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Most transcriptome studies involve sequencing and quantification of steady-state mRNA by isolating and sequencing poly (A) RNA. Although this type of sequencing data is informative to determine steady-state mRNA levels, it does not provide information on transcriptional output and thus may not always reflect changes in transcriptional regulation of gene expression . Furthermore, sequencing poly (A) RNA may miss transcribed regions of the genome not usually modified by polyadenylation which includes many long non-coding RNAs including enhancer RNA (eRNA). Here, we describe nuclear RNA sequencing (nucRNA-seq) which investigates the transcriptional landscape through sequencing and quantification of nuclear RNAs which are both unspliced and spliced transcripts for protein-coding genes and nuclear-retained long non-coding RNAs.

Most transcriptome studies involve sequencing and quantification of steady-state mRNA by isolating and sequencing poly (A) RNA. Although this type of sequencing data is informative to determine steady-state mRNA levels, it does not provide information on transcriptional output and thus may not always reflect changes in transcriptional regulation of gene expression . Furthermore, sequencing poly (A) RNA may miss transcribed regions of the genome not usually modified by polyadenylation which includes many long non-coding RNAs including enhancer RNA (eRNA). Here, we describe nuclear RNA sequencing (nucRNA-seq) which investigates the transcriptional landscape through sequencing and quantification of nuclear RNAs which are both unspliced and spliced transcripts for protein-coding genes and nuclear-retained long non-coding RNAs.

Embryonic stem (ES) cells are regulated by a network of transcription factors that maintain the pluripotent state. Differentiation relies on down-regulation of pluripotency transcription factors disrupting this network. While investigating transcriptional regulation of the pluripotency transcription factor Kruppel-like factor 4 (Klf4), we observed that homozygous deletion of distal enhancers caused a 17-fold decrease in Klf4 transcript but surprisingly decreased protein levels by less than twofold, indicating that posttranscriptional control of KLF4 protein overrides transcriptional control. The lack of sensitivity of KLF4 to transcription is due to high protein stability (half-life >24 h). This stability is context-dependent and is disrupted during differentiation, as evidenced by a shift to a half-life of

Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells.

The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers—SRR18, SRR107, and SRR111—that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus129 × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.

Chromosomes segregate into discrete regions of the nucleus during interphase, which are referred to as “chromosome territories”; however, the level of independence (or conversely, the level of interaction) between the territories is vigorously debated. Chromosome conformation and capture techniques, such as Hi-C, can provide detailed information on the organization of the genome. From an analysis of Hi-C data, we have found that the chromosomes of both mouse and human embryonic stem (ES) cells tend to interact much less than the chromosomes of differentiated cells. DNA FISH experiments with chromosome paints support the Hi-C data showing that chromosome territories in ES cells tend to be farther apart; in fact, a global decrease in inter-chromosomal interactions correlated with an increase in average nuclear size. Surprisingly, the primary transcriptional hardware, RNAPII, did not show clear organizational changes upon cellular differentiation. Direct stochastic optical reconstruction (dSTORM) microscopy in both ES and differentiated cell nuclei showed that RNAPII maintain a constant density and level of clustering. Our data reveal a structural difference in genome organisation between ES cells and differentiated cells and suggest that the genome undergoes a fundamental reorganization after cellular differentiation.

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.