Christine Roden

Novel determinants of mammalian primary microRNA processing revealed by systematic evaluation of hairpin-containing transcripts and human genetic variation

Mature microRNAs (miRNAs) are processed from hairpin-containing primary miRNAs (pri-miRNAs). However, rules that distinguish pri-miRNAs from other hairpin-containing transcripts in the genome are incompletely understood. By developing a computational pipeline to systematically evaluate 30 structural and sequence features of mammalian RNA hairpins, we report several new rules that are preferentially utilized in miRNA hairpins and govern efficient pri-miRNA processing. We propose that a hairpin stem length of 36 ± 3 nt is optimal for pri-miRNA processing. We identify two bulge-depleted regions on the miRNA stem, located ∼16-21 nt and ∼28-32 nt from the base of the stem, that are less tolerant of unpaired bases. We further show that the CNNC primary sequence motif selectively enhances the processing of optimal-length hairpins. We predict that a small but significant fraction of human single-nucleotide polymorphisms (SNPs) alter pri-miRNA processing, and confirm several predictions experimentally including a disease-causing mutation. Our study enhances the rules governing mammalian pri-miRNA processing and suggests a diverse impact of human genetic variation on miRNA biogenesis.

In vivo mutagenesis of miRNA gene families using a scalable multiplexed CRISPR/Cas9 nuclease system

A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten, and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and in vivo. Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms.

A Molecular Chipper technology for CRISPR sgRNA library generation and functional mapping of noncoding regions

Clustered regularly-interspaced palindromic repeats (CRISPR)-based genetic screens using single-guide-RNA (sgRNA) libraries have proven powerful to identify genetic regulators. Applying CRISPR screens to interrogate functional elements in noncoding regions requires generating sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. Here we present a Molecular Chipper technology for generating dense sgRNA libraries for genomic regions of interest, and a proof-of-principle screen that identifies novel cis-regulatory domains for miR-142 biogenesis. The Molecular Chipper approach utilizes a combination of random fragmentation and a type III restriction enzyme to derive a densely covering sgRNA library from input DNA. Applying this approach to 17 microRNAs and their flanking regions and with a reporter for miR-142 activity, we identify both the pre-miR-142 region and two previously unrecognized cis-domains important for miR-142 biogenesis, with the latter regulating miR-142 processing. This strategy will be useful for identifying functional noncoding elements in mammalian genomes.

The DNA Methylcytosine Dioxygenase Tet2 Sustains Immunosuppressive Function of Tumor-Infiltrating Myeloid Cells to Promote Melanoma Progression

&NA; Ten‐Eleven‐Translocation‐2 (Tet2) is a DNA methylcytosine dioxygenase that functions as a tumor suppressor in hematopoietic malignancies. We examined the role of Tet2 in tumor‐tissue myeloid cells and found that Tet2 sustains the immunosuppressive function of these cells. We found that Tet2 expression is increased in intratumoral myeloid cells both in mouse models of melanoma and in melanoma patients and that this increased expression is dependent on an IL‐1R‐MyD88 pathway. Ablation of Tet2 in myeloid cells suppressed melanoma growth in vivo and shifted the immunosuppressive gene expression program in tumor‐associated macrophages to a proinflammatory one, with a concomitant reduction of the immunosuppressive function. This resulted in increased numbers of effector T cells in the tumor, and T cell depletion abolished the reduced tumor growth observed upon myeloid‐specific deletion of Tet2. Our findings reveal a non‐cell‐intrinsic, tumor‐promoting function for Tet2 and suggest that Tet2 may present a therapeutic target for the treatment of non‐hematologic malignancies. Graphical Abstract Figure. No caption available. HighlightsDeletion of Tet2 in myeloid cells reduces melanoma tumor burdenTet2 expression is induced via the IL‐1R‐MyD88 axis in tumor‐associated macrophagesTet2 maintains myeloid immunosuppressive function and the associated genetic programMyeloid‐specific Tet2 deletion results in higher numbers of tumor‐infiltrating T cells &NA; The DNA methylcytosine dioxygenase Tet2 functions as a tumor suppressor in multiple contexts, including hematopoietic malignancies. Pan et al. now reveal a tumor‐promoting role for Tet2, whereby Tet2 functions to sustain an immunosuppressive program in myeloid cells that in turn dampens the anti‐tumor T cell response.

miR-125b promotes MLL-AF9–driven murine acute myeloid leukemia involving a VEGFA-mediated non–cell-intrinsic mechanism

The hematopoietic stem cell-enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia (AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9 By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9-driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non-cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2 Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non-cell-intrinsic leukemia-promoting effects by an oncogenic miRNA.

MicroRNAs in Control of Stem Cells in Normal and Malignant Hematopoiesis

Studies on hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) have helped to establish the paradigms of normal and cancer stem cell concepts. For both HSCs and LSCs, specific gene expression programs endowed by their epigenome functionally distinguish them from their differentiated progenies. MicroRNAs (miRNAs), as a class of small non-coding RNAs, act to control post-transcriptional gene expression. Research in the past decade has yielded exciting findings elucidating the roles of miRNAs in control of multiple facets of HSC and LSC biology. Here, we review recent progresses on the functions of miRNAs in HSC emergence during development, HSC switch from a fetal/neonatal program to an adult program, HSC self-renewal and quiescence, HSC aging, HSC niche, and malignant stem cells. While multiple different miRNAs regulate a diverse array of targets, two common themes emerge in HSC and LSC biology: miRNA-mediated regulation of epigenetic machinery and cell signaling pathways. In addition, we propose that miRNAs themselves behave like epigenetic regulators, as they possess key biochemical and biological properties that can provide both stability and alterability to the epigenetic program. Overall, the studies of miRNAs in stem cells in the hematologic contexts not only provide key understandings to post-transcriptional gene regulation mechanisms in HSCs and LSCs but also will lend key insights for other stem cell fields.

microRNA Expression Profiling: Technologies, Insights, and Prospects

Since the early days of microRNA (miRNA) research, miRNA expression profiling technologies have provided important tools toward both better understanding of the biological functions of miRNAs and using miRNA expression as potential diagnostics. Multiple technologies, such as microarrays, next-generation sequencing, bead-based detection system, single-molecule measurements, and quantitative RT-PCR, have enabled accurate quantification of miRNAs and the subsequent derivation of key insights into diverse biological processes. As a class of ~22 nt long small noncoding RNAs, miRNAs present unique challenges in expression profiling that require careful experimental design and data analyses. We will particularly discuss how normalization and the presence of miRNA isoforms can impact data interpretation. We will present one example in which the consideration in data normalization has provided insights that helped to establish the global miRNA expression as a tumor suppressor. Finally, we discuss two future prospects of using miRNA profiling technologies to understand single cell variability and derive new rules for the functions of miRNA isoforms.

Single-cell microRNA/mRNA co-sequencing reveals non-genetic heterogeneity and novel regulatory mechanisms

Co-measurement of multiple omic profiles from the same single cells opens up the opportunity to decode molecular regulation that underlie intercellular heterogeneity in development and disease. Here, we present co-sequencing of microRNAs and mRNAs in the same single cells using a half-cell genomics approach. This method demonstrates good robustness (~95% success rate) and reproducibility (R2=0.93 for both miRNAs and mRNAs), and yields paired half-cell miRNA and mRNA profiles that could be independently validated. Linking the level of miRNAs to the expression of predicted target mRNAs across 19 single cells that are phenotypically identical, we observe that the predicted targets are significantly anti-correlated with the variation of abundantly expressed miRNAs, suggesting that miRNA expression variability alone may lead to non-genetic cell-to-cell heterogeneity. Genome-scale analysis of paired miRNA-mRNA co-profiles further allows us to derive and validate new regulatory relationships of cellular pathways controlling miRNA expression and variability.

Molecular Chipper: Functional Mapping of the Non-Coding Genome with CRISPR

Only ~2% of the human genome encodes proteins. The functions of the vast majority of non-coding sequences, transcribed or non-transcribed, remain largely unknown. Powered by next generation sequencing and evolving high through-put biochemical technologies, continuing efforts have been devoted to the biochemical annotation of genomic regions based on their transcription activity, DNase-I hypersensitivity, transcription factor binding, chromatin modifications and replication domains. However, biochemical annotation does not equal functional roles in molecular control or cellular behavior. While these genome-scale biochemical data pave the way for deciphering the noncoding genome, high-throughput mapping technologies are required to assign functional roles to specific noncoding elements. Here, we review the use of CRISPR-based technologies in functional screens of noncoding genomic elements, with a focus on the Molecular Chipper technology.