Redundant regulation

Control of gene activity through transcriptional regulatory elements is a major driving force in human evolution. A new study measures nascent transcription directly and shows that sequence, activity and three-dimensional organization of transcriptional regulatory elements all contribute to the evolution of gene activity within primate CD4(+) T cells

A Preliminary Study of DBH (Encoding Dopamine Beta-Hydroxylase) Genetic Variation and Neural Correlates of Emotional and Motivational Processing in Individuals With and Without Pathological Gambling

Background and aims Corticostriatal-limbic neurocircuitry, emotional and motivational processing, dopaminergic and noradrenergic systems and genetic factors have all been implicated in pathological gambling (PG). However, allelic variants of genes influencing dopaminergic and noradrenergic neurotransmitters have not been investigated with respect to the neural correlates of emotional and motivational states in PG. Dopamine beta-hydroxylase (DBH) converts dopamine to norepinephrine; the T allele of a functional single-nucleotide polymorphism rs1611115 (C-1021T) in the DBH gene is associated with less DBH activity and has been linked to emotional processes and addiction. Here, we investigate the influence of rs1611115 on the neural correlates of emotional and motivational processing in PG and healthy comparison (HC) participants. Methods While undergoing functional magnetic resonance imaging, 18 PG and 25 HC participants, all European Americans, viewed gambling-, sad-, and cocaine-related videotapes. Analyses focused on brain activation differences related to DBH genotype (CC/T-carrier [i.e., CT and TT]) and condition (sad/gambling/cocaine). Results CC participants demonstrated greater recruitment of corticostriatal-limbic regions, relative to T-carriers. DBH variants were also associated with altered corticostriatal-limbic activations across the different videotape conditions, and this association appeared to be driven by greater activation in CC participants relative to T-carriers during the sad condition. CC relative to T-carrier subjects also reported greater subjective sadness to the sad videotapes. Conclusions Individual differences in genetic composition linked to aminergic function contribute significantly to emotional regulation across diagnostic groups and warrant further investigation in PG

Determinants of promoter and enhancer transcription directionality in metazoans

Divergent transcription from promoters and enhancers is pervasive in many species, but it remains unclear if it is a general feature of all eukaryotic cis regulatory elements. To address this, here we define cis regulatory elements in C. elegans, D. melanogaster and H. sapiens and investigate the determinants of their transcription directionality. In all three species, we find that divergent transcription is initiated from two separate core promoter sequences and promoter regions display competition between histone modifications on the +1 and -1 nucleosomes. In contrast, promoter directionality, sequence composition surrounding promoters, and positional enrichment of chromatin states, are different across species. Integrative models of H3K4me3 levels and core promoter sequence are highly predictive of promoter and enhancer directionality and support two directional classes, skewed and balanced. The relative importance of features to these models are clearly distinct for promoters and enhancers. Differences in regulatory architecture within and between metazoans are therefore abundant, arguing against a unified eukaryotic model

Determinants of transcription initiation directionality in metazoans

Divergent transcription from promoters and enhancers is pervasive in many species, but it remains unclear if it is a general and passive feature of all eukaryotic cis regulatory elements. To address this, we define promoters and enhancers in C. elegans, D. melanogaster and H. sapiens using ATAC-Seq and investigate the determinants of their transcription initiation directionalities by analyzing genome-wide nascent, cap-selected, polymerase run-on assays. All three species initiate divergent transcription from separate core promoter sequences. Sequence asymmetry downstream of forward and reverse initiation sites, known to be important for termination and stability in H. sapiens, is unique in each species. Chromatin states of divergent promoters are not entirely conserved, but in all three species, the levels of histone modifications on the +1 nucleosome are independent from those on the -1 nucleosome, arguing for independent initiation events. This is supported by an integrative model of H3K4me3 levels and core promoter sequence that is highly predictive of promoter directionality and of two types of promoters: those with balanced initiation directionality and those with skewed directionality. Lastly, D. melanogaster enhancers display variation in chromatin architecture depending on enhancer location, and D. melanogaster promoter regions with dual enhancer/promoter potential are enriched for divergent transcription. Our results point to a high degree of variation in regulatory element transcription initiation directionality within and between metazoans, and to non-passive regulatory mechanisms of transcription initiation directionality in those species

Language at rest: A longitudinal study of intrinsic functional connectivity in preterm children

AbstractBackgroundPreterm (PT) children show early cognitive and language deficits and display altered cortical connectivity for language compared to term (T) children. Developmentally, functional connectivity networks become more segregated and integrated, through the weakening of short-range and strengthening of long-range connections.MethodsLongitudinal intrinsic connectivity distribution (ICD) values were assessed in PT (n=13) compared to T children (n=12) at ages 8 vs. 16 using a Linear Mixed Effects model. Connectivity values in regions generated by the group×age interaction analysis were then correlated to scores on full IQ (FSIQ), verbal IQ (VIQ), verbal comprehension IQ (VCIQ), performance IQ (PIQ), Peabody picture vocabulary test—revised (PPVT­R), and Rapid Naming Composite (RDRL_Cmp).ResultsNine regions were generated by the group×age interaction analysis. PT connectivity significantly increased over time in all but two regions, and they ultimately displayed greater relative connectivity at age 16 than Ts in all areas except the left occipito-temporal cortex (OTC). PTs underwent significant connectivity reductions in the left OTC, which corresponded with worse performance on FSIQ, VIQ, and PIQ. These findings differed from Ts, who did not undergo any significant changes in connectivity over time.ConclusionsThese findings suggest that the developmental alterations in connectivity in PT children at adolescence are both pervasive and widespread. The persistent and worsening cognitive and language deficits noted in the PT subjects may be attributed to the loss of connections in the left OTC

FACT sets a barrier for cell fate reprogramming in Caenorhabditis elegans and human cells

The chromatin regulator FACT (facilitates chromatin transcription) is essential for ensuring stable gene expression by promoting transcription. In a genetic screen using Caenorhabditis elegans, we identified that FACT maintains cell identities and acts as a barrier for transcription factor-mediated cell fate reprogramming. Strikingly, FACT's role as a barrier to cell fate conversion is conserved in humans as we show that FACT depletion enhances reprogramming of fibroblasts. Such activity is unexpected because FACT is known as a positive regulator of gene expression, and previously described reprogramming barriers typically repress gene expression. While FACT depletion in human fibroblasts results in decreased expression of many genes, a number of FACT-occupied genes, including reprogramming-promoting factors, show increased expression upon FACT depletion, suggesting a repressive function of FACT. Our findings identify FACT as a cellular reprogramming barrier in C. elegans and humans, revealing an evolutionarily conserved mechanism for cell fate protection

A splicing-dependent transcriptional checkpoint associated with prespliceosome formation

There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 protein (Prp5p) is an RNA-stimulated adenosine triphosphatase (ATPase) required for prespliceosome formation in yeast. We demonstrate through in vivo RNA labeling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at Ser5 of the largest subunit’s heptad repeat accumulates over introns and that this defect requires Cus2 protein. A similar accumulation of polymerase was observed when prespliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with prespliceosome formation during cotranscriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in wildtype and cloche/npas4l mutant zebrafish embryos

DNA accessibility of cis regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many of the genes that regulate embryonic development have been described, the underlying CRE dynamics controlling their expression remain largely unknown. To address this, we applied single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq) to whole 24 hours post fertilization (hpf) stage zebrafish embryos and developed a new computational tool, ScregSeg, that selects informative genome segments and classifies complex accessibility dynamics. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization, expanding knowledge of regulatory principles between chromatin modalities. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos revealed novel cellular roles for this hemato-vascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work constitutes a valuable resource for future studies in developmental, molecular, and computational biology

A BBP–Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast

The 3′ end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP–Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpΔ56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpΔ56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpΔ56. We propose that a BBP–Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association

Proteomic analysis of the U1 snRNP of Schizosaccharomyces pombe reveals three essential organism-specific proteins

Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30–60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing