Renal L-2-hydroxyglutarate dehydrogenase activity promotes hypoxia tolerance and mitochondrial metabolism in Drosophila melanogaster.

Objectives: The mitochondrial enzyme L-2-hydroxyglutarate dehydrogenase (L2HGDH) regulates the abundance of L-2-hydroxyglutarate (L-2HG), a potent signaling metabolite capable of influencing chromatin architecture, mitochondrial metabolism, and cell fate decisions. Loss of L2hgdh activity in humans induces ectopic L-2HG accumulation, resulting in neurodevelopmental defects, altered immune cell function, and enhanced growth of clear cell renal cell carcinomas. To better understand the molecular mechanisms that underlie these disease pathologies, we used the fruit fly Drosophila melanogaster to investigate the endogenous functions of L2hgdh.

Generating and testing the efficacy of reagents for CRISPR/Cas9 homology directed repair-based manipulations in Tribolium.

CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation.

Glycolytic Disruption Triggers Interorgan Signaling to Nonautonomously Restrict Larval Growth.

larval growth requires efficient conversion of dietary nutrients into biomass. Lactate Dehydrogenase (Ldh) and Glycerol-3-phosphate dehydrogenase (Gpdh1) support larval biosynthetic metabolism by maintaining NAD/NADH redox balance and promoting glycolytic flux. Consistent with the cooperative functions of Ldh and Gpdh1, the loss of both enzymes, but neither single enzyme, induces a developmental arrest.

Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies and compassionate xenograft use have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model.

Involvement of the SAGA and TFIID coactivator complexes in transcriptional dysregulation caused by the separation of core and tail Mediator modules.

Regulation of RNA polymerase II transcription requires the concerted efforts of several multisubunit coactivator complexes, which interact with the RNA polymerase II preinitiation complex to stimulate transcription. We previously showed that separation of the Mediator core from Mediator's tail module results in modest overactivation of genes annotated as highly dependent on TFIID for expression. However, it is unclear if other coactivators are involved in this phenomenon.

Sufficient principal component regression for pattern discovery in transcriptomic data.

Motivation: Methods for the global measurement of transcript abundance such as microarrays and RNA-Seq generate datasets in which the number of measured features far exceeds the number of observations. Extracting biologically meaningful and experimentally tractable insights from such data therefore requires high-dimensional prediction. Existing sparse linear approaches to this challenge have been stunningly successful, but some important issues remain.

Genome-Wide Profiling of Transcription Initiation with STRIPE-seq.

Transcription start site (TSS) usage is a critical factor in the regulation of gene expression. A number of methods for global TSS mapping have been developed, but barriers of expense, technical difficulty, time, and/or cost have limited their broader adoption. To address these issues, we developed Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq).

Generating and testing the efficacy of transgenic Cas9 in Tribolium castaneum.

CRISPR/Cas9 genome editing has now expanded to many insect species, including Tribolium castaneum. However, compared to Drosophila melanogaster, the CRISPR toolkit of T. castaneum is limited.

The more the merrier: Simultaneous mapping of multiple chromatin components in a single sample.

Gopalan et al. (2021) present multi-CUT&Tag, a modification of cleavage under targets and tagmentation (CUT&Tag) that enables simultaneous genome-wide mapping of multiple chromatin-associated targets in a single sample.

Global approaches for profiling transcription initiation.

Transcription start site (TSS) selection influences transcript stability and translation as well as protein sequence. Alternative TSS usage is pervasive in organismal development, is a major contributor to transcript isoform diversity in humans, and is frequently observed in human diseases including cancer. In this review, we discuss the breadth of techniques that have been used to globally profile TSSs and the resulting insights into gene regulation, as well as future prospects in this area of inquiry.

Connection of core and tail Mediator modules restrains transcription from TFIID-dependent promoters.

The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear.

Genome-Wide Profiling of Protein-DNA Interactions with Chromatin Endogenous Cleavage and High-Throughput Sequencing (ChEC-Seq ).

Interactions between regulatory proteins and specific genomic regions are critical for all chromatin-based processes, including transcription, DNA replication, and DNA repair. Genome-wide mapping of such interactions is most commonly performed with chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), but a number of orthogonal methods employing targeted enzymatic activity have also been introduced. We previously described a genome-wide implementation of chromatin endogenous cleavage (ChEC-Seq), wherein a protein of interest is fused to micrococcal nuclease (MNase) to enable targeted, calcium-dependent genomic cleavage.

Flexible analysis of TSS mapping data and detection of TSS shifts with TSRexploreR.

Heterogeneity in transcription initiation has important consequences for transcript stability and translation, and shifts in transcription start site (TSS) usage are prevalent in various developmental, metabolic, and disease contexts. Accordingly, numerous methods for global TSS profiling have been developed, including most recently Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a method to profile transcription start sites (TSSs) on a genome-wide scale with significant cost and time savings compared to previous methods. In anticipation of more widespread adoption of STRIPE-seq and related methods for construction of promoter atlases and studies of differential gene expression, we built TSRexploreR, an R package for end-to-end analysis of TSS mapping data.

LSD1 and Aberrant DNA Methylation Mediate Persistence of Enteroendocrine Progenitors That Support -Mutant Colorectal Cancer.

Despite the connection of secretory cells, including goblet and enteroendocrine (EEC) cells, to distinct mucus-containing colorectal cancer histologic subtypes, their role in colorectal cancer progression has been underexplored. Here, our analysis of The Cancer Genome Atlas (TCGA) and single-cell RNA-sequencing data demonstrates that EEC progenitor cells are enriched in -mutant colorectal cancer patient tumors, cell lines, and patient-derived organoids. In -mutant colorectal cancer, EEC progenitors were blocked from differentiating further by DNA methylation and silencing of NEUROD1, a key gene required for differentiation of intermediate EECs.

Early satellite cell communication creates a permissive environment for long-term muscle growth.

Using muscle stem cell (satellite cell)-specific extracellular vesicle (EV) tracking, satellite cell depletion, cell culture, and single-cell RNA sequencing, we show satellite cells communicate with other cells in skeletal muscle during mechanical overload. Early satellite cell EV communication primes the muscle milieu for proper long-term extracellular matrix (ECM) deposition and is sufficient to support sustained hypertrophy in adult mice, even in the absence of fusion to muscle fibers. Satellite cells modulate chemokine gene expression across cell types within the first few days of loading, and EV delivery of miR-206 to fibrogenic cells represses expression required for appropriate ECM remodeling.

Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.

Key Points: Ribosome biogenesis and MYC transcription are associated with acute resistance exercise (RE) and are distinct from endurance exercise in human skeletal muscle throughout a 24 h time course of recovery. A PCR-based method for relative ribosomal DNA (rDNA) copy number estimation was validated by whole genome sequencing and revealed that rDNA dosage is positively correlated with ribosome biogenesis in response to RE. Acute RE modifies rDNA methylation patterns in enhancer, intergenic spacer and non-canonical MYC-associated regions, but not the promoter.

Architectural Mediator subunits are differentially essential for global transcription in Saccharomyces cerevisiae.

Mediator is a modular coactivator complex involved in the transcription of the majority of RNA polymerase II-regulated genes. However, the degrees to which individual core subunits of Mediator contribute to its activity have been unclear. Here, we investigate the contribution of two essential architectural subunits of Mediator to transcription in Saccharomyces cerevisiae.

Simple and efficient profiling of transcription initiation and transcript levels with STRIPE-seq.

Accurate mapping of transcription start sites (TSSs) is key for understanding transcriptional regulation. However, current protocols for genome-wide TSS profiling are laborious and/or expensive. We present Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a simple, rapid, and cost-effective protocol for sequencing capped RNA 5' ends from as little as 50 ng total RNA.

CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention.

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning.

Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in -Mutant Colorectal Cancer.

Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis.

Community-Oriented Epidemic Preparedness and Response to the Jerusalem 2018-2019 Measles Epidemic.

Measles epidemics are still a public health challenge worldwide, necessitating a rapid response. The Jerusalem District Health Office applied a community-oriented intervention during the 2018-2019 epidemic (2150 notified cases). Program development targeted the socioeconomic and cultural characteristics of high-incidence Jewish ultraorthodox communities.

Expression in Ovarian Cancer Precursor Cells Alters the CTCF Cistrome and Enhances Invasiveness through .

High-grade serous carcinoma (HGSC) is the most aggressive and predominant form of epithelial ovarian cancer and the leading cause of gynecologic cancer-related death. We have previously shown that (also known as , rother f the egulator of mprinted ites) is expressed in most ovarian cancers, and is associated with global and promoter-specific DNA hypomethylation, advanced tumor stage, and poor prognosis. To explore its role in HGSC, we expressed in human fallopian tube secretory epithelial cells (FTSEC), the presumptive cells of origin for HGSC.

A Cyclin A-Myb-MuvB-Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles.

Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. In this study, we use integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs).

Mediator Is Essential for Small Nuclear and Nucleolar RNA Transcription in Yeast.

Eukaryotic RNA polymerase II (RNAPII) transcribes mRNA genes and non-protein-coding RNA (ncRNA) genes, including those encoding small nuclear and nucleolar RNAs (sn/snoRNAs). In metazoans, RNAPII transcription of sn/snoRNAs is facilitated by a number of specialized complexes, but no such complexes have been discovered in yeast. It has been proposed that yeast sn/snoRNA and mRNA expression relies on a set of common factors, but the extent to which regulators of mRNA genes function at yeast sn/snoRNA genes is unclear.

Distinct patterns of histone acetyltransferase and Mediator deployment at yeast protein-coding genes.

The transcriptional coactivators Mediator and two histone acetyltransferase (HAT) complexes, NuA4 and SAGA, play global roles in transcriptional activation. Here we explore the relative contributions of these factors to RNA polymerase II association at specific genes and gene classes by rapid nuclear depletion of key complex subunits. We show that the NuA4 HAT Esa1 differentially affects certain groups of genes, whereas the SAGA HAT Gcn5 has a weaker but more uniform effect.