Characterization of oxidized methylcytosine binding protein activities in the mammalian brain and stem cells

5-Methylcytosine embedded in mammalian DNA represses local transcription by recruiting modification-specific binding partners. Its active removal is initiated by sequential oxidation of the 5-methyl group by TET enzymes to produce three oxidized species, collectively referred to as [ox]mC. Although rare, the distribution of [ox]mC modifications is tissue-, gene-, and coding strand-specific and distinct from 5-methylcytosine, suggesting unique functions. To examine this possibility, I fractionated mammalian brain extracts to discover, isolate and characterize binding partners specific for [ox]mC. This purification reveals remarkably specific factors that are selective for each of the three oxidation states and sensitive to the 5-modification state on each strand. I demonstrate that one such factor, WDR76, is a highly 5-hydroxymethylcytosine-specific binding protein. I have begun to lay the foundation for further mechanistic studies of these specific binding proteins in mouse embryonic stem cells and leukemia. My results provide an essential bridge from studies of the distribution of [ox]mC and the effects of TET knockouts, to the possible functions of [ox]mC recognition in gene regulation or chromatin signaling

A fluorescence viewer for rapid molecular assay readout in space and low-resource terrestrial environments.

Fluorescence-based assays provide sensitive and adaptable methods for point of care testing, environmental monitoring, studies of protein abundance and activity, and a wide variety of additional applications. Currently, their utility in remote and low-resource environments is limited by the need for technically complicated or expensive instruments to read out fluorescence signal. Here we describe the Genes in Space Fluorescence Viewer (GiS Viewer), a portable, durable viewer for rapid molecular assay readout that can be used to visualize fluorescence in the red and green ranges. The GiS Viewer can be used to visualize any assay run in standard PCR tubes and contains a heating element. Results are visible by eye or can be imaged with a smartphone or tablet for downstream quantification. We demonstrate the capabilities of the GiS Viewer using two case studies-detection of SARS-CoV-2 RNA using RT-LAMP and quantification of drug-induced changes in gene expression via qRT-PCR on Earth and aboard the International Space Station. We show that the GiS Viewer provides a reliable method to visualize fluorescence in space without the need to return samples to Earth and can further be used to assess the results of RT-LAMP and qRT-PCR assays on Earth

foxF-1 Controls Specification of Non-body Wall Muscle and Phagocytic Cells in Planarians

Planarians are flatworms capable of regenerating any missing body part in a process requiring stem cells and positional information. Muscle is a major source of planarian positional information and consists of several types of fibers with distinct regulatory roles in regeneration. The transcriptional regulatory programs used to specify different muscle fibers are poorly characterized. Using single-cell RNA sequencing, we define the transcriptomes of planarian dorsal-ventral muscle (DVM), intestinal muscle (IM), and pharynx muscle. This analysis identifies foxF-1, which encodes a broadly conserved Fox-family transcription factor, as a master transcriptional regulator of all non-body wall muscle. The transcription factors encoded by nk4 and gata4/5/6-2 specify two different subsets of DVM, lateral and medial, respectively, whereas gata4/5/6-3 specifies IM. These muscle types all express planarian patterning genes. Both lateral and medial DVM are required for medial-lateral patterning in regeneration, whereas medial DVM and IM have a role in maintaining and regenerating intestine morphology. In addition to the role in muscle, foxF-1 is required for the specification of multiple cell types with transcriptome similarities, including high expression levels of cathepsin genes. These cells include pigment cells, glia, and several other cells with unknown function. cathepsin+ cells phagocytose E. coli, suggesting these are phagocytic cells. In conclusion, we describe a regulatory program for planarian muscle cell subsets and phagocytic cells, both driven by foxF-1. FoxF proteins specify different mesoderm-derived tissues in other organisms, suggesting that FoxF regulates formation of an ancient and broadly conserved subset of mesoderm derivatives in the Bilateria.National Institutes of Health (U.S.) (Grant R01GM080639

Raw image for gel presented in Fig 4A, without labeling.

Sample order: 1–3. Space control. 4–6: Space 6hpe. 7–9: Ground control. 10–12: Ground 6hpe. (TIFF)</p

Rapid readout of gene expression studies using the GiS Viewer.

A.18S rRNA RT-PCR reaction with 2-fold dilution series of input liver cDNA run in miniPCR® and imaged in GiS Viewer, shown at cycle 21. Left to right: 10 μM fluorescein, water, cDNA 2-fold dilution series ranging from 0.03125 μl per reaction to 1 μl per reaction. B. Cyp4a14 RT-PCR run in the miniPCR and imaged in GiS Viewer shown at cycle 21, samples 1–3: control liver cDNA from three separate mice, samples 4–6: 6hpe APAP-treated liver cDNA from three separate mice, sample 5: water control, sample 6: 10 μM fluorescein. C. Mt2 RT-PCR run in miniPCR and imaged in GiS Viewer shown at cycle 29. samples 1–3: control liver cDNA from three separate mice, samples 4–6: 6hpe APAP-treated liver cDNA from three separate mice, sample 5: water control, sample 6: 10 μM fluorescein. D. Quantification of fluorescence intensity in A., n = 3 per sample. We were able to distinguish a 3–4 fold difference in gene expression and up to a 16-fold dilution of input cDNA. E. Quantification of fluorescence intensity in B. F. Quantification of fluorescence intensity in C. APAP: acetaminophen. hpe: hours post-exposure. Error bars are mean + SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Blank and positive control are not included in quantification of fluorescence intensity.</p

RT-LAMP detection of SARS-CoV-2 RNA.

Samples containing 1000, 500, or 100 copies of SARS-CoV-2 RNA were used as template for RT-LAMP reactions. Reactions were monitored continuously in the GiS Viewer and maximum signal was observed at 30 minutes. Samples 1–2: 1000 copies of RNA, samples 3–4: 500 copies of RNA, samples 5–6: 100 copies RNA, samples 7–8: no template control. Limit of detection for this assay was 100 copies within 15 minutes.</p

RT-PCR primer sequences.

Fluorescence-based assays provide sensitive and adaptable methods for point of care testing, environmental monitoring, studies of protein abundance and activity, and a wide variety of additional applications. Currently, their utility in remote and low-resource environments is limited by the need for technically complicated or expensive instruments to read out fluorescence signal. Here we describe the Genes in Space Fluorescence Viewer (GiS Viewer), a portable, durable viewer for rapid molecular assay readout that can be used to visualize fluorescence in the red and green ranges. The GiS Viewer can be used to visualize any assay run in standard PCR tubes and contains a heating element. Results are visible by eye or can be imaged with a smartphone or tablet for downstream quantification. We demonstrate the capabilities of the GiS Viewer using two case studies–detection of SARS-CoV-2 RNA using RT-LAMP and quantification of drug-induced changes in gene expression via qRT-PCR on Earth and aboard the International Space Station. We show that the GiS Viewer provides a reliable method to visualize fluorescence in space without the need to return samples to Earth and can further be used to assess the results of RT-LAMP and qRT-PCR assays on Earth.</div

Demonstration of the heating capacity of the GiS Viewer onboard the ISS.

AT oligos (1.5 μM, 0.375 μM, 0.09375 μM) were heated to 72C for 10 minutes and then allowed to cool to room temperature for 10 minutes. Video speed is 100X real time. From left to right samples are two replicates of a 1:4 dilution series of AT oligos (1.5 μM, 0.375 μM, 0.09375 μM) followed by a water control and 10 μM fluorescein. (MP4)</p