J. Mauro Calabrese

Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells

MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.

Divergent Transcription from Active Promoters

Transcription initiation by RNA polymerase II (RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein-encoding gene promoters. Transcription start site–associated RNAs (TSSa-RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa-RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter-associated RNAPII and H3K4-trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa-RNA positions; however, H3K79-dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation.

Quantitative analysis of Argonaute protein reveals microRNA-dependent localization to stress granules

Argonaute proteins associate with microRNAs (miRNAs) that bind mRNAs through partial base-pairings to primarily repress translation in animals. A fraction of Argonaute proteins and miRNAs biochemically cosediment with polyribosomes, yet another fraction paradoxically accumulates in ribosome-free processing bodies (PBs) in the cytoplasm. In this report, we give a quantitative account of the Argonaute protein localization and dynamics in living cells in different cellular states. We find that the majority of Argonaute is distributed diffusely in the cytoplasm, and, when cells are subjected to stress, Argonaute proteins accumulate to newly assembled structures known as stress granules (SGs) in addition to PBs. Argonaute proteins displayed distinct kinetics at different structures: exchange faster at SGs and much slower at PBs. Further, miRNAs are required for the Argonaute protein localization to SGs but not PBs. These quantitative kinetic data provide insights into miRNA-mediated repression.

RNA sequence analysis defines Dicer's role in mouse embryonic stem cells

Short RNA expression was analyzed from Dicer-positive and Dicer-knockout mouse embroyonic stem (ES) cells, using high-throughput pyrosequencing. A correlation of miRNA quantification with sequencing frequency estimates that there are 110,000 miRNAs per ES cell, the majority of which can be accounted for by six distinct miRNA loci. Four of these miRNA loci or their human homologues have demonstrated roles in cell cycle regulation or oncogenesis, suggesting that a major function of the miRNA pathway in ES cells may be to shape their distinct cell cycle. Forty-six previously uncharacterized miRNAs were identified, most of which are expressed at low levels and are less conserved than the set of known miRNAs. Low-abundance short RNAs matching all classes of repetitive elements were present in cells lacking Dicer, although the production of some SINE- and simple repeat-associated short RNAs appeared to be Dicer-dependent. These and other Dicer-dependent sequences resembled miRNAs. At a depth of sequencing that approaches the total number of 5′ phosphorylated short RNAs per cell, miRNAs appeared to be Dicers only substrate. The results presented suggest a model in which repeat-associated miRNAs serve as host defenses against repetitive elements, a function canonically ascribed to other classes of short RNA.

Topoisomerases facilitate transcription of long genes linked to autism

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

Site-Specific Silencing of Regulatory Elements as a Mechanism of X Inactivation

The inactive X chromosomes (Xi) physical territory is microscopically devoid of transcriptional hallmarks and enriched in silencing-associated modifications. How these microscopic signatures relate to specific Xi sequences is unknown. Therefore, we profiled Xi gene expression and chromatin states at high resolution via allele-specific sequencing in mouse trophoblast stem cells. Most notably, X-inactivated transcription start sites harbored distinct epigenetic signatures relative to surrounding Xi DNA. These sites displayed H3-lysine27-trimethylation enrichment and DNaseI hypersensitivity, similar to autosomal Polycomb targets, yet excluded Pol II and other transcriptional hallmarks, similar to nontranscribed genes. CTCF bound X-inactivated and escaping genes, irrespective of measured chromatin boundaries. Escape from X inactivation occurred within, and X inactivation was maintained exterior to, the area encompassed by Xist in cells subject to imprinted and random X inactivation. The data support a model whereby inactivation of specific regulatory elements, rather than a simple chromosome-wide separation from transcription machinery, governs gene silencing over the Xi.

A Latent Pro-Survival Function for the Mir-290-295 Cluster in Mouse Embryonic Stem Cells

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.

Detection of RNA-Protein Interactions in Living Cells with SHAPE

SHAPE-MaP is unique among RNA structure probing strategies in that it both measures flexibility at single-nucleotide resolution and quantifies the uncertainties in these measurements. We report a straightforward analytical framework that incorporates these uncertainties to allow detection of RNA structural differences between any two states, and we use it here to detect RNA-protein interactions in healthy mouse trophoblast stem cells. We validate this approach by analysis of three model cytoplasmic and nuclear ribonucleoprotein complexes, in 2 min in-cell probing experiments. In contrast, data produced by alternative in-cell SHAPE probing methods correlate poorly (r = 0.2) with those generated by SHAPE-MaP and do not yield accurate signals for RNA-protein interactions. We then examine RNA-protein and RNA-substrate interactions in the RNase MRP complex and, by comparing in-cell interaction sites with disease-associated mutations, characterize these noncoding mutations in terms of molecular phenotype. Together, these results reveal that SHAPE-MaP can define true interaction sites and infer RNA functions under native cellular conditions with limited preexisting knowledge of the proteins or RNAs involved.

SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells.

Significance Long noncoding RNAs (lncRNAs) are important regulators of gene expression, but their structural features are largely unknown. We used structure-selective chemical probing to examine the structure of the Xist lncRNA in living cells and found that the RNA adopts well-defined and complex structures throughout its entire 18-kb length. By looking for changes in reactivity induced by the cellular environment, we were able to identify numerous previously unknown hubs of protein interaction. We also found that the Xist structure governs specific protein interactions in multiple distinct ways. Our results provide a detailed structural context for Xist function and lay a foundation for understanding structure–function relationships in all lncRNAs. The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global structural architecture, cell-induced conformational changes, and protein–RNA interactions within Xist are poorly understood. We used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex well-defined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning one-half of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeat-containing elements adopt accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable interactions. This work creates a broad quantitative framework for understanding structure–function interrelationships for Xist and other lncRNAs in cells.

The mouse DXZ4 homolog retains Ctcf binding and proximity to Pls3 despite substantial organizational differences compared to the primate macrosatellite

BackgroundThe X-linked macrosatellite DXZ4 is a large homogenous tandem repeat that in females adopts an alternative chromatin organization on the primate X chromosome in response to X-chromosome inactivation. It is packaged into heterochromatin on the active X chromosome but into euchromatin and bound by the epigenetic organizer protein CTCF on the inactive X chromosome. Because its DNA sequence diverges rapidly beyond the New World monkeys, the existence of DXZ4 outside the primate lineage is unknown.ResultsHere we extend our comparative genome analysis and report the identification and characterization of the mouse homolog of the macrosatellite. Furthermore, we provide evidence of DXZ4 in a conserved location downstream of the PLS3 gene in a diverse group of mammals, and reveal that DNA sequence conservation is restricted to the CTCF binding motif, supporting a central role for this protein at this locus. However, many features that characterize primate DXZ4 differ in mouse, including the overall size of the array, the mode of transcription, the chromatin organization and conservation between adjacent repeat units of DNA sequence and length. Ctcf binds Dxz4 but is not exclusive to the inactive X chromosome, as evidenced by association in some males and equal binding to both X chromosomes in trophoblast stem cells.ConclusionsCharacterization of Dxz4 reveals substantial differences in the organization of DNA sequence, chromatin packaging, and the mode of transcription, so the potential roles performed by this sequence in mouse have probably diverged from those on the primate X chromosome.