Braveheart, a Long Noncoding RNA Required for Cardiovascular Lineage Commitment

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.Damon Runyon Cancer Research Foundation (DRG 2032-09)Damon Runyon Cancer Research Foundation (DFS 04-12)European Molecular Biology Organization (Long-term Fellowship)National Heart, Lung, and Blood Institute. Bench to Bassinet Program (U01HL098179)National Heart, Lung, and Blood Institute. Bench to Bassinet Program (U01HL098188)Smith Family FoundationPew Charitable Trusts. Program in the Biomedical Science

H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z[superscript AP3]) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z[superscript AP3] interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z[superscript AP3] was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z[superscript AP3] ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z[superscript AP3] ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z[superscript AP3] displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z[superscript AP3] mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.Massachusetts Life Sciences Center (David H. Koch Institute for Integrative Cancer Research at MIT Core Grant P30-CA14051)National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (Grant CBET-0939511)MIT Faculty Start-up FundMassachusetts Institute of Technology. Computational and Systems Biology Initiative (Merck & Co. Postdoctoral Fellowship

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Braveheart, a Long Noncoding RNA Required for Cardiovascular Lineage Commitment

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.Stem Cell and Regenerative Biolog

H2A.Z acidic patch is necessary for ESC differentiation.

<p>(A) Surface rendering of the H2A (left) and H2A.Z (right) nucleosome center. The H2A (orange) and H2A.Z (light brown) structures are shown with H2B (red), H3 (blue), and H4 (green) as indicated by the labels. The divergent residues (teal) are highlighted with arrows. The images were generated in Pymol using the following PDB files: 1AOI for canonical H2A-containing nucleosome structure and 1F66 for H2A.Z nucleosome structure. Below, sequence alignment of C terminal docking domain of H2A and H2A.Z. The acidic patch region is highlighted in a grey box. The bold, italicized and underlined residues indicate the divergent H2A.Z residues replaced in our study to the corresponding H2A residues. (B) Schematic diagram depicting the system used in this study to investigate the function of the H2A.Z acidic patch. (C) qRT-PCR representing the relative levels of endogenous H2A.Z transcript in H2A.Z^WT and H2A.Z^AP3 dox-inducible transgenic ESCs in the presence (+) and absence (−) of H2A.Z 3′UTR-specific shRNA. Transcript levels were normalized relative to Tubb5. Error bars represents standard deviation calculated from three independent biological replicates. (D) (Top) Western blot using H2A.Z antibodies on whole cell lysates isolated from dox-induced and uninduced (−/+) H2A.Z^WT and H2A.Z^AP3 transgenic ESC lines in the presence (+) and absence (−) of the H2A.Z 3′UTR-specific shRNA. Titrations of the dox-induced samples (25 µg and 50 µg of whole cell lysates) were performed to demonstrate comparable expression of H2A.Z-YFP transgene in H2A.Z^WT and H2A.Z^AP3 ESCs. H3 levels were used as a load control (lower exposure). Densitometric measurements were used to determine the ratio of transgene signal to H3 for the indicated samples (bottom) using ImageJ. Error bars represent standard deviations from a triplicate set of experiments. ESC colony morphology and OCT4 staining for H2A.Z^WT, H2A.Z^KD, (E) and H2A.Z^AP3 (G) expressing ESC. Embryoid bodies (EBs) were generated from H2A.Z^WT, H2A.Z^KD (F), and H2A.Z^AP3 (H) expressing ESCs. The left panels show bright field images of EBs cultured for 10 days in the absence of LIF. The right panels show hematoxylin and eosin stained sections of day 10 EBs for indicated cell lines. (I) qRT-PCR analyses showing mRNA levels of indicated pluripotency (<i>Pou5f1/Oct4</i> and <i>Nanog</i>) and differentiation markers (<i>Nestin</i>, <i>Cdx2</i>, <i>Isl1</i>) in Day 0 and Day 10 EBs generated from unmodified ESCs, H2A.Z^WT, H2A.Z^KD, and H2A.Z^AP3 ESCs. Student's t-test was performed to generate indicated p values. ***p<0.005, **p<0.01, *p<0.05. Error bars represent standard deviations from a triplicate set of experiments.</p

H2A.Z acidic patch couples chromatin dynamics to gene expression regulation during ESC differentiation.

<p>Schematic depicting H2A.Z dynamics in H2A.Z^WT (A) and H2A.Z^AP3 (B) ESCs and its consequence on gene expression of developmental genes and ultimately lineage commitment. The black arrow represents similar on rate for H2A.Z -containing dimers in H2A.Z^WT and H2A.Z^AP3 ESCs while a bold black arrow represents a greater off rate for H2A.Z^AP3-H2B dimers whereas the hashed black arrow represents weaker off rate for H2A.Z-H2B dimers in H2A.Z^WT. The model demonstrates how the divergent acidic patch domain functions to couple H2A.Z dynamics with the regulation of gene expression programs during ESC differentiation.</p

H2A.Z acidic patch is necessary for regulation of bivalent genes during ESC differentiation.

<p>(A) Heat map displaying genes with absolute fold-changes of >1.5 fold at D0 or D3 in H2A.Z^AP3 EBs relative to H2A.Z^WT. Log2-fold changes in expression of these genes were hierarchically clustered using a Euclidean distance metric. Gene Ontology analysis of the clusters was performed using DAVID. (B) Venn diagrams representing overlap of differentially-regulated genes (>1.5 fold) in H2A.Z^AP3 relative to H2A.Z^WT (green) and genes with the bivalent chromatin mark within +/−2 kb of its TSS (blue) at D0 (top) and D3 (bottom). The p values were calculated by hypergeometric tests. (C) Heat map showing changes in transcript levels of a representative subset of genes (including developmental regulators, pluripotency factors, chromatin regulators and house keeping genes) using Nanostring in H2A.Z^WT, H2A.Z^KD, and H2A.Z^AP3 ESCs at D0 and D3 of EB differentiation. The heat map was generated using log counts from each experiment, which were normalized to the geometric mean of the expression of housekeeping genes <i>Gapdh</i>, <i>Tubb5</i>, and <i>Cltc</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003725#pgen.1003725.s008" target="_blank">Table S3</a>). Genes were then normalized across the 6 experiments. Yellow represents genes that are up-regulated 2 fold or over and blue represents genes down-regulated by 2 fold or more. (D) Boxplots representing the median expression levels of H2A.Z^WT, H2A.Z^KD, and H2A.Z^AP3 D0 and D3 EBs were generated using log transformed, normalized gene expression values of all genes represented in the Nanostring probe set (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003725#pgen.1003725.s008" target="_blank">Table S3</a>). P values were calculated by unpaired student t-test. *p<0.008, **p<0.0008, and ***p<0.0002. The false discovery rate for a p value 0.0074 (<0.008) was generated using Monte Carlo simulation and found to be 3.9% from 10,000 random iterations, suggesting a small but significant change in median expression levels between Day 3 H2A.Z^WT and H2A.Z^KD EBs. qPCR was performed on ChIP DNA generated in H2A.Z^WT and H2A.Z^AP3 ESCs using H3K4me3 (E) and H3K27me3 (F) antibodies. (G) Quantitative qPCR was performed on ChIP-DNA enriched for H3.3 and H3.1 (Flag-HA tagged) using a mixture of Flag and HA antibodies in H2A.Z^WT and H2A.Z^AP3 containing HA-Flag tagged H3.3 and H3.1 transgenes independently. The ratio of H3.3 enrichment relative to H3.1 was plotted for active (<i>Zfpm2, NagK, Thra</i>) and bivalent (<i>Hoxa11, Foxa2, Bmp2</i>) H2A.Z target promoters. Relative enrichment ratios were also plotted for control regions- <i>Pou5f1/Oct4</i> promoter, coding regions for <i>Pou5f1</i> (Pou5f1-CR) and <i>Bmp2</i> (Bmp2-CR) and heterochromatin elements (LINE L1, SINE B2). Student's t-test was performed to generate indicated p values. *p<0.05. Error bars represent standard deviations from a triplicate set of experiments. NS indicates p values >0.5.</p

H2A.Z acidic patch influences chromatin stability and H2A.Z dynamics.

<p>(A) Mean recovery curves generated from independent FRAP experiments performed on H2A, H2A.Z^WT and H2A.Z^AP3 expressing ESCs. Curves were generated by plotting mean fluorescence intensity of the bleached region measured every 30 secs for a duration of approximately 15 mins. (B) Recovery curves were used to determine percentage mobile fractions. (C–E) Similar recovery curves were generated from FRAP analyses performed on H2A.Z^WT, H2A, and H2A.Z^AP3 ESCs subjected to retinoic acid (RA)-induced differentiation for 5 days. (F) Graph representing the estimated mobile fractions of H2A, H2A.Z^WT and H2A.Z^AP3 in ESCs, and RA differentiated cells. *p<0.02, **p<0.004. NS indicates p values >0.6. P values were calculated using the standard unpaired Student t-test. Mean recovery curves were generated from individual curves from n>14 distinct cells for each cell type and condition. (G) Graph representing the estimated mobile fractions of H2A-mCherry in H2A.Z^WT and H2A.Z^AP3 (YFP transgenes) ESCs and day 5 RA differentiated cells. *p<0.04, **p<0.004. P values were calculated using the standard unpaired Student t-test. Mean recovery curves were generated from individual curves from n>12 distinct cells for each cell type and condition.</p

H2A.Z acidic patch is incorporated at lower levels at target genes.

<p>ChIP-Seq analysis of H2A.Z in ESCs shows that the divergent acidic patch residues are required for stable incorporation of H2A.Z (A) Density map of H2A.Z^WT (dark blue), H2A.Z^AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) enrichment at all H2A.Z target genes ordered from most H3K27me3 enriched genes to least H3K27me3 enriched genes in ESCs within the region −4 kb to +4 kb relative to the TSS. The right panel representing the expression levels of the corresponding genes in ESCs generated from RNA-Seq data. Red to white gradient represents genes with high to low expression levels respectively. (B) Average enrichment patterns of H2A.Z^WT, H2A.Z^AP3, H3K4me3, H3K27me3, and RNAP2-Ser5P +/−2 kb around the TSS at bivalent (top) and H3K4me3 (H3K27me3 negative) only promoters (bottom). H2A.Z^WT, H2A.Z^AP3, and H3K27me3 are plotted on the primary axis (right). H3K4me3 and RNAP2-Ser5P are plotted on the secondary axis (left). (C) Average read density plots comparing binding profiles of H2A.Z^WT, H2A.Z^AP3, and input at all H2A.Z target gene promoters in ESCs plotted +/−2 kb relative to TSS. The ChIP-Seq datasets for H2A.Z^WT and H2A.Z^AP3 were generated using GFP antibodies against the YFP transgene. (D) Genome profile of ChIP-Seq reads showing the distribution of H2A.Z^WT (dark blue), H2A.Z^AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) across the HoxA locus- a representative set of H2A.Z target genes. (E) Semi-quantitative western blot of H2A.Z^WT and H2A.Z^AP3 chromatin fractions probed with GFP and H3 (load control) using a range of DNA concentrations (top). Graph quantifying the ratio of transgene levels relative to H3 at the indicated DNA concentrations shows ∼1.85 fold more H2A.Z^WT in chromatin fractions compared to H2A.Z^AP3 (bottom). Fold change was calculated from the average ratio of each transgene to H3. Ratios for H2A.Z^WT/H3 (0.439) and H2A.Z^AP3/H3 (0.255) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) for replicate 1 (R1) were used to calculate the 1.72 (0.439/0.255) fold change between H2A.Z^WT and H2A.Z^AP3. Similar results were obtained for an independent replicate (R2). Ratios for H2A.Z^WT/H3 (0.439) and H2A.Z^AP3/H3 (0.219) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) were used to calculate the 2.0 (0.439/0.219) for R2. Thus, the levels of H2A.Z^WT were on average 1.85-fold higher in chromatin-associated fractions relative to H2A.Z^AP3. (F) Graph showing the ratio of SRCAP and RUVBL1 signal to their respective input signal, from co-immunoprecipitation analyses performed in H2A.Z^WT and H2A.Z^AP3 ESCs (in the endogenous H2A.Z knockdown background). Densitometric measurements of the western blots were performed in ImageJ. The standard deviations were generated from triplicates data points. (G) Nuclei isolated from H2A.Z^WT and H2A.Z^AP3 expressing ESCs were subjected to increasing salt concentrations as indicated. Histones were extracted at these salt concentrations and resolved by SDS-PAGE. Histones were detected by immunoblotting with GFP antibodies.</p