Histone variant H2BE enhances chromatin accessibility in neurons to promote synaptic gene expression and long-term memory.

Histone proteins affect gene expression through multiple mechanisms, including through exchange with histone variants. Recent findings link histone variants to neurological disorders, yet few are well studied in the brain. Most notably, widely expressed variants of H2B remain elusive. We applied recently developed antibodies, biochemical assays, and sequencing approaches to reveal broad expression of the H2B variant H2BE and defined its role in regulating chromatin structure, neuronal transcription, and mouse behavior. We find that H2BE is enriched at promoters, and a single unique amino acid allows it to dramatically enhance chromatin accessibility. Further, we show that H2BE is critical for synaptic gene expression and long-term memory. Together, these data reveal a mechanism linking histone variants to chromatin accessibility, transcriptional regulation, neuronal function, and memory. This work further identifies a widely expressed H2B variant and uncovers a single histone amino acid with profound effects on genomic structure.

Histone variant H2BE enhances chromatin accessibility in neurons to promote synaptic gene expression and long-term memory.

Regulation of histone proteins affects gene expression through multiple mechanisms including exchange with histone variants. However, widely expressed variants of H2B remain elusive. Recent findings link histone variants to neurological disorders, yet few are well studied in the brain. We applied new tools including novel antibodies, biochemical assays, and sequencing approaches to reveal broad expression of the H2B variant H2BE, and defined its role in regulating chromatin structure, neuronal transcription, and mouse behavior. We find that H2BE is enriched at promoters and a single unique amino acid allows it to dramatically enhance chromatin accessibility. Lastly, we show that H2BE is critical for synaptic gene expression and long-term memory. Together, these data reveal a novel mechanism linking histone variants to chromatin regulation, neuronal function, and memory. This work further identifies the first widely expressed H2B variant and uncovers a single histone amino acid with profound effects on genomic structure.

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 in vivo labeling of nascent transcripts. Cells are lysed, total RNA is collected, and biotin is conjugated to EU-labeled RNAs. Custom biotin RNAs are added and biotinylated RNAs are isolated for generation of cDNA libraries. The sequencing data are normalized to controls for quantitative assessment of the nascent transcriptome. The protocol takes 4 days, not including sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Palozola et al. (2017).

CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters.

Validation of gene transfer vectors containing tissue-specific promoters in cell-based functional assays poses a formidable challenge for gene therapy product development. Here, we describe a novel approach based on CRISPR/dCas9 transcriptional activation to achieve robust transgene expression from transgene cassettes containing tissue or cell type-specific promoters after infection with AAV vectors in cell-based systems. Guide RNA sequences targeting two promoters that are highly active within mammalian photoreceptors were screened in a novel promoter activation assay. Using this screen, we generated and characterized stable cell lines that co-express dCas9.VPR and top-performing guide RNA candidates. These cells exhibit potent activation of proviral plasmids after transfection or after infection with AAV vectors delivering transgene cassettes carrying photoreceptor-specific promoters. In addition, we interrogated mechanisms to optimize this platform through the addition of multiple guide RNA sequences and co-expression of the universal adeno-associated virus receptor (AAVR). Collectively, this investigation identifies a rapid and broadly applicable strategy to enhance in vitro expression and to evaluate potency of AAV vectors that rely upon cell or tissue-specific regulatory elements.

A changing paradigm of transcriptional memory propagation through mitosis.

The highly reproducible inheritance of chromosomes during mitosis in mammalian cells involves nuclear envelope breakdown, increased chromatin compaction, loss of long-range intrachromosomal interactions, loss of enhancer-promoter proximity, displacement of many transcription regulators from the chromatin and a marked decrease in RNA synthesis. Despite these dramatic changes in the mother cell, daughter cells are able to faithfully re-establish the parental chromatin and gene expression features characteristic of the cell type. Pioneering studies of mitotic chromatin signatures showed that despite global repression of transcription, the Hsp70 gene promoter retains an open chromatin conformation, which was proposed to allow the reactivation of the Hsp70 gene upon completion of mitosis - a phenomenon termed mitotic bookmarking. It was later shown that various cell-type-specific transcription factors, such as GATA-binding factor 1 (GATA1) in erythroblasts and forkhead box protein A1 (FOXA1) in hepatocytes, remain bound at a subset of their interphase binding sites in mitosis. Such bookmarking transcription factors remain on chromosomes in mitosis and have been shown to enable a subset of genes to be reactivated in a timely fashion upon mitotic exit. In addition, sensitive new methods to measure transcription revealed that mitotic cells retain residual transcription at a large number of genes. Furthermore, genes recover their interphase level of transcription in distinct waves. Thus, gene expression is precisely regulated as cells pass through mitosis to ensure faithful propagation of cell identity and function through cellular generations.

Mitotic transcription and waves of gene reactivation during mitotic exit.

Although the genome is generally thought to be transcriptionally silent during mitosis, technical limitations have prevented sensitive mapping of transcription during mitosis and mitotic exit. Thus, the means by which the interphase expression pattern is transduced to daughter cells have been unclear. We used 5-ethynyluridine to pulse-label transcripts during mitosis and mitotic exit and found that many genes exhibit transcription during mitosis, as confirmed with fluorescein isothiocyanate-uridine 5'-triphosphate labeling, RNA fluorescence in situ hybridization, and quantitative reverse transcription polymerase chain reaction. The first round of transcription immediately after mitosis primarily activates genes involved in the growth and rebuilding of daughter cells, rather than cell type-specific functions. We propose that the cell's transcription pattern is largely retained at a low level through mitosis, whereas the amplitude of transcription observed in interphase is reestablished during mitotic exit.

Low-Level, Global Transcription during Mitosis and Dynamic Gene Reactivation during Mitotic Exit.

Mitosis is thought to be a period of transcriptional silence due to the compact nature of mitotic chromosomes and the apparent exclusion of RNA Pol II and many transcription factors from mitotic chromatin. Yet accurate reactivation of a cell's specific gene expression program is needed to reestablish functional cell identity after mitosis. The majority of studies on protein regulation and localization during mitosis have relied extensively on antibodies and cross-linking-based approaches that are known to artifactually exclude proteins from mitotic chromatin. Here we show that RNA Pol II localization in mitosis is antibody- and fixation-dependent, and that direct assessment of transcription by pulse-labeling nascent RNA reveals global, low-level mitotic transcription. We also find a hierarchy of gene reactivation as the cells transition from mitosis to their interphase amplitude of gene expression. Resetting of gene transcription during mitotic exit is coincident with enhancer transcription. Our work thus shifts focus from assessing mitotic exit as a binary transcription switch to a more nuanced concert of transcription amplitude and enhancer usage. We suggest that understanding how gene expression patterns are conserved during mitosis rests upon deciphering how transcription is maintained by promoters.

BET Inhibitors Suppress ALDH Activity by Targeting ALDH1A1 Super-Enhancer in Ovarian Cancer.

The emergence of tumor cells with certain stem-like characteristics, such as high aldehyde dehydrogenase (ALDH) activity due to ALDH1A1 expression, contributes to chemotherapy resistance and tumor relapse. However, clinically applicable inhibitors of ALDH activity have not been reported. There is evidence to suggest that epigenetic regulation of stem-related genes contributes to chemotherapy efficacy. Here, we show that bromodomain and extraterminal (BET) inhibitors suppress ALDH activity by abrogating BRD4-mediated ALDH1A1 expression through a super-enhancer element and its associated enhancer RNA. The clinically applicable small-molecule BET inhibitor JQ1 suppressed the outgrowth of cisplatin-treated ovarian cancer cells both in vitro and in vivo Combination of JQ1 and cisplatin improved the survival of ovarian cancer-bearing mice in an orthotopic model. These phenotypes correlate with inhibition of ALDH1A1 expression through a super-enhancer element and other stem-related genes in promoter regions bound by BRD4. Thus, targeting the BET protein BRD4 using clinically applicable small-molecule inhibitors, such as JQ1, is a promising strategy for targeting ALDH activity in epithelial ovarian cancer. Cancer Res; 76(21); 6320-30. ©2016 AACR.

An allele of sequoia dominantly enhances a trio mutant phenotype to influence Drosophila larval behavior.

The transition of Drosophila third instar larvae from feeding, photo-phobic foragers to non-feeding, photo-neutral wanderers is a classic behavioral switch that precedes pupariation. The neuronal network responsible for this behavior has recently begun to be defined. Previous genetic analyses have identified signaling components for food and light sensory inputs and neuropeptide hormonal outputs as being critical for the forager to wanderer transition. Trio is a Rho-Guanine Nucleotide Exchange Factor integrated into a variety of signaling networks including those governing axon pathfinding in early development. Sequoia is a pan-neuronally expressed zinc-finger transcription factor that governs dendrite and axon outgrowth. Using pre-pupal lethality as an endpoint, we have screened for dominant second-site enhancers of a weakly lethal trio mutant background. In these screens, an allele of sequoia has been identified. While these mutants have no obvious disruption of embryonic central nervous system architecture and survive to third instar larvae similar to controls, they retain forager behavior and thus fail to pupariate at high frequency.