Chengqi Xin

Genome sequence of the date palm Phoenix dactylifera L

Date palm (Phoenix dactylifera L.) is a cultivated woody plant species with agricultural and economic importance. Here we report a genome assembly for an elite variety (Khalas), which is 605.4 Mb in size and covers >90% of the genome (~671 Mb) and >96% of its genes (~41,660 genes). Genomic sequence analysis demonstrates that P. dactylifera experienced a clear genome-wide duplication after either ancient whole genome duplications or massive segmental duplications. Genetic diversity analysis indicates that its stress resistance and sugar metabolism-related genes tend to be enriched in the chromosomal regions where the density of single-nucleotide polymorphisms is relatively low. Using transcriptomic data, we also illustrate the date palm’s unique sugar metabolism that underlies fruit development and ripening. Our large-scale genomic and transcriptomic data pave the way for further genomic studies not only on P. dactylifera but also other Arecaceae plants.

A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing

To compare the two RNA-sequencing protocols, ribo-minus RNA-sequencing (rmRNA-seq) and polyA-selected RNA-sequencing (mRNA-seq), we acquired transcriptomic data-52 and 32 million alignable reads of 35 bases in length-from the mouse cerebrum, respectively. We found that a higher proportion, 44% and 25%, of the uniquely alignable rmRNA-seq reads, is in intergenic and intronic regions, respectively, as compared to 23% and 15% from the mRNA-seq dataset. Further analysis made an additional discovery of transcripts of protein-coding genes (such as Histone, Heg1, and Dux), ncRNAs, snoRNAs, snRNAs, and novel ncRNAs as well as repeat elements in rmRNA-seq dataset. This result suggests that rmRNA-seq method should detect more polyA- or bimorphic transcripts. Finally, through comparative analyses of gene expression profiles among multiple datasets, we demonstrated that different RNA sample preparations may result in significant variations in gene expression profiles.

Transcriptome profiling of the developing postnatal mouse testis using next-generation sequencing

Mammalian testis development is a complex and highly sophisticated process. To study the dynamic change of normal testis development at the transcriptional level, we investigated mouse testes at three postnatal ages: 6 days postnatal, 4 weeks old, and 10 weeks old, representing infant (PN1), juvenile (PN2), and adult (PN3) stages, respectively. Using ultra high-throughput RNA sequencing (RNA-seq) technology, we obtained 211 million reads with a length of 35 bp. We identified 18837 genes that were expressed in mouse testes, and found that genes expressed at the highest level were involved in spermatogenesis. The gene expression pattern in PN1 was distinct from that in PN2 and PN3, which indicates that spermatogenesis has commenced in PN2. We analyzed a large number of genes related to spermatogenesis and somatic development of the testis, which play important roles at different developmental stages. We also found that the MAPK, Hedgehog, and Wnt signaling pathways were significantly involved at different developmental stages. These findings further our understanding of the molecular mechanisms that regulate testis development. Our study also demonstrates significant advantages of RNA-seq technology for studying transcriptome during development.

A novel mechanism of epigenetic regulation: nucleosome-space occupancy.

Nucleosome positioning around the gene space (or transcriptional unit) plays a crucial role for gene regulation but we do not know if the spatial organization--nucleosome-space occupancy or nucleosome density in a defined sequence unit length--contributes to the regulation complexity of mammalian gene expression. Using our own rmRNA-Seq (ribosomal RNA-minus RNA sequencing) and publically available ChIP-Seq (H3) data from mouse stem cells, we discovered a non-random distribution of nucleosomes along chromosomes, and further genome-wide studies on histone modifications, DNA methylation, transcriptional activity, gene density, and base compositional dynamics, demonstrated that nucleosome-space occupancy of genomic regions--clustered genes and their intergenic spaces--show distinctive features, where a high occupancy coincides with active transcription, intensive histone modifications, poor DNA methylation, and higher GC contents as compared to the nucleosome-poor regions. We therefore proposed that nucleosome-space occupancy as a novel mechanism of epigenetic gene regulation, creating a vital environment for transcriptional activation.

Comparative analyses of H3K4 and H3K27 trimethylations between the mouse cerebrum and testis.

The global features of H3K4 and H3K27 trimethylations (H3K4me3 and H3K27me3) have been well studied in recent years, but most of these studies were performed in mammalian cell lines. In this work, we generated the genome-wide maps of H3K4me3 and H3K27me3 of mouse cerebrum and testis using ChIP-seq and their high-coverage transcriptomes using ribominus RNA-seq with SOLiD technology. We examined the global patterns of H3K4me3 and H3K27me3 in both tissues and found that modifications are closely-associated with tissue-specific expression, function and development. Moreover, we revealed that H3K4me3 and H3K27me3 rarely occur in silent genes, which contradicts the findings in previous studies. Finally, we observed that bivalent domains, with both H3K4me3 and H3K27me3, existed ubiquitously in both tissues and demonstrated an invariable preference for the regulation of developmentally-related genes. However, the bivalent domains tend towards a “winner-takes-all” approach to regulate the expression of associated genes. We also verified the above results in mouse ES cells. As expected, the results in ES cells are consistent with those in cerebrum and testis. In conclusion, we present two very important findings. One is that H3K4me3 and H3K27me3 rarely occur in silent genes. The other is that bivalent domains may adopt a “winner-takes-all” principle to regulate gene expression.

Thousands of Novel Transcripts Identified in Mouse Cerebrum, Testis, and ES Cells Based on ribo-minus RNA Sequencing

The high-throughput next-generation sequencing technologies provide an excellent opportunity for the detection of less-abundance transcripts that may not be identifiable by previously available techniques. Here, we report a discovery of thousands of novel transcripts (mostly non-coding RNAs) that are expressed in mouse cerebrum, testis, and embryonic stem (ES) cells, through an in-depth analysis of rmRNA-seq data. These transcripts show significant associations with transcriptional start and elongation signals. At the upstream of these transcripts we observed significant enrichment of histone marks (histone H3 lysine 4 trimethylation, H3K4me3), RNAPII binding sites, and cap analysis of gene expression tags that mark transcriptional start sites. Along the length of these transcripts, we also observed enrichment of histone H3 lysine 36 trimethylation (H3K36me3). Moreover, these transcripts show strong purifying selection in their genomic loci, exonic sequences, and promoter regions, implying functional constraints on the evolution of these transcripts. These results define a collection of novel transcripts in the mouse genome and indicate their potential functions in the mouse tissues and cells.

Sequence Signatures of Nucleosome Positioning in Caenorhabditis elegans

Our recent investigation in the protist Trichomonas vaginalis suggested a DNA sequence periodicity with a unit length of 120.9 nt, which represents a sequence signature for nucleosome positioning. We now extended our observation in higher eukaryotes and identified a similar periodicity of 175 nt in length in Caenorhabditis elegans. In the process of defining the sequence compositional characteristics, we found that the 10.5-nt periodicity, the sequence signature of DNA double helix, may not be sufficient for cross-nucleosome positioning but provides essential guiding rails to facilitate positioning. We further dissected nucleosome-protected sequences and identified a strong positive purine (AG) gradient from the 5′-end to the 3′-end, and also learnt that the nucleosome-enriched regions are GC-rich as compared to the nucleosome-free sequences as purine content is positively correlated with GC content. Sequence characterization allowed us to develop a hidden Markov model (HMM) algorithm for decoding nucleosome positioning computationally, and based on a set of training data from the fifth chromosome of C. elegans, our algorithm predicted 60%-70% of the well-positioned nucleosomes, which is 15%-20% higher than random positioning. We concluded that nucleosomes are not randomly positioned on DNA sequences and yet bind to different genome regions with variable stability, well-positioned nucleosomes leave sequence signatures on DNA, and statistical positioning of nucleosomes across genome can be decoded computationally based on these sequence signatures.

Hydroxyurea-induced global transcriptional suppression in mouse ES cells

Hydroxyurea (HU), as a therapeutic medicine, has been extensively used clinically. To further survey molecular mechanisms of HU treatment, we analyzed global transcriptomic alteration of mouse ES cells in response to the treatment using high-throughput sequencing. We show that the global transcriptional activity is significantly suppressed as cells are exposed to HU treatment and alters multiple key cellular pathways, including cell cycle, apoptosis and DNAs. HU treatment also alters alternative splicing mechanisms and suppresses non-coding RNA expression. Our result provides novel clues for the understanding of how cells respond to HU and further suggests that high-throughput sequencing technology provides a powerful tool to study mechanisms of clinical drugs at the cellular level.

Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development.

MicroRNAs (miRNAs) play crucial roles in multiple stages of plant development and regulate gene expression at posttranscriptional and translational levels. In this study, we first identified 238 conserved miRNAs in date palm (Phoenix dactylifera) based on a high-quality genome assembly and defined 78 fruit-development-associated (FDA) miRNAs, whose expression profiles are variable at different fruit development stages. Using experimental data, we subsequently detected 276 novel P. dactylifera-specific FDA miRNAs and predicted their targets. We also revealed that FDA miRNAs function mainly in regulating genes involved in starch/sucrose metabolisms and other carbon metabolic pathways; among them, 221 FDA miRNAs exhibit negative correlation with their corresponding targets, which suggests their direct regulatory roles on mRNA targets. Our data define a comprehensive set of conserved and novel FDA miRNAs along with their expression profiles, which provide a basis for further experimentation in assigning discrete functions of these miRNAs in P. dactylifera fruit development.

The association between H3K4me3 and antisense transcription.

Histone H3 lysine 4 trimethylation (H3K4me3) is well known to occur in the promoter region of genes for transcription activation. However, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3′ end of actively transcribed (sense) genes, named as 3′-H3K4me3. 3′-H3K4me3 is associated with ∼15% of protein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II (RNAPII) binding sites and 5′-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3′-H3K4me3 is associated with the initiation of antisense transcription. Furthermore, 3′-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3′-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3′ end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3′-H3K4me3 modification. More importantly, we observed the 3′-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3′-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in vertebrate. Therefore, we speculate that these 3′-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3′-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.