL3/4 complexes. MiDAC and the MLL3/4 complexes co-localize at many genomic regions, that are enriched for H4K20ac and the enhancer marks H3K4me1, H3K4me2 and H3K27ac. We find that MiDAC antagonizes the recruitment of the MLL3/4 complexes to negatively regulate H4K20ac, H3K4me2 and H3K27ac resulting in transcriptional attenuation of associated genes. In summary, our findings provide a paradigm how the opposing roles of chromatin-modifying components, such as MiDAC and the MLL3/4 complexes, balance the transcriptional output of specific gene expression programs.

Absolute Scaling of Single-Cell Transcriptomes Reveals Pervasive Hypertranscription in Adult Stem and Progenitor Cells

Hypertranscription facilitates biosynthetically demanding cellular state transitions through global upregulation of the nascent transcriptome. Despite its potential widespread relevance, documented examples of hypertranscription remain few and limited predominantly to early development. This limitation is in large part due to the fact that modern sequencing approaches, including single-cell RNA sequencing (scRNA-seq), generally assume similar levels of transcriptional output per cell. Here, we use molecule counting and spike-in normalization to develop absolute scaling of single-cell RNA sequencing data. Absolute scaling enables an estimation of total transcript abundances per cell, which we validate in embryonic stem cell (ESC) and germline data and apply to adult mouse organs at steady-state or during regeneration. The results reveal a remarkable dynamic range in transcriptional output among adult cell types. We find that many different multipotent stem and progenitor cell populations are in a state of hypertranscription, including in the hematopoietic system, intestine and skin. Hypertranscription marks cells with multilineage potential in adult organs, is redeployed in conditions of tissue injury, and can precede by 1-2 days bursts of proliferation during regeneration. In addition to the association between hypertranscription and the stem/progenitor cell state, we dissect the relationship between transcriptional output and cell cycle, ploidy and secretory behavior. Our analyses reveal a common set of molecular pathways associated with hypertranscription across adult organs, including chromatin remodeling, DNA repair, ribosome biogenesis and translation. Our findings introduce an approach towards maximizing single-cell RNA-seq profiling. By applying this methodology across a diverse collection of cell states and contexts, we put forth hypertranscription as a general and dynamic cellular program that is pervasively employed during development, organ maintenance and regeneration.

Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3′-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins—the spliceosome NineTeen Complex (NTC) and related proteins (NTR)—and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

Transcription recycling assays identify PAF1 as a driver for RNA Pol II recycling

RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states.

Transcriptional burst kinetics are linked to short term transcriptional memory

Transcriptional bursting is thought to be a stochastic process that allows the dynamic regulation of most genes. The random telegraph model assumes the existence of two states, ON and OFF. However recent studies indicate the presence of additional ON states, suggesting that bursting kinetics and their regulation can be quite complex. We have developed a system to study transcriptional bursting in the context of p53 biology using the endogenous p21 gene tagged with MS2 in human cells. Remarkably, we find that transcriptional bursts from the p21 gene contain multiple ON and OFF states that can be regulated by elevation of p53 levels. Distinct ON states are characterized by differences in burst duration, classified as Short and Long, with long bursts associated with higher Pol II initiation rates. Importantly, the different ON states display memory effects that allow us to predict the likelihood of properties of future bursting events. Long bursting events result in faster re-activation, longer subsequent bursts and higher transcriptional output in the future compared to short bursts. Bursting memory persists up to 2 hours suggesting a stable inheritable promoter architecture. Bursting memory at the p21 gene is the strongest under basal conditions and is suppressed by UV and inhibition of H3K9me1/2, which also increase transcriptional noise. Stabilization of p53 by Nutlin-3a partially reverses suppression of bursting memory suggesting that higher p53 levels may be a key in enforcing memory under conditions of cellular stress. Overall our data uncover a new found bursting property termed Short-Term Transcriptional Memory (STTM) that has the potential to fine-tune transcriptional output at the p21 gene.

Noncoding RNAs link metabolic reprogramming to immune microenvironment in cancers

Altered metabolic patterns in tumor cells not only meet their own growth requirements but also shape an immunosuppressive microenvironment through multiple mechanisms. Noncoding RNAs constitute approximately 60% of the transcriptional output of human cells and have been shown to regulate numerous cellular processes under developmental and pathological conditions. Given their extensive action mechanisms based on motif recognition patterns, noncoding RNAs may serve as hinges bridging metabolic activity and immune responses. Indeed, recent studies have shown that microRNAs, long noncoding RNAs and circRNAs are widely involved in tumor metabolic rewiring, immune cell infiltration and function. Hence, we summarized existing knowledge of the role of noncoding RNAs in the remodeling of tumor metabolism and the immune microenvironment, and notably, we established the TIMELnc manual, which is a free and public manual for researchers to identify pivotal lncRNAs that are simultaneously correlated with tumor metabolism and immune cell infiltration based on a bioinformatic approach.

One Omics Approach Does Not Rule Them All: The Metabolome and the Epigenome Join Forces in Haematological Malignancies

Aberrant DNA methylation, dysregulation of chromatin-modifying enzymes, and microRNAs (miRNAs) play a crucial role in haematological malignancies. These epimutations, with an impact on chromatin accessibility and transcriptional output, are often associated with genomic instability and the emergence of drug resistance, disease progression, and poor survival. In order to exert their functions, epigenetic enzymes utilize cellular metabolites as co-factors and are highly dependent on their availability. By affecting the expression of metabolic enzymes, epigenetic modifiers may aid the generation of metabolite signatures that could be utilized as targets and biomarkers in cancer. This interdependency remains often neglected and poorly represented in studies, despite well-established methods to study the cellular metabolome. This review critically summarizes the current knowledge in the field to provide an integral picture of the interplay between epigenomic alterations and the cellular metabolome in haematological malignancies. Our recent findings defining a distinct metabolic signature upon response to enhancer of zeste homolog 2 (EZH2) inhibition in multiple myeloma (MM) highlight how a shift of preferred metabolic pathways may potentiate novel treatments. The suggested link between the epigenome and the metabolome in haematopoietic tumours holds promise for the use of metabolic signatures as possible biomarkers of response to treatment.

Building regulatory landscapes: enhancer recruits cohesin to create contact domains, engage CTCF sites and activate distant genes

ABSTRACTDevelopmental gene expression is often controlled by distal tissue-specific enhancers. Enhancer action is restricted to topological chromatin domains, typically formed by cohesin-mediated loop extrusion between CTCF-associated boundaries. To better understand how individual regulatory DNA elements form topological domains and control expression, we used a bottom-up approach, building active regulatory landscapes of different sizes in inactive chromatin. We demonstrate that transcriptional output and protection against gene silencing reduces with increased enhancer distance, but that enhancer contact frequencies alone do not dictate transcription activity. The enhancer recruits cohesin to stimulate the formation of local chromatin contact domains and activate flanking CTCF sites for engagement in chromatin looping. Small contact domains can support strong and stable expression of distant genes. The enhancer requires transcription factors and mediator to activate genes over all distance ranges, but relies on cohesin exclusively for the activation of distant genes. Our work supports a model that assigns two functions to enhancers: its classic role to stimulate transcription initiation and elongation from target gene promoters and a role to recruit cohesin for the creation of contact domains, the engagement of flanking CTCF sites in chromatin looping, and the activation of distal target genes.